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Italian Journal of Zoology

ISSN: 1125-0003 (Print) 1748-5851 (Online) Journal homepage: http://www.tandfonline.com/loi/tizo20

Physiological responses to heavy metals and adaptation to increased oxygen partial pressure in Antarctic fish and protozoa Vincenzo Albergoni , Arnaldo Cassini , Olimpia Coppellotti , Noemi Favero , Paola Irato , Ester Piccinni & Gianfranco Santovito To cite this article: Vincenzo Albergoni , Arnaldo Cassini , Olimpia Coppellotti , Noemi Favero , Paola Irato , Ester Piccinni & Gianfranco Santovito (2000) Physiological responses to heavy metals and adaptation to increased oxygen partial pressure in Antarctic fish and protozoa, Italian Journal of Zoology, 67:S1, 1-11, DOI: 10.1080/11250000009356349 To link to this article: http://dx.doi.org/10.1080/11250000009356349

Published online: 28 Jan 2009.

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Date: 28 January 2016, At: 17:04

Ital. J. Zool., SUPPLEMENT 1: 1-11 (2000)

Physiological responses to heavy metals and adaptation to increased oxygen partial pressure in Antarctic fish and protozoa VINCENZO ALBERGONI ARNALDO CASSINI OLIMPIA COPPELLOTTI NOEMI FAVERO PAOLA IRATO ESTER PICCINNI GIANFRANCO SANTOVITO

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Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, I-35131 Padova (Italy)

ABSTRACT In the first part of this study, metal bioaccumulation and metallothioneins were investigated in various organs of the red-blooded teleost, Trematomus bernacchii, and the haemoglobinless Chionodraco hamatus. Hepatic metallothionein correlated positively with Cd, Cu, and Zn concentrations in T. bernacchii, whereas in C. hamatus it showed a positive correlation only with Cd. A metal-linking protein with characteristics typical of metallothioneins was identified in the liver of C. hamatus as well as of T. bernacchii. In the second part, data on the amino acid sequence of the enzyme Superoxide dismutase (SOD) were analysed and compared with SOD sequences from other animals. In the third part, a low degree of tolerance against the toxic effects of copper was recorded in two cilates, Pleuronema coronatum and Euplotes rariseta, collected from Terra Nova Bay and cultured in the laboratory. KEY WORDS: Fish - Heavy metals - Metallothionein - Oxygen Protozoa - Superoxide dismutase. ACKNOWLEDGEMENTS This research was supported by the Italian National Program for Antarctic Research (PNRA).

INTRODUCTION Some metals are natural components of sea water and their presence does not normally influence the health of marine organisms. Essential trace metals are required in various physiological and metabolic processes but, at excessive concentrations, they become toxic. Non-essential metals may be toxic even at low concentrations and may be present in the environment as contaminants. Natural occurrences of elevated Cd have been reported in the coastal marine ecosystem around Antarctica. Surface sediments along the coast of Terra Nova Bay have Cd contents ranging from 0.05 to 0.49 pg/g dry wt (Bargagli et al., 1996). As a consequence of highly elevated Cd levels, organisms may accumulate the metal, which penetrates their tissues by means of various mechanisms according to chemical speciation. : Metallothioneins (MTs), a group of ubiquitous low molecular weight metal-binding proteins, characterised by an unusually high cysteine content (30%), may be induced in tissues by various stimuli, especially heavy metals or various stress conditions! They have numerous functions, e.g., regulation of essential metal contents and detoxification of essential and non-essential metals (Kägi, 199D. Tissue accumulation of MTs in fish primarily occurs in liver, kidney and gills. There is currently great interest in the use of specific biomarkers for analysis of environmental contamination, and the induction of MTs by heavy metals is a potential candidate for assessment of heavy metal bioavailability and environmental impact. In this paper, we report the concentrations of three metals (Cd, Cu, Zn) and correlations between tissue levels and chelating molecules, measured as MTs in various tissues of two Antarctic teleosts, Trematomus bernacchii, and the icefish Chionodraco hamatus, collected during the 10th (1994-95) Italian Antarctic Expedition. Identification of a metal-binding protein similar to MT from C. hamatus is also reported. In previous works, responses to heavy metals such· as cadmium, copper, and nickel were examined in some ciliates collected in Terra Nova Bay, Antarctica, and compared with related species belonging to the same genera coming from various geographical regions (Coppellotti, 1994; Coppellotti & De Gabrieli, 1995; Albergoni et al., 1997). As regards a typical species of Antarctic microbiotecton, Euplotes focardii (Ciliophora, Hypotrichia) revealed resistance against the toxic effect of exposure to cadmium (up to 15 pg Cd/ml), comparable to that against nickel (18 pg Ni/ml) but much higher than that against copper (0.08 pg Cu/ml). Here the effects of copper exposure in cultures of two other ciliates, E. rariseta (Ciliophora, Hypqtrichia) and Pleuronema coronatum (Ciliophora, Hymenostomatia), collected during the 11th Italian Antarctic Expedition, were examined, considering that copper in Antarctic sea waters is recovered in remarkable concentrations in the range between 2.0 and 2.9 nM (127.1-184.3 ng/1), as reported by

