Cadmium accumulation, metallothionein and glutathione levels, and ...

ISSN 0963-9292, Volume 19, Number 6

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Ecotoxicology (2010) 19:1066–1073 DOI 10.1007/s10646-010-0488-x

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Cadmium accumulation, metallothionein and glutathione levels, and histopathological changes in the kidneys and liver of magpie (Pica pica) from a zinc smelter area Tadeusz Włostowski • Krzysztof Dmowski El_zbieta Bonda-Ostaszewska

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Accepted: 16 March 2010 / Published online: 27 March 2010 Ó Springer Science+Business Media, LLC 2010

Abstract The objective of this study was to examine a relationship between cadmium (Cd) accumulation and histopathological changes in the kidneys and liver of magpies (Pica pica) from a zinc smelter area. The concentrations of metallothionein (MT) and glutathione (GSH) that are linked to a protective effect against Cd toxicity were also determined. There was a positive correlation between the concentration of Cd (2.2–17.9 lg/g) and histopathological changes (interstitial inflammation and tubular cell degeneration) in the kidneys (Rs = 0.87, P = 0.0000). The renal Cd also positively correlated with apoptosis (Rs = 0.72, P = 0.0005) but the metal did not affect lipid peroxidation. Notably, the average concentration of Cd in the kidneys exceeded MT capacity by about 7 lg/g which is thought to produce renal injury. Importantly, GSH level in the kidneys of magpies from the polluted area dropped to 38% of that observed in the reference birds, probably potentiating Cd toxicity. On the contrary, the liver accumulation of Cd was relatively small (0.88–3.38 lg/g), the hepatic MT capacity exceeded the total concentration of Cd and no association between the hepatic Cd and histopathology was found despite the fact that GSH level was only half that observed in the reference birds. The data suggest that Cd intoxication may be responsible for histopathological changes occurring in the kidneys of free-ranging magpies and that the pathology T. Włostowski (&) E. Bonda-Ostaszewska Institute of Biology, University of Białystok, S´wierkowa 20B, 15-950 Białystok, Poland e-mail: [email protected] K. Dmowski Department of Ecology, University of Warsaw, Banacha 2, 02-097 Warsaw, Poland e-mail: [email protected]

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may be associated with inappropriate amount of renal MT and GSH. Keywords Cadmium Metallothionein Glutathione Nephrotoxicity Apoptosis Magpie

Introduction Cadmium (Cd) is an industrial and environmental pollutant that is toxic to humans and animals (Goering et al. 1995; Satarug et al. 2003). The major source of Cd intake is food and most of Cd that is absorbed after oral exposure preferentially accumulates in the kidneys and liver where it can cause renal and hepatic dysfunction (Lehman and Klaassen 1986; Włostowski et al. 2000). Chronic Cd exposure produces damage primarily to the kidneys, including tubular degeneration, interstitial inflammation, apoptosis and glomerular swelling (Groten et al. 1994; Liu et al. 1998a; Nicholson et al. 1983). Importantly, during chronic exposure, the Cd-induced nephrotoxicity is dependent on renal Cd concentration. For instance, in humans, laboratory rodents as well as in some birds renal injury occurs when the Cd concentration exceeds 100 lg/g wet weight (Elliott et al. 1992; Groten et al. 1994; Larison et al. 2000; Liu et al. 1998a; Satarug et al. 2003). In contrast, in the free-living mammals such as roe deer, moose, Algerian mice, yellownecked mice and bank voles from an industrialized area, the renal injury occurs at the Cd level lower than 20 lg/g wet weight (Beiglbock et al. 2002; Damek-Poprawa and Sawicka-Kapusta 2003, 2004; Leffler and Nyholm 1996; Pereira et al. 2006). These reports suggest differences in Cd thresholds that cause renal injury among mammals and birds. Still, the reason for this difference in susceptibility to Cd toxicity is not known and remains to be elucidated.

