Function of metallothioneins in carp Cyprinus carpio from two field sites in Western Ukraine

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 1425–1432

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Function of metallothioneins in carp Cyprinus carpio from two field sites in Western Ukraine$ Halina I. Falfushynska, Oksana B. Stoliar  Department of Chemistry, Ternopil National Pedagogical University, M. Kryvonosa Street 2, Ternopil 46027, Ukraine

a r t i c l e in fo

abstract

Article history: Received 15 September 2008 Received in revised form 28 January 2009 Accepted 14 February 2009 Available online 7 April 2009

The aim of the present study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MT) from the liver and gills of carp Cyprinus carpio L. in rural (R) and industrial (I) sites in Western Ukraine. The MT is represented by two chromatographic forms, the features of which exhibit seasonal rather than spatial dependence. The pronounced differences between the sites were due to the lower levels of Zn in the liver and the higher levels of Zn in MT of carp from site I, providing evidence of the higher overall anthropogenic impact here that leads to the distortion of this essential metal accumulation and to the activation of metal-binding function of MT. In spring, higher levels of Cu and Cd in MT and in the tissues were reflected at site R probably as the result of the permitting pollution here. The principal component analysis demonstrated the correlation of MT-bound metal levels to their levels in water and the absence of such relation for general tissue metal levels for Zn. & 2009 Elsevier Inc. All rights reserved.

Keywords: Environmental stress Carp Heavy metals Metallothioneins

1. Introduction Heavy metals constitute a major problem with regard to environmental contamination because they are toxic and tend to accumulate in living organisms (Hogstrand and Haux, 1991; Bury et al., 2003). Therefore, determination of the content of these metals in aquatic animals is an important part of water-quality monitoring. On the other hand, the levels of metals in an organism may reflect not only the water pollution caused by these metals but also the general dependence on the metabolic state and, therefore, the effect of other pollutants. Particularly, essential metals may be substituted by nonessential metals in fish taken from polluted areas (Hanson, 1997). There is little information about metal accumulation in the biota of Ukrainian water bodies (Lukashev, 2006; Stolyar et al., 2008). The important role in the intracellular binding of Zn, Cu, and cadmium (Cd) is attributed to low molecular weight, thermostable, sulfhydryl-rich proteins metallothioneins(MT) (Kagi and Schaffer, 1988). The synthesis of MT is often induced when organisms are exposed to heavy metals, especially Cd, both in the laboratory and in the field. Therefore, the MT are proposed to be a specific ‘‘biomarker’’ responsive to metal pollution (Roesijadi, 1992; Viarengo et al., 1999; Fernandes et al., 2008). The recent

$ Funding sources: This work was granted by Ministry of Education and Science of Ukraine (Ukrainian-Greece Scientific and Technical Cooperation Joint Project #M-65/2006). All studies were conducted in accordance with national and institutional guidelines for the protection of animal welfare.  Corresponding author. Fax: +380 352 436055. E-mail address: [email protected] (O.B. Stoliar).

0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.02.013

data confirm that MT may also be used as biomarkers in case of contamination with some pesticides or other substances that provoke oxidative stress (Cavaletto et al., 2002; Paris-Palacios et al., 2003). At the same time, in some polluted areas the MT induction in fish was not observed (Rotchell et al., 2001) or their content was not elevated even when MT gene transcription was increased (Hansen et al., 2006). With a view to including MT in biomonitoring programs, the organisms most commonly studied are bivalves (Roesijadi, 1992; Viarengo et al., 1999). Several studies of metal accumulation by MT of fish in field conditions have been reported (Olsvik et al., 2001; Hansen et al., 2006; Fernandes et al., 2008). Fish are interesting objects for biomonitoring because they are unique among the vertebrates, as they have two routes of metal acquisition: from their diet and from water through the gills (Bury et al., 2003). Moreover, since fish provide a valuable source of dietary nutrients for people, the study of the level of contaminants in fish is of great practical importance. Although the current ecological conditions in the studied area (Western Ukraine) are generally regarded as favorable, both the presence of the small factories and farms, as well as the poorly controlled urban waste disposal practices, represents a potential danger for the surface water. The effect of site dependent and seasonal aquatic pollution in this area was confirmed by our recent studies of heavy metal accumulation and of MT state in frog (Stolyar et al., 2008; Falfushinska et al., 2008) and of oxidative stress biomarkers in carp (Falfushynska and Stolyar, 2008). The purpose of this study was to ascertain heavy metal fluctuations and the participation of MT in the binding of metals in the tissues of a popular commercial fish carp Cyprinus carpio in

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different seasons and at two sites. To our knowledge, this is the first study to use the features of fish MT for the estimation of the quality of aquatic environment of Ukraine.