V. ALBERGONI, Α. CASSINI, O. COPPELLOTTI, Ν. FAVERO, P. IRATO, Ε. PICCINNI, G. SANTOVITO

Westerlund & Öhman (1991). The degree of tolerance to copper with respect to growth rate and morphology was examined in both ciliates, but metal, total acid-soluble thiol and glutathione contents were only analysed in E. rariseta, which could be cultured in larger amounts.

MATERIALS AND METHODS

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Fish Specimens of T. bernacchii and C. hamatus were sampled from Terra Nova Bay, Ross Sea (74°42'S, 164°7'E). Fish were sectioned and tissues (liver, gills, muscle, heart, blood) were removed and frozen at -80° C. Plasma was frozen after centrifugation of blood for 20 min at 1,500 g. Tissues were homogenised with a Polytron in 4 vol/g of tissue of 0.5 M sucrose, 20 mM Tris-HCl buffer, pH 8.6, with the addition of 0.006 mM leupeptin, 0.5 mM PMSF (phenylmethylsulphonyl fluoride) and 0.01% ß-mercaptoethanol. Aliquots of homogenates were used for heavy metal determination; other aliquots were centrifuged at 48,000 g at 4° C for 60 min. The resulting supernatants were used for heavy metal and MT quantification. The remaining liver homogenates were also treated according to Kimura et al. (1979) with ethanol and chloroform (1.05 : 0.08, for each vol of homogenate) and centrifuged at 48,000 g at 4° C for 60 min. The resulting aliquots were used for purification of MTs. For heavy metal determination, samples of homogenate and cell-free extracts were digested with AristaR nitric acid in a mod. MDS-2000 CEM microwave. Cd, Cu and Zn contents were determined by atomic absorption ñame or graphite spectrophotometer (Perkin-Elmer mod. 5100) according to metal concentration. Data refer to dry weight for homogenates and to total protein concentrations assayed by Gornall's method (Gornall et al., 1949) for cell-free extracts. Metallothionein concentrations in cell-free extracts of various tissues were determined by the silver saturation method (Scheuhammer & Cherian, 1991). Data refer to total proteins. Differences between mean values were tested for significance by Student's t-test or analysis of variance, followed by the StudentNewman-Keuls test. Linear regression was carried out to estimate the correlation between MTs and metal concentrations. Metallothioneins were purified by a combination of gel-filtration, ion-exchange and reverse-phase chromatography. The first purification step was carried out in a Bio-Gel P-60 column. Subsequently, metal-linking fractions were pooled, lyophilised, dialysed, and applied to a Pharmacia FPLC (Pharmacia mod. LCC 500) equipped with a Mono Q ion-exchange column (Pharmacia mod. HR 5/5). The resulting peaks were assayed for Cd, Cu, and Zn contents with the atomic absorption spectrophotometer previously described. Folin phenol reagent (Lowry etal., 1951) and the silver saturation method were also used. Metal-containing fractions were dialysed against 5 mM Tris HC1, pH 7.5, and applied to an HPLC system (Pharmacia). Chromatography was performed on a Cjg (Vydac) reverse-phase column. Horse MT (Sigma) was eluted by reverse-phase column in the same conditions. Absorbance was monitored at 214 and 254 nm. UV spectral analyses were performed on a Perkin-Elmer Lambda 10 UV/V1S spectrometer during the various purification steps. The proteins eluted from the reverse-phase column were assayed for homogeneity by electrophoresis in denaturing conditions (SDS/PAGE) in mini-slab gels, based on the Laemmli method (1970). Stacking gels were 0.1% SDS and 4% polyacrylamide; separating gels were 0.05% SDS and 15% polyacrylamide. Electrophoresis was performed at 20 mA constant for about 2 hours. Gels were stained by the silver-stain method of Morrissey (1981). To prevent MT aggregation, proteins were carboxymethylated as follows. An aliquot of sample was mixed with 5 μΐ of a solution containing 200 mM Tris HC1 (pH 8.8), 8% SDS, 50% glycerol,