Cadmium and histopathology in magpies


It is well known that susceptibility to Cd toxicity increases dramatically in animals that are unable to synthesize metallothionein (MT), a low-molecular-weight protein that is induced by and bound to the metal (Klaassen et al. 1999; Nordberg and Nordberg 2000). Notably, in MT-null animals the kidney and liver injury occurs at the Cd concentration lower than 10 lg/g (Habeebu et al. 2000; Liu et al. 1998b). It is commonly thought that an appropriate amount of MT is required to provide protection against Cd-induced tissue injury. When the amount of Cd in the kidneys and liver exceeds the binding capability of MT, the non-MT-bound Cd ions are believed to cause nephro- and hepatotoxicity (Goyer et al. 1989; Nomiyama and Nomiyama 1998). One mechanism by which these ions can produce injury is thought to be through generation of reactive oxygen species and lipid peroxidation, which in turn depress renal and hepatic functions (Shaikh et al. 1999; Thevenod and Friedmann 1999). Another molecule that is known to modify the susceptibility to Cd toxicity is glutathione (GSH) (Kang and Euger 1987; Nzengue et al. 2008; Singhal et al. 1987). This sulfhydryl-rich tripeptide can effectively bind with Cd, thereby altering metal uptake and elimination as well as preventing nucleophilic interaction of the metal ions with essential cellular structures; it can also protect against Cd-induced oxidative stress (Almazan et al. 2000; Nzengue et al. 2008; Wang et al. 2009). Thus, any decrease in the level of tissue GSH is expected to enhance Cd toxicity (Chan and Cherian 1992; Maracine and Segner 1998; Nzengue et al. 2008). Still, it is unknown whether the tissue levels of GSH and MT are sufficiently high to protect against Cd toxicity in the animals free-living in an industrialized area (Vanparys et al. 2008). It is also unknown whether birds inhabiting such an environment exhibit increased susceptibility to Cd toxicity (like mammals) or they are resistant to toxic effects of this metal. Therefore, to address these questions, in the present work we examined the levels of Cd, MT and GSH as well as evaluated the toxicity by measuring histopathology, apoptosis and lipid peroxidation in the kidneys and liver of magpies (Pica pica) from a zinc smelter area. The magpie appeared to be a suitable species for these studies due to its resident behaviour and relatively small individual territory (Birkhead et al. 1992); in fact, the birds have been used frequently for the assessment of heavy metal pollution (Dmowski 1997).

Materials and methods Birds, and tissue sampling and processing The adult magpies were captured, using traps with a magpie as a bait, in April/May 2007 in the vicinity

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(0.3–2 km) of a zinc smelter (Szopienice near Katowice, southern Poland) and in Kampinos National Park near Warsaw (central Poland) treated as a reference site (Dmowski 1997). Due to a small sample size the females and males were treated jointly in further analysis. Previous studies demonstrated that the magpies are permanent residents in the area surrounding the zinc smelter and that their external tail feathers (R6) before moulting are suitable analytical material for the assessment of heavy metal pollution (Dmowski 1997). Multi-element analysis of the magpie’s feathers had revealed that the birds from the zinc smelter area were intoxicated primarily by Cd (the content of this metal was 80 times that of the reference birds); the intoxication by lead (Pb) and zinc (Zn) appeared to be 20 and 4 times higher, respectively, than that in the reference birds and no intoxication by mercury (Hg) was found (Dmowski 1997). The birds were killed by decapitation and the kidneys and liver were excised. A portion of the organs was fixed in 10% phosphate-buffered formalin for histological examination. The remaining portions of the kidneys and liver were frozen and kept at -80°C until analysis. After thawing, a portion (about 250 mg) of the kidneys and liver (in duplicate) was transferred to 1.0 ml chilled 0.25 M sucrose and homogenized with a Teflon pestle in a glass homogenizer. Aliquots (0.1 and 0.5 ml) of the homogenate were taken for determination of lipid peroxidation and Cd concentration, respectively. The remaining homogenate was centrifuged at 200009g for 20 min at 4°C, and the resulting supernatant was removed for MT and GSH assays. All experimental procedures were approved by the Local Committee for the Ethics of Animal Experimentation in Warsaw (permission to K.D.) and were compatible with the standards of the Polish Law on Experimenting on Animals, which implements the European Communities Council Directive (86/609/EEC). Cadmium determination Cd determination was carried out as described elsewhere (Włostowski et al. 2004). The homogenate (0.5 ml) was placed in a glass tube with 2.0 ml of concentrated nitric acid. After 20 h of sample digestion at room temperature, 72% perchloric acid (0.5 ml) was added and the mixture was heated at 100°C for 3 h. Finally, the temperature was raised to 150–180°C and digestion continued for another 4 h. Deionized water was added to the residue after digestion to a volume of 3 ml (first solution). A portion of the first solution (200 ll) was evaporated to dryness in a quartz crucible at 130°C, and the residue was redissolved in an appropriate amount of deionized water (second solution). Cd analysis of these solutions was carried out

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by electrothermal atomic absorption spectrophotometry (AAS) using an AAS3 Zeiss Jena instrument with an EA3 furnace attachment. The conditions for Cd determination were as follows: wavelength 228.8 nm, drying at 110°C for 15 s, ashing at 250°C for 5 s, and atomizing at 1200°C for 5 s. Quality assurance procedures included the analysis of reagent blanks and appropriate standard reference material (NIST bovine liver 1577b). The recovery of Cd was 88–95%. The precision expressed as relative standard deviation of ten measurements of the same sample was 8%.

distilled water were added and vortexed. The reaction mixture was placed in a water bath at 95°C for 1 h. After cooling, 1.0 ml of distilled water and 5.0 ml of butanol/ pyridine mixture (15:1 v/v) were added and vortexed. After centrifugation, absorbance of the organic phase was determined at 532 nm. Tetraethoxypropane was used to prepare a calibration curve. The results were expressed as TBAreacting substances (RS) (nmol/g wet weight).