2. Materials and methods 2.1. Experimental groups The experiments were carried out during three seasons (May (spring), July (summer), and September (autumn)) of year 2007. Two-year-old male specimens of carp (Cyprinus carpio L.) of 2372 cm length with weight 276724 g were collected from two ponds, one was the rural pond (R) near the springs of the river Seret (village Zalizci, 491490 N, 251230 E) and the other was the pond in the site with the complex industrial and urban pollution (I) located in the mid stream in the same river after the city Ternopil with a population of about 2,20,000 (village Ostriv, 491330 N, 251370 E) (Fig. 1). This pond receives urban wastes. The motorway and railway lines with intense traffic, as well as a number of different manufactures and repair shops, are situated in the surrounding area. The sampling for analysis was carried out simultaneously in both groups. About 20 individuals from each site were transported to the laboratory in 40 L cages with aerated native water (dissolved oxygen levels were 8.6770.5 mg/L) and analyzed within a day after the sampling procedure. The condition factor (CF) of fish was calculated from the following equation: CF ¼ [body weight (g)/total length3 (cm)]  100. The mortality was not observed until dissection. Water samples were also collected in plastic containers, sealed and transferred to the laboratory in iced packs to determine physicochemical properties. All studies were conducted in accordance with national and institutional guidelines for the protection of animal welfare. The project was approved by the Institutional Animal Care and Use Committee. The six fish in each group were killed by a blow to the head and the spinal cord severed. Liver and gills were immediately removed and analyzed. Each procedure of tissue analysis was carried out at a temperature around 4 1C. Serum albumin, cytochrome c, insulin, phenylmethylsulfonyl fluoride (PMSF), b-mercaptoethanol, metallothionein from rabbit liver, DEAE-cellulose, and Sephadex G-75 were purchased from Sigma. All other chemicals were of analytical grade.

2.2. Metallothioneins analysis MT were obtained as thermostable proteins by consequent steps of sizeexclusion on Sephadex G-75 and anion-exchange liquid chromatography on DEAEcellulose (Brouwer et al., 1995). A 5% homogenate (w/v) from 1.0 g of tissue, joint in aliquot quality from five individuals of group, was prepared in ice-cold 10 mM Tris–HCl buffer, pH 8.0, containing 10 mM 2-mercaptoethanol and 0.1 mM PMSF for the inhibition of proteolysis. The homogenate was centrifuged at 10,000g for 45 min at 4 1C. The supernatant was incubated under 85 1C for 5 min and subsequently centrifuged at 10,000g for 45 min at 4 1C. The obtained thermostable supernatant in a volume 3 mL was applied to MT purification. Chromatography on a Sephadex G-75 column (1.5 cm  50 cm) was carried out in the same buffer containing 0.02% NaN3 at a flow rate of 0.33 mL/min. Fractions (5 mL) were collected and analyzed for absorbance at 280 and 254 nm. Column calibration was

achieved by applying a mixture of the following standards: serum albumin (67.0 kDa), cytochrome c (12.3 kDa), and insulin (5.8 kDa). The fractions of peak with high absorbance at 254 nm and comparative high density ratio D254/D280 identified as MT-containing peak (Kagi and Schaffer, 1988) were pooled (total 10 mL) and applied to anion-exchange chromatography on a column (1.5 cm  50 cm), packed with DEAE-cellulose. After removing of non-bound proteins (in the volume of buffer 70 mL) elution was carried out in gradient mode with 0–1.0 M NaCl in a 10 mM Tris–HCl (pH 8.0) containing 10 mM 2-mercaptoethanol and 1 mM 2-isopropanol at a flow rate of 0.5 mL/min. The fractions (5 mL) of each peak with high absorbance at 254 nm were pooled (total 10 mL) for the ultraviolet (UV) absorption spectra and metal determination.

2.3. Heavy metal determination To determine the metal concentration in the water, samples of water (50 mL, acidified with 50 mL of HNO3 and concentrated 10 times) were digested with 5 mL HNO3 for 3 h at 105 1C. The determination of metals in fresh weighted tissues (500 mg) and pooled eluate of each MT fraction (10 mL) in each group of animals was also carried out after their digesting with 5 mL HNO3 for 3 h at 105 1C, using hermetic acid-cleaned Teflon bomb. The concentration of zinc (Zn) and copper (Cu) was analyzed by an atomic absorption spectrophotometer (spectrophotometer C-115, ‘‘Lomo’’, Russia), while the cadmium concentration was analyzed by graphite furnace atomic absorption spectrometry (S-600, ‘‘Selmi’’, Ukraine). Detection limits for metals were 0.1 mg/g fresh weight (FW) for tissue and 1 mg/L for water. Quality control was assessed by Quality Control Sample for trace metal and method of Standard Addition (http://www.dentalmercury.com/245_1.pdf). The hardness of water was measured by a standard analytical test (www.epa.ie/ rivermap/docs/Parameters.pdf). Metal concentration in the water was expressed as mg/L and that in the tissues and MT – as mg/g (fresh weight, FW), in MT also as nmol/g (FW). 2.4. Statistical analysis Water and all MT analyses on the joint samples were carried out in triplicate and all the other measurements on 6 animals and were expressed as means7standard deviation (SD). Data were tested for normality and homogeneity of variance using the Kolmogorov–Smirnov and the Levene tests, respectively. Twoway analysis of variance (ANOVA) with the post-hoc Tukey HSD test was used to determine whether there were differences in the individual biochemical variables among sites and seasons and the interaction between two factors. For detection of correlation, Pearson’s correlation test was also performed. Data were subjected to a principal component analysis (PCA) to determine the relation between metal levels in the water, tissues and MT. All statistical calculations were performed on the separate data from each individual with SPSS 15.0 software, Statistica v 6.0 and Excel for Windows-2000.