1 μΐ 0.4 M DTE, and 1 μΐ 0.4 M EDTA. The mixture was boiled for 5 min and carboxymethylated by adding 4 μΐ of 1 M iodoacetic acid at 50° C. Proteins thus treated were then assayed by SDS/PAGE electrophoresis. Gels were stained by the silver-stain method of Otsuka et al. (1988). To detect SH groups electrophoretically, samples were treated with thiolyte (monobromobimane-MB), a soluble yellowish compound which specifically binds SH groups to protein, giving rise to strong fluorescence (Viarengo et al., 1997). Some samples were labelled with a 6-mM thiolyte solution and separated by SDS electrophoresis; then the gels were maintained in methanol, acetic acid and water at a ratio of 45:10:45 v/v. The fluorescence of the protein bands in the gel was highlighted with a UV transilluminator and photographed. Apo-MT and Cd substitution were obtained by the method of Vasák (1991), slightly modified. The MT, obtained by means of FPLC, was treated with a few drops of HO 6 M (final pH 2.0), incubated for 1 h at room temperature, cooled on ice, and separated from the dissociated metal ions by dialysis with HC1 10 mM overnight at 4° C. Samples were rendered oxygen-free by a stream of nitrogen (about 20 min). Excess of CdCl2 at the same pH was added. The solution was titrated with 0.5 M Tris HCl to pH 8.6. Purified SOD was digested with different proteolytic enzymes: pepsin, thermolysin and V8. The resulting enzymes were separated by HPLC with C18 (Vydac) reverse-phase column. Molecular weights of the peptides were estimated by MALDITOFF mass spectrometry. The amino acid sequences of N-terminal and other peptides were determined by Automated Edman degradation of protein on an Applied Biosystems sequencer equipped with an online phenylthiohydantoin (PTH) amino acid analyzer. Sequence analysis was conducted according to the standard programme of Applied Biosystems. Polybrene was used as a carrier for protein samples. Sequences were aligned using the CLUSTAL V programme (Pearson & Lipman, 1988). The phylogenetic tree was reconstructed with the neighbor-joining method (Saitou & Nei, 1987). Distances were estimated with the Poisson correction (Kimura, 1983). Ciliates Pleuronema coronatum and Euplotes rariseta were isolated and later identified from samples of sea water and sediments collected in Terra Nova Bay, Ross Sea (74°42'S, l64°06'E) in January 1996 during the 11th Italian Antarctic Expedition. Cultures from a single cell of P. coronatum and E. rariseta were established in the laboratory at 0-2° C in natural sea water (pH 7.8-8.0, salinity 35%o) filtered through 0.22 Sterivex-GS (Millipore) filters, supplemented with vitamins, extra salts and 0.002% (w/v) proteose-peptone. Euplotes rariseta cells were also fed with Chlorella sp. Copper was added to sea water as CuCl2-2H2O at a range of final concentrations between 0.05 and 2.50 μg Cu/ml, corresponding to 0.8-40 μΜ. Cells were inoculated in fresh culture medium, up to 25 ml in plastic Petri dishes for growth curves and up to 500 ml in flasks for biochemical investigations, to give an initial density of 40 cells/ml for P. coronatum and 300 cells/ml for E. rariseta, respectively. Growth was monitored daily up to day 8 by counting fixed cells under a Wild M8 Leitz stereomicroscope. For other experiments, cells were collected by centrifugation, washed in sea water, resuspended in 50 mM Tris-HCl buffer, pH 7.5, for metal content determination or cold 5% trichloroacetic acid for acid-soluble thiol and glutathione determination, and subsequently homogenised in the cold by sonication. The Tris-HCl buffer homogenate was centrifuged at 46,000 g for 30 min at 2° C. Metal contents (Cu and Zn) were measured by atomic absorption spectroscopy (Perkin-Elmer mod. 4000) without further processing of the supernatants, and after digestion with 1 ml of AristaR HNO3 in a MDS-2000 CEM microwave oven, as indicated above, in homogenates and sediments. The resulting extracts were made up to 4 ml with milliQ water and submitted to analysis. Values are expressed as μg metal/g dry wt. The trichloroacetic acid homogenate was centrifuged at 22,000 g for 30 min at 2° C. Supernatants were used for determination of acid-soluble thiols (AST)