Metallothionein assay

The fixed portions of the kidneys and liver were dehydrated in ethanol and xylene, embedded in paraffin, cut into 6 lm sections, and stained with hematoxylin and eosin for microscopic examination. For statistical purposes the severity of histopathological changes was measured on a semi-quantitative scale scored in four categories according to the intensity of alterations: without alteration (0), slightly altered (1), intermediately altered (2), and strongly altered (3).

MT capacity in the kidneys and liver was determined by a Cd-saturation method (Włostowski et al. 2004). Briefly, a 0.1-ml sample was incubated in a 1.5-ml vial for 10 min at room temperature with 1.0 ml Tris–HCl buffer (0.03 M, pH 7.8) containing 1.0 lg Cd/ml. To remove non-MTbound Cd, bovine hemoglobin (Sigma) (0.1 ml of a 5% solution in H2O) was added and the sample was heated for 1.5 min at 95°C, cooled and centrifuged for 5 min at 100009g. Addition of hemoglobin, heating, and centrifugation of the sample was repeated three times. Cd bound to MT in the resulting clear supernatant was determined by electrothermal AAS. MT capacity was expressed in lg Cd bound to MT/g wet weight. Glutathione assay The total GSH (reduced ? oxidized) was measured in the postmitochondrial fraction according to the method of Tietze (1969) by using NWLSS Glutathione Assay Kit (Vancouver, WA, USA). Briefly, an aliquot of the supernatant (50 ll) was deproteinized by adding 100 ll of an aqueous solution of 5% metaphosphoric acid. After centrifugation an aliquot (25 ll) of the supernatant was diluted by adding 500 ll of Assay Buffer. To 400 ll of this solution 400 ll of Assay Buffer, 50 ll of 5,50 -dithiobis-2nitrobenzoic acid (DTNB) and 50 ll of glutathione reductase in Assay Buffer were added and incubated for 2 min and 30 s at room temperature. Subsequently 50 ll of NADPH solution was added and the reduction rate of DTNB into 5-thio-2-nitrobenzoic acid (TNB) was measured spectrophotometrically at 412 nm for 3 min. GSH was expressed as lmol/g wet weight. Lipid peroxidation assay Lipid peroxidation was assessed by measuring malondialdehyde (MDA) formation, using the thiobarbituric acid (TBA) assay (Ohkawa et al. 1979). To 0.1 ml of the tissue homogenate, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid, 1.5 ml of 0.8 TBA and 0.6 ml of

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Histological examinations

In situ apoptosis detection Apoptosis in the kidneys and liver was demonstrated in situ by the TUNEL (TdT-mediated dUTP-fluorescein Nick End Labeling) assay, using a kit from Roche Diagnostics (Mannheim, Germany) according to their instructions. Briefly, sections were dewaxed in xylene, hydrated in graded alcohol series and permeabilized in 0.1% Triton X-100/0.1% sodium citrate for 8 min. Terminal deoxynucleotidyl transferase (TdT) enzyme and fluorescein-labeled nucleotides were applied to the sections for 60 min at 37°C. Sections were washed with PBS and treated with alkaline phosphatase-conjugated anti-fluorescein antibody for 30 min at 37°C. They were next treated with substrate (NBT/BCIP) for 10 min in the dark. The numbers of apoptotic cells were determined in ten random microscopic fields for each bird, using a 409 objective, and apoptosis was expressed as the mean of the number of apoptotic cells/microscopic field. Statistical analysis Data were expressed as means ± SD and medians, and analyzed by the non-parametric Mann–Whitney U-test. The non-parametric Spearman correlation was performed to examine the relationship between the tissue concentration of Cd and histopathology, apoptosis and GSH (SPSS 14.0).