3. Results 3.1. Chemical analysis of water The analysis of the metal content in the water taken from the two sites pointed out its fluctuations between seasons and sites (Table 1). The lowest Cu and Zn levels and the highest Cd level were reflected in summer. The hardness at the R site in two Table 1 Chemical parameters of water samples from two sites, M7SD, N ¼ 3.

Fig. 1. Localization of the sampling sites of the carp Cyprinus carpio in Ternopil region. R – rural site and I – industrial site. The map from ukrainainkognita.org.ua/ pzf/Ternopil.htm was utilized.

Parameters

Site

Spring

Hardness (mM CaCO3)

R I

6.270.9 5.770.6

Cu (mg/L)

R I

13.271.2 13.671.1

Zn (mg/L)

R I

289.6718.4 79.675.4b

Cd (mg/L)

R I

o1.0 o1.0

Summer

Autumn

11.671.2a 5.670.4b

14.171.8a 5.570.5b

4.870.3a 1.670.1a,b

6.370.5a 15.371.3b

26.771.9a 75.775.2b

30.172.9a 84.074.8b

2.170.2 2.870.1b

1.170.1 1.170.1

a Between seasons statistically significant differences compared to spring values. b Between sites statistically significant differences. Always po0.05.

ARTICLE IN PRESS H.I. Falfushynska, O.B. Stoliar / Ecotoxicology and Environmental Safety 72 (2009) 1425–1432

seasons and Cu levels (except at site I in summer) exceeded the quantity recommended for water intended for human consumption (Council Directive 98/83/EC).

3

1427

R-site I-site

2.5

CF of the fish at site R was 1.770.3, 1.870.2, and 2.770.3, and at I site 2.070.3, 2.070.1, and 2.270.2 in spring, summer, and autumn, respectively (differences between data were significant (po0.05) for autumn/spring in both sites and for two sites in autumn). Seasonal dependence was reflected for all metal concentrations in the carp tissues (Table 2) with similar regularity in both sites (r40.53, po0.05). The Zn and Cd contents demonstrated the prominent fluctuation from spring to summer/autumn. The Cu content had another dynamic with the lowest mean value in summer. The differences between sites were observed in separate seasons in both tissues. The lower Zn content in the tissues of carp from site I during summer–autumn and the higher Cu and Cd contents (as compared with the counterpart site) in the tissues of carp from site R in spring were the most prominent among them. According to ANOVA, the overall dependence on the site was reflected for Cu, Zn, and Cd contents in both tissues (po0.05); however, only the Cd content in both tissues and the Zn content in the liver demonstrated the site  season interaction (po0.05).

Absorbance. 254 nm

3.2. Morphological indices of carp and metal concentrations in the tissues

2

1.5

1

0.5

0 0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

1.9

2.1

2.3

2.5

Ve/Vo

2.5 R-site I-site

3.3. The separation of metallothioneins Gel-filtration of the thermostable solutions from both liver and gills of carp revealed low molecular weight peak, which had apparent molecular mass of 7 kDa (Fig. 2). It was identified as the MT-containing peak based upon its spectral features (comparatively high density ratio D254/D280), thermostability, and molecular weight (Kagi and Schaffer, 1988). The density ratio D254/D280 was 1.370.1 for the high molecular weight peak and 1.970.2 for the MT-containing peak. Table 2 The distribution of Cu, Zn, and Cd between the metallothioneins and other tissue compounds (mg/g FW). Metal Site Liver

9.471.2 6.671.0b 115.0728.4 86.3722.1 0.470.1 0.270.0b

Summer Cu R I Zn R I Cd R I

4.570.4a 4.570.4a 603.3791.7a 365.1742.5a,b 0.370.0a 0.370.0a

Autumn Cu R I Zn R I Cd R I

4.670.5a 4.370.3a 213.578.7a 168.7720.8a,b 0.870.1a 0.870.1a

1.5

1

0.5

Gills

Total content Metallothioneins Spring Cu R I Zn R I Cd R I

Absorbance. 254 nm

2

1.770.1 (18.1%) 1.270.1b (18.2%) 9.970.8 (8.6%) 21.371.9b (24.7%) 0.370.0 (75.0%) 0.170.0b (50.0%)

Total content Metallothioneins

2.470.7 3.070.2 172.0727.1 127.9732.8 0.570.1 0.270.0b

1.170.1(45.8%) 0.970.1 (30.0%) 13.571.2 (7.8%) 16.971.4 (13.2%) 0.370.0 (60.0%) 0.170.0b (50.0%)

0.670.1a (13.3%) 1.670.3a 0.770.1a (15.6%) 2.170.4a 3.170.2a (0.5%) 421.3769.4a 4.970.2a,b (1.3%) 319.2737.3a,b 0.270.0a (67.0%) 0.470.0 0.370.0a,b (100.0%) 0.370.0a 1.370.1 (28.3%) 1.870.2 (41.9%) 8.670.7 (4.0%) 15.171.2b (9.0%) 0.670.0 (77.0%) 0.770.0b (88.0%)