RESPONSES TO HEAVY METALS AND ADAPTATION TO INCREASED pO 2 and total, both reduced and oxidized (GSH+GSSG), glutathione contents. AST contents were measured according to the DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] mehod (Moron et al., 1979). Glutathione contents were determined enzymatically by the recycling procedure following Anderson (1985). Values are expressed as pmoles/g dry wt and were the mean of six experiments.

tic fish. Metallothionein had statistically higher values in liver than in all other tissues, in both fish species (group a). In the other tissues, the MT values of the two teleosts were very similar (0.2-0.5 pg/mg protein in T. bernacchii, 0.4-0.5 pg/mg protein in C. hamatus); only T. bernacchii heart had a higher value than plasma and muscle.

RESULTS

Correlations between metals and MT concentrations

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Heavy metal contents in cell-free extracts and plasma

Metal concentrations (pg/mg of total protein) in cellfree extracts and plasma of the two species are listed in Table I. In T. bernacchii, Cd was present in the statistically highest amounts in liver (group a); Cu was equally concentrated in liver and plasma (again group a), with values statistically greater than in other tissues. Zn levels were statistically higher in plasma (group b), than in all other tissues. In C. hamatus, Cd accumulated in statistically higher amounts in muscle (group b); Cu in plasma was statistically higher than in liver and gills (group tí). Zn distributions did not show any statistically significant differences in the various tissues. Comparing the values of the three metals by Student's t-test, between the supernatants from the same organs of the two species, T. bernacchii liver accumulated all metals significantly more than C. hamatus liver (P < 0.001), whereas C. hamatus gills and muscle accumulated more Cu and Zn, respectively (P < 0.05), than the same organs of T. bernacchii. MT concentrations Table I shows MT concentrations, expressed as pg/mg of protein, determined in various tissues of red-blooded (T. bernacchii) and white-blooded (C. hamatus) Antarc-

Significant linear correlations between metals and MT concentrations were found only in liver and gills (Fig. 1). Trematomus bernacchii liver shows correlations for all three metals (r = 0.937 for Cd, r = 0.740 for Cu, r = 0.529 for Zn), whereas in C. hamatus liver a linear correlation was observed only between Cd and MT, with r = 0.667. Correlations were also found between Cd and MT in the gills of both species, with r = 0.799 for T. bernacchii and r = 0.925 for C. hamatus. Identification of metal-binding proteins in Chionodraco hamatus and Trematomus bernacchii As the greatest MT concentrations were found in liver, proteins were purified using this organ. In the first separation step by gel-filtration chromatography (Bio-Gel P-60), supernatant from C. hamatus showed the presence of a metal peak eluting at a Ve/V0 value of 2.14. A similar result was obtained for T. bernacchii liver supernatant, the elution profile of which shows a metal-linking peak eluting at a Ve/V0 value of 2.26, very similar to that of C. hamatus. Both peaks were further purified using ion-exchange and reverse-phase chromatography. As regards C. hamatus, the elution profile from ion-exchange chromatography shows two main peaks eluting at 0.051 (peak 1) and 0.061 M (peak 2) of NaCl. The UV absorp-

TABLE I - Concentrations of Cd, Cu, Zn and MTs (ßg/mg protein) in cell-free extracts from tissues of Trematomus bernacchii and Chionodraco hamatus.