0.370.0a (18.8%) 0.570.1a,b (23.8%) 3.070.2a (0.7%) 3.870.3a,b (1.2%) 0.270.0a (50.0%) 0.370.0a,b (100%)

1.870.2 0.670.1a (33.3%) 2.670.3b 1.170.1b (42.3%) 255.0730.1 5.570.4a (2.2%) 276.0743.5a 8.070.7a,b (2.9%) 1.070.2a 0.270.0a (20.0%) 0.870.1 0.770.1a,b (88.0%)

M7SD, N ¼ 6 for total content, and N ¼ 3 for metallothioneins.

0 0.9

1.1

1.3

1.5

1.7 Ve/Vo

Fig. 2. Elution profile on Sephadex G-75 of the thermostable extract from the liver (A) and gills (B) of carp. Arrows highlight the elution volume of markers: 67.0, 12.3, and 5.8 kDa. MT-contained fraction, which is indicated by the bar, was pooled for ion-exchange chromatography.

The ion-exchange chromatography of the MT-containing fraction demonstrated two peaks (Fig. 3), which were rather stable both seasonally and spatially (0.23–0.25 M NaCl for MT-1 and 0.37–0.40 M NaCl for MT-2), with the exception of autumn, when the earlier elution (MT-2a) or division into two peaks was observed for MT-2. The obtained profile of elution was similar to that of standard rabbit MT. All forms of MT demonstrated similar UV-spectra with a high level of absorption in the middle of the UV-spectrum, indicating the presence of characteristic metal– thiolate clusters (Kagi and Schaffer, 1988). These spectra were different from the spectra of high molecular weight fraction obtained by gel-filtration (Fig. 4).

ARTICLE IN PRESS H.I. Falfushynska, O.B. Stoliar / Ecotoxicology and Environmental Safety 72 (2009) 1425–1432

1.2

0.45

1.0

0.40 0.35

1.2 0.30 0.25

0.8

0.20

Absorbance. 254 nm

0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10

MT-1 MT-2

1.2 1.0 0.8 0.6 0.4 0.2

R-site

I-site

C(NaCl) V. ml

0.0 65 2.0

90

0.20 0.15 65

1.6 1.4

0.25

120

150

90

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2

C(NaCl) V. ml

120

150

0.10

180 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10

MT-1 MT-2

R-site

65

180

90

I-site

C(NaCl) V. ml

120

150

180

1.8

0.55

MT-1

I-site

-0.2

180

C(NaCl), M

150

Absorbance. 254 nm

120

0.30

0.4

R-site

0.10 90

0.35 0.6

C(NaCl) V. ml

0.0 65

0.40

0.8

0.0

0.15 I-site

0.45

MT-2

0.2

0.4 R-site

0.50

MT-1

0.55 0.50

0.40 1.2

0.35 MT-2a

0.8

0.30

MT-2

0.25

C(NaCl), M

Absorbance. 254 nm

0.45

0.20

0.4 R-site

I-site

C(NaCl) V. ml

0.10 90

120

150

1.4

0.45 0.40

1.0

0.35

MT-2a MT-2

MT-1

0.6

0.30 0.25 0.20

0.2

0.15

0.0 65

Absorbance. 254 nm

0.50 1.6

C(NaCl), M

Absorbance. 254 nm

1.6

0.50

C(NaCl), M

MT-2

Absorbance. 254 nm

MT-1

2.0

0.55

1.4

0.55

C(NaCl), M

2.4

R-site

I-site

0.15

C(NaCl) V. ml

0.10

-0.2 65

180

C(NaCl), M

1428

90

120

150

180

Fig. 3. DEAE-cellulose chromatography profiles of metallothioneins from carp tissues (A, C, E, liver; B, D, F, gills) in spring (A, B), summer (C, D) and autumn (E, F). Elution profiles of proteins are shown by the solid lines, a gradient of elution buffer – by the dotted line. Arrows indicate the elution volume of standard metallothionein from rabbit liver.

3.4. The participation of metallothioneins in metal binding

HMW

MT-1

MT-2

1.8 1.6 Absorbance

1.4

R site

1.2

I site

1 0.8 0.6 0.4 0.2 220 240 260 280 300 320

225 245 265 285 305

0 210 230 250 270 290 310

The analysis of the distribution of metals between MT and other compounds in the tissues (Table 2, Fig. 5) demonstrates that their average rate in MT was decreasing in the range Cd4Cu4Zn. The comparison of three metal levels in MT reflects that Zn was the most abundant metal. The Cu content in MT was about 5–19 times less. The molar ratio Cd:(Zn+Cu) in MT separate forms is less than 1:13. The differences between sites were reflected in the Zn content in MT with the higher level at site I for the liver in all seasons and for the gills in autumn. The Cu and Cd levels in MT were higher in spring at site R; however, in other seasons they were higher at site I or were the same for both sites. ANOVA confirms the overall effect of site and season and their interaction (po0.01) for all metal indices in MT. According to the PCA, the two main principal components explained 77.8% of the total variation of indices for the liver and 74.7% for the gills (Fig. 6). The first principal component was positively correlated with the Cu and Zn content in MT and in the water (with the one exception), as well as with the Cu content in the tissue, and negatively correlated with the Zn

2

Wavelength. nm Fig. 4. UV-spectra of hepatic proteins of carp from the R and I sites in spring. HMW – high molecular weight fraction obtained by chromatography on Sephadex G-75; MT-1 and MT-2 – forms obtained by chromatography of metallothioneins on DEAE-cellulose.

ARTICLE IN PRESS H.I. Falfushynska, O.B. Stoliar / Ecotoxicology and Environmental Safety 72 (2009) 1425–1432

35 Summer

a,b

20 15

b

10 a a R

400

I

Summer

Spring

Autumn

MT-2a/2

MT-1

a,b b

10 a a,b

5

a a

a

a,b

a,b

0

I

R

Summer

15

aa,b

a,b

R

Cu (MT), nmol/g FW

MT-2a/2

MT-1

25

0

R

I

250

Autumn

I

R

Spring

I

R

Summer

I

Autumn

350 300

b

MT-1

MT-2a/2

250 b

200 150 a

100 50

b

0

a

I

R 7

a,b a

a

Spring

I

R

a

a,b

MT-1

a

4 3 a,b a,b

1

BDL

a

0 R

I

a,b

R

MT-2a/2

100

a,b a

b

50

a,b a

a b

I

R

7

Autumn

MT-2a/2

2

MT-1

150

I

6 5

200

0 R

Summer

Zn (MT), nmol/g FW

Zn (MT), nmol/g FW

Spring

30

5

Cd (MT), nmol/g FW

20

Autumn

Cd (MT), nmol/g FW

Cu (MT), nmol/g FW

Spring

1429

a

R

Spring

I

a

a,b

I

R

Summer

Autumn

6 MT-1

5

MT-2a/2 a,b

4 3 2

a,b a

a

1

BDL

a,b

a

0 I

R

I

R

I

R

I

R

I

Fig. 5. The distribution of Cu (A, B), Zn (C, D), and Cd (E, F) between metallothioneins separate forms MT-1 and MT-2/2a in the carp liver (A, C, E) and gills (B, D, F), nmol/g FW: (a) Between seasons statistically significant differences compared with spring values; (b) between sites statistically significant differences. BDL – below detection limit. Always po0.05.

content in the tissue and with hardness of water (in the gills). The second principal component was negatively correlated with CF of fish.

4. Discussion Western Ukraine, unlike eastern and central regions of Ukraine, does not host large-scale industries; therefore, the environmental quality as a whole is characterized as clean or slightly polluted (MedEcoPortal http://www.health.gov.ua). Our data, presented both in the present and in another recent study (Stolyar et al., 2008), also did not reflect any considerable pollution in the investigated area, with the exception of elevated Cu levels and water hardness in some cases. The measuring of the pH, nitrite, nitrate, and phosphate content, oxidizability in the same sites (Falfushynska and Stolyar, 2008) demonstrated that their levels were within acceptable parameters for human consumption (Council Directive 98/83/EC) levels for the quality of water intended for human consumption (http://www.emwis.org/IFP/ law_EU.htm), with the exception of oxidizability, which was

significantly elevated in spring at both sites and in summer at site R. In various studies, significant correlations have been observed between exposure to contaminants, both natural and experimental, and metal levels in the tissues (Hogstrand and Haux, 1991; Wong et al., 2001; Farombi et al., 2007; Atli and Canli, 2007; Costa et al., 2008; Fernandes et al., 2008). However, the data describing the metal levels in the feral aquatic animals are connected mostly to heavily polluted sites and reflected the changes of Cd levels, while no alteration of the distribution of Zn and Cu was demonstrated in the tissues (Olsson and Haux, 1986; Dethloff et al., 1999; Rotchell et al., 2001; Reynders et al., 2008). For example, the variations in the heavy metal load of bream Abramis brama L. collected from Lake Balaton could be attributed to the seasonal change in the condition factor of fish rather than to variations in the pollutant load of the site (Farkas et al., 2003). Ko¨ck et al. (1996) also demonstrated that the seasonal patterns of metal accumulation in arctic char Salvelinus alpinus were related to increasing metabolic rates during summer and did not correlate with seasonal variations in the metal concentrations in lake water or in the diet.

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1.0

Zn-w Cu-t

0.5 Factor 2 : 22.73%

Zn-t

0.0 Cu-w

Hd

Cu-MT Zn-MT

-0.5 CF

-1.0 -1.0

-0.5

0.0

0.5

1.0

Factor 1 : 52.05% 1.0

Zn-w

Factor 2 : 16.93%

0.5 Zn-t

0.0

Zn-MT Cu-MT Cu-w Cu-t

Hd

-0.5

CF

-1.0

-1.0

-0.5

0.0 Factor 1 : 57.43%

0.5

1.0

Fig. 6. Principal component analysis (PCA) of the parameters data set for the liver (A) and for the gills (B). Parameters: the metal content in the tissue (Cu-t), in MT (Cu-MT), and in water (Cu-w); hardness (Hd), conditional factor (CF). The arrows indicate parameters having significant factorial weights 40.7.