η

Trematomus bernacchii Liver Plasma Muscle Gills Heart Chionodraco hamatus Liver Plasma Muscle Gills Heart

15 12 10

9 10

16 11 10

9 10

Cd

Cu

0.057±0.044 0.013±0.018 0.021+0.011 0.006±0.003 0.010±0.009

a b b b b

0.042+0.028 0.043±0.037 0.011±0.009 0.005±0.002 0.012±0.009

0.010±0.006 0.009±0.010 0.037+0.024 0.010±0.009 0.005+0.005

a a b a a

0.00810.005 a 0.026±0.026 b 0.012+0.005 ab 0.008±0.003 a 0.018+0.014 ab

a a b b b

Zn

MT

0.271+0.061 a 0.625±0.571 b O.O45±O.O3O a 0.20710.065 a 0.158+0.061 a

1.78+0.94 a 0.17+0.15 b 0.26+0.08 b 0.49+0.45 be 1.15+1.13 c

0.164+0.031 0.385+0.507 0.102+0.062 0.214+0.065 0.175+0.076

1.27+0.24 0.41+0.51 0.4310.10 0.48+0.71 0.4210.34

a a a a a

Values are means 1 SD. Means with different letters {a, b, c) are significantly different at Ρ < 0.05; η, number of samples.

a b b b b

V. ALBERGONI, Α. CASSINI, O. COPPELLOTTI, Ν. FAVERO, P. IRATO, Ε. PICCINNI, G. SANTOVITO

4

T. bernacchii liver

C. hamatus liver correlation MT vs Cd 2.0 -,

correlation MT vs Cd

I

r = 0.937 Ρ < 0.001

4

I

3 2-

r = 0.667 Ρ < 0.01

1 0

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0.00

0.04

0.08

0.12

0.16

0.000 0.005 0.010 0.015 0.020

Cd ^g/mg prot)

Cd (Hg/mg prot)

T. bernacchii liver correlation MT vs Cu

T. bernacchii gills correlation MT vs Cd o OH

1.6-1 1.2-

00

i

r = 0.799 Ρ < 0.05

/

o

/ /o

0.80.4nη.

0.00

0.03

0.06

0.09

0.000 0.004 0.008 0.012 0.016

0.12

Cu

Cd

C. hamatus gills correlation MT vs Cd

7! bernacchii liver correlation MT vs Zn 5 i

4

i

2.5

r =0.529 Ρ < 0.05

Δ

|2.0-

3 -

6

L

5

'•

1.0 -

2 -

\

1 -

Η 0.5 0.0

0 0.00

r =0.925 Ρ < 0.001

0.10

0.20

0.30

0.40

Zn ^g/mg prot)

0.000 0.006 0.012 0.018 0.024 Cd

Fig. 1 - Linear correlation of metallothionein versus metal concentrations in soluble fraaion of liver and gills in T. bernacchii and C. hamatus.

tion spectra of both peaks, obtained after acidification and reconstitution with Cd, were typical of classic MTs, showing low absorbance at 280 nm and a shoulder at 254 nm, characteristic of Cd++ thiolate co-ordination (Fig. 2). As regards T. bernacchii, ion-exchange chromatography resulted in two main metal-linking peaks

eluting at 0.051 (peak 1) and 0.058 M (peak 2) of NaCl. An example of the UV spectrum is shown in Figure 3 and is similar for both peaks. Subsequently, C. hamatus MTs were applied to a C 8 reverse-phase column. Peaks 1 and 2 eluted with a single peak at 43.0 and 30.9% of acetonitrile, respectively

RESPONSES TO HEAVY METALS AND ΑΟΑΡΤΑΉΟΝ TO INCREASED p O 2

1.00 —

1.00-