In our study, the general Cu levels in the tissues reflect its levels in the water, confirmed by PCA (Fig. 6). Despite Cd content being close to the detection limit, its high ability for accumulation from the surroundings is demonstrated. On the other hand, the metal characteristics were not related to CF. The most probable explanation for the higher levels of Cu and Cd detected in spring at site R is that the effluents with fungicides that include Cu and Cd in their formula are usually sprayed mid- to late winter and the rains or thawed waters of this season transfer them to the wetlands. The liver is the main organ for Cu and Cd storage; therefore, the intermittent pollution by these metals may provoke the rise of their concentration in liver tissue. However, in summer and in autumn, the levels of Cu and Cd reflect higher pollution at

site I. Our previous data about the hepatic metal contents in the liver of frog from the same sites also demonstrated higher levels of Cu at site R in spring and higher concentration of Cu and Cd at the urban site in summer and autumn (Stolyar et al., 2008). On the other hand, it seems that Zn levels in the tissues cannot reflect its levels in the water. In the study of Reynders et al. (2008), no pronounced differences in Zn concentration in the tissues of four roach populations taken from locations with different levels of contamination were revealed, while the differences for Cd were significant. Based on our recent data from fish and frog, we concluded that continuous environmental pressure is exerted at site I (Falfushinska et al., 2008; Falfushynska and Stolyar, 2008; Stolyar et al., 2008), distorting the bioavailability of the essential metals. The facts concerning oxidative stress in carp and the neurotoxicity of water environment in summer (Falfushynska and Stolyar, 2008) deserve particular attention. Similar observations were made based on piscine liver in polluted marine areas (Hanson, 1997). Organic contaminants such as polycyclic aromatic hydrocarbons and various chlorinated pesticides are shown to be the disruptive agents of hepatic metabolism in this report. Our data are also supported by the results of Eastwood and Couture (2002), who observed that the yellow perch Perca flavescens from the most metal-contaminated lakes exhibited lower indicators of physical condition than fish from cleaner lakes. As we can see in our study, the relation between the accumulation of metals and morphological indices (CF) is also absent despite CF being included in the set of significant characteristics by PCA for both tissues. The absence or positive relation between Zn content in the tissues and the hardness of the water observed in our study (Fig. 6) confirms also an ineffective regulation of the Zn content in the tissues, because usually hardness and Zn content appear to be negatively correlated (Eastwood and Couture, 2002). The intracellular storage of Zn, Cu, and Cd is connected to the function of MT. The extremely high ability of MT to bind Cd is well known (Hogstrand and Haux, 1991; Olsvik et al., 2000). In our study, the elevation of the content and/or the rate of Cd in MT, especially at site R in spring and at site I in summer and autumn, probably reflect the selective affinity and the low ability to eliminate this metal (Kraemer et al., 2006). On the other hand, even at the highest rate in autumn at site I, the tissues of carp contain much less than one ion of Cd per molecule of MT, according to the stoichiometry of MT metal composition (Kagi and Schaffer, 1988). So, in our situation, the level of Cd is not ecologically dangerous; however, the possibility of its bioaccumulation in the food chain and the sensitivity of carp make the study of Cd accumulation in this animal an important part of biomonitoring. For Zn and Cu, the close correlation of its content in the water and MT of both tissues was demonstrated by PCA. Moreover, the site  season interaction was reflected for Zn and Cu content in MT in contrary to its content in tissue (except for Zn in the liver). In fact, the metal content in MT of carp is more suitable for the appreciation of the pollution than its general content in the tissues, especially for Zn. The absence of a correlation between Zn content in the tissue and MT was demonstrated also for another lower vertebrate, turtle, despite positive correlation between content in the tissues and MT for Cu and Cd being found (Andreani et al., 2008). The activation of Zn-binding function of MT in fish at site I through three seasons, despite its lower levels in the liver, may reflect the response to compounds other than heavy metal compounds typical for the mixed urban and industrial pollution (Cavaletto et al., 2002; Paris-Palacios et al., 2003). The particular situation concerning increased Cu and Cd binding was observed for MT in spring at site R. Therefore, we may conclude that the elevation of MT metal-binding capacity is an adaptive answer to

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the overall pollution at site I and to the permitted pollution by Cu and Cd at site R in spring. It must be underlined that Cu is one of the most toxic metals for fish (Couture and Rajotte, 2003; Handy, 2003). Therefore, the Cu content in MT of carp may be a suitable marker of this popular source of aquatic pollution. The high diversity of MT forms in invertebrates is well known and explored in the study of the biomonitoring purposes (Brouwer et al., 1995; Dallinger et al., 2000). The attempts to explain the differences between the MT forms obtained by ion-exchange chromatography for the evaluation of their function in fish have been also performed (Olsson and Hogstrand, 1987; Muto et al., 1999; Lacorn et al., 2001; Van Campenhout et al., 2004). Some authors indicate that the MT-1 and MT-2 chromatographic forms in vertebrate aquatic animals bind metals selectively (Kito et al., 1982; Olsvik et al., 2000; Das et al., 2002). However, in our study, the difference between MT profiles of carp from different sites and distribution of metals between them is not so clear as to recommend this approach for biomonitoring purposes in the conditions of low level of metal pollution. The comparison of two tissues allows us to conclude that the liver is the most appropriate tissue for studying metal accumulation or MT response in carp. The important differences between the two sites in Cu and Zn accumulation and binding with MT were observed only in this tissue.

5. Conclusions In summary, the seasonal difference of the metal-binding ability of metallothioneins in carp was reflected at two comparatively unpolluted sites according to our data. The specific accumulation of Cu- and Cd-containing substances at the rural site in spring and high overall impact at the industrial site through three seasons were observed. Only the measuring of metallothioneins binding ability but not the metal levels in the tissues, especially in the liver, gave the important information about the health status of the carp and the levels of waterborne metals.

Acknowledgments This work was granted by the Ministry of Education and Science of Ukraine(Ukrainian-Greece scientific and technical cooperation joint Project #M-65/2006). The authors are grateful to Dr N.S. Loumbourdis for commenting on the manuscript, the Post-graduate students H. Levandovich and N. Burmas for the technical assistance, T. Semyrozum for assistance with the preparation, B. Pechenyak for the language correction and Prof. L. Drobot for the linguistic and phraseological improvement of this manuscript. References Andreani, G., Santoro, M., Cottignoli, S., Fabbri, M., Carpene`, E., Isani, G., 2008. Metal distribution and metallothionein in loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles. Sci. Total Environ. 390, 287–294. Atli, G., Canli, M., 2007. Natural occurrence of metallothioneinlike proteins in liver tissues of four fish species from the northeast Mediterranean Sea. Water Environ. Res. 79, 958–963. Brouwer, M., Enghild, J., Hoexum-Brouwer, T., Thogersen, I., Truncali, A., 1995. Primary structure and tissue-specific expression of blue crab (Callinectes sapidus) metallothionein isoforms. Biochem. J. 31, 617–622. Bury, N.R., Walker, P.A., Glover, C.N., 2003. Nutritive metal uptake in Teleost fish. J. Exp. Biol. 206, 11–23. Cavaletto, M., Ghezzi, A., Burlando, B., Evangelisti, V., Ceratto, N., Viarengo, A., 2002. Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis. Comp. Biochem. Physiol. 131C, 447–455.

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Costa, P.M., Repolho, T., Caeiro, S., Diniz, M.E., Moura, I., Costa, M.H., 2008. Modelling metallothionein induction in the liver of Sparus aurata exposed to metal-contaminated sediments. Ecotoxicol. Environ. Saf. 71, 117–124. Council Directive 98/83/EC levels for the quality of water intended for human consumption. /http://www.emwis.org/IFP/law_EU.htmS (accessed 07.2008). Couture, P., Rajotte, J.W., 2003. Morphometric and metabolic indicators of metal stress in wild yellow perch (Perca flavescens) from Sudbury, Ontario: a review. J. Environ. Monitor. 5, 216–221. Dallinger, R., Berger, B., Gruber, C., Hunziker, P., Sturzenbaum, S., 2000. Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers. Cell Mol. Biol. 46, 331–346. Das, K., Debacker, V., Bouquegneau, J.M., 2002. White-sided dolphin metallothioneins: purification, characterisation and potential role. Comp. Biochem. Physiol. 131C, 245–252. Dethloff, G.M., Schlenk, D., Hamm, J.T., Bailey, H.C., 1999. Alterations in physiological parameters of rainbow trout (Oncorhynchus mykiss) with exposure to copper and copper/zinc mixtures. Ecotoxicol. Environ. Saf. 42, 253–264. Eastwood, S., Couture, P., 2002. Seasonal variations in condition and liver metal concentrations of yellow perch (Perca flavescens) from a metal-contaminated environment. Aquat. Toxicol. 58, 43–56. Farkas, A., Salanki, J., Specziar, A., 2003. Age- and size-specific patterns of heavy metals in the organs of freshwater fish Abramis brama L. populating a lowcontaminated site. Water Res. 37, 959–964. Farombi, E.O., Adelowo, O.A., Ajimoko, Y.R., 2007. Biomarkers of oxidative stress and heavy metal levels as indicators of environmental pollution in african cat fish (Clarias gariepinus) from Nigeria Ogun River. Int. J. Environ. Res. Public Health 4, 158–165. Falfushinska, H.I., Romanchuk, L.D., Stolyar, O.B., 2008. Different responses of biochemical markers in frogs (Rana ridibunda) from urban and rural wetlands to the effect of carbamate fungicide. Comp. Biochem. Physiol. 148C, 223–229. Falfushynska, H., Stolyar, O., 2008. Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western Ukraine. Ecotoxicol. Environ. Saf. Fernandes, D., Bebianno, M.J., Porte, C., 2008. Hepatic levels of metal and metallothioneins in two commercial fish species of the Northern Iberian shelf. Sci. Total Environ. 391, 159–167. Handy, R.D., 2003. Chronic effects of copper exposure versus endocrine toxicity: two sides of the same toxicological process? Comp. Biochem. Physiol. 135A, 25–38. Hansen, B.H., Romma, S., Softeland, L.I., Olsvik, P.A., Andersen, R.A., 2006. Induction and activity of oxidative stress-related proteins during waterborne Cu-exposure in brown trout (Salmo trutta). Chemosphere 65, 1707–1714. Hanson, P.J., 1997. Response of hepatic trace element concentrations in fish exposed to elemental and organic contaminants. Estuaries 20, 659–676. Hogstrand, C., Haux, C., 1991. Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein. Comp. Biochem. Physiol. 100C, 137–141. Kagi, J.H.R., Schaffer, A., 1988. Biochemistry of metallothionein. Biochemistry 27, 8509–8515. Kito, H., Ose, Y., Mizuhira, V., Sato, T., Ishikawa, T., Tazawa, T., 1982. Separation and purification of (Cd, Cu, Zn)-metallothionein in carp hepatopancreas. Comp. Biochem. Physiol. C73, 121–127. Ko¨ck, G., Triendl, M., Hofer, R., 1996. Seasonal patterns of metal accumulation in Arctic char (Salvelinus alpinus) from an oligotrophic Alpine lake related to temperature. Can. J. Fish. Aquat. Sci. 53, 780–786. Kraemer, L.D., Campbell, P.G., Hare, L., 2006. Seasonal variations in hepatic Cd and Cu concentrations and in the sub-cellular distribution of these metals in juvenile yellow perch (Perca flavescens). Environ. Pollut. 142, 313–325. Lacorn, M., Lahrssen, A., Rotzoll, N., Simat, T.J., Steinhart, H., 2001. Quantification of metallothionein isoforms in fish liver and its implications for biomonitoring. Environ. Toxicol. Chem. 20, 140–145. Lukashev, D.V., 2006. Monitoring of contamination of the ecosystem of the Dnieper River within the town of Kiev by heavy metals using freshwater molluscs. Hydrobiol. J. 42, 77–88. MedEcoPortal. /www.health.gov.uaS (accessed 07.2008). Muto, N., Ren, H.-W., Hwang, G.-S., Tominaga, S., Itoh, N., Tanaka, K., 1999. Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air pumping stress, dexamethasone, and metals. Comp. Biochem. Physiol. C122, 75–82. Olsson, P.E., Hogstrand, C., 1987. Improved separation of perch liver metallothionein by fast protein liquid chromatography. J. Chromatogr. 402, 293–299. Olsson, P.E., Haux, C., 1986. Increased hepatic metallothionein content correlates to cadmium accumulation in environmentally exposed perch (Perca fluviatilis). Aquat. Toxicol. 9, 231–242. Olsvik, P.A., Gundersen, P., Andersen, R.A., Zachariassen, K.E., 2000. Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode. Aquat. Toxicol. 50, 301–316. Olsvik, P.A., Gundersen, P., Andersen, R.A., Zachariassen, K.E., 2001. Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn. Comp. Biochem. Physiol. 128C, 189–201.

ARTICLE IN PRESS 1432

H.I. Falfushynska, O.B. Stoliar / Ecotoxicology and Environmental Safety 72 (2009) 1425–1432

Paris-Palacios, S., Biagianti-Risbourg, S., Vernet, G., 2003. Metallothionein induction related to hepatic structural perturbations and antioxidative defences in roach (Rutilus rutilus) exposed to the fungicide procymidone. Biomarkers 8, 128–141. Reynders, H., Bervoets, L., Gelders, M., De Coen, W.M., Blust, R., 2008. Accumulation and effects of metals in caged carp and resident roach along a metal pollution gradient. Sci. Total Environ. 391, 82–95. Roesijadi, G., 1992. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22, 81–114. Rotchell, J.M., Clarke, K.R., Newton, L.C., Bird, D.J., 2001. Hepatic metallothionein as a biomarker for metal contamination: age effects and seasonal variation in European flounders (Pleuronectes flesus) from the Severn Estuary and Bristol Channel. Mar. Environ. Res. 52, 151–171.

Stolyar, O.B., Loumbourdis, N.S., Falfushinska, H.I., Romanchuk, L.D., 2008. Comparison of metal bioavailability in frogs from urban and rural sites of Western Ukraine. Arch. Environ. Contam. Toxicol. 54, 107–113. Van Campenhout, K., Infante, H.G., Adams, F., Blust, R., 2004. Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLCICP-TOFMS. Toxicol. Sci. 80, 276–287. Viarengo, A., Burlando, B., Dondero, F., Marro, A., Fabbri, R., 1999. Metallothionein as a tool in biomonitoring programmes. Biomarkers 4, 455–466. Wong, C.K., Wong, P.P., Chu, L.M., 2001. Heavy metal concentrations in marine fishes collected from fish culture sites in Hong Kong. Arch. Environ. Contam. Toxicol. 40, 60–69. /http://www.dentalmercury.com/245_1.pdfS.