Results The concentrations of Cd, MT, GSH and TBARS as well as apoptosis in the kidneys and liver of magpies from polluted



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Table 1 Cadmium, metallothionein and glutathione concentrations as well as lipid peroxidation (TBARS) and apoptosis in the kidneys of magpies from polluted and reference sites Polluted site (Szopienice) n = 11

Reference site (Kampinos) n=8

Significance of difference P value (Mann–Whitney U-test)

Mean ± SD

9.16 ± 5.17

0.38 ± 0.07

0.0003

Median (min–max)

7.58 (2.17–17.94)

0.37 (0.30–0.52)

Cadmium (lg/g wet weight)

Metallothionein (lg Cd/g wet wt) Mean ± SD

2.06 ± 1.28

Median (min–max) 1.47 (0.48–4.98) Glutathione (lmol/g wet weight)

0.85 ± 0.28

0.0208

0.90 (0.46–1.35)

Mean ± SD

0.35 ± 0.05

0.91 ± 0.28

Median (min–max)

0.35 (0.30–0.40)

0.95 (0.50–1.30)

Mean ± SD

173 ± 51

161 ± 21

Median (min–max)

163 (108–255)

158 (130–204)

Mean ± SD

11.5 ± 2.0

4.5 ± 0.9

Median (min–max)

11.0 (7.0–15.0)

4.2 (3.5–6.0)

0.0003

TBARS (nmol/g wet weight) 0.8688

Apoptotic cells/microscopic field 0.0003

Table 2 Cadmium, metallothionein and glutathione concentrations as well as lipid peroxidation (TBARS) and apoptosis in the liver of magpies from polluted and reference sites Polluted site (Szopienice) n = 11

Reference site (Kampinos) n=8

Significance of difference P value (Mann–Whitney U-test)

1.98 ± 0.81 1.91 (0.88–3.38)

0.14 ± 0.04 0.12 (0.08–0.20)

0.0003

0.0039

Cadmium (lg/g wet weight) Mean ± SD Median (min–max)

Metallothionein (lg Cd/g wet weight) Mean ± SD

3.41 ± 1.35

1.53 ± 0.32

Median (min–max)

3.16 (1.35–5.34)

1.45 (1.20–2.18)

Mean ± SD

1.90 ± 1.00

3.94 ± 0.76

Median (min–max)

1.92 (0.50–3.20)

3.97 (2.40–4.85)

Mean ± SD

153 ± 35

144 ± 14

Median (min–max)

138 (97–204)

143 (126–165)

Mean ± SD

9.6 ± 2.6

3.2 ± 0.9

Median (min–max)

9.0 (5.0–14.0)

3.2 (2.0–4.5)

Glutathione (lmol/g wet weight) 0.0005

TBARS (nmol/g wet weight) 0.7102

Apoptotic cells/microscopic field

and reference sites are presented in Tables 1 and 2. An average concentration of Cd in the kidneys of magpies from a zinc smelter area (Szopienice) was 24 times higher than that of the birds from a reference site (Kampinos) (Table 1). Although MT capacity in the kidneys of magpies from Szopienice was significantly (P = 0.0208) higher than that in birds from Kampinos, the Cd-binding capacity of MT in the birds from the polluted area was only 23% that of the total Cd concentration. As result, the renal Cd

0.0003

exceeded MT capacity by about 7 lg/g (Table 1). In addition, the GSH level in the kidneys of magpies from the polluted area dropped to 38% of that observed in the birds from the reference site. Notably, there was no significant difference (P = 0.8688) in the renal lipid peroxidation between the two groups of birds. However, the number of apoptotic cells increased 2.5-fold (P = 0.0003) in the kidneys of magpies from Szopienice in comparison with the reference birds (Table 1, Fig. 1). Likewise,

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Fig. 1 Representative photomicrographs of kidney section from a reference magpies and b magpies living in the vicinity of zinc smelter (interstitial inflammation and tubular cell degeneration

(arrows) are seen). c Immunohistochemical demonstration of apoptotic cells (arrows) in kidney by TUNEL technique. Scale bar, 20 lm

histopathological changes (mainly interstitial inflammation as well as tubular cell degeneration, Fig. 1) occurred exclusively in all birds captured in the zinc smelter area. Furthermore, the Spearman analysis revealed a strong positive correlation between the renal Cd concentration and histopathological changes (P = 0.0000) as well as apoptosis (P = 0.0005) (Table 3), suggesting an involvement of Cd in the kidney pathology. The accumulation of Cd in the liver of magpies from Szopieniece was 14 times higher than that in birds from

Kampinos (Table 2). In contrast to the kidney, the hepatic Cd-binding capacity of MT in birds from the zinc smelter area exceeded the total concentration of Cd by 1.4 lg/g, suggesting that potentially all Cd could be bound with the protein. However, the hepatic GSH level in the magpies from the polluted area was only half that observed in the reference birds; despite this, no significant difference (P = 0.7102) in the hepatic lipid peroxidation between the two groups of birds was found. Although the number of apoptotic cells in the liver increased significantly (P = 0.0003) in the Szopienice magpies (Table 2), only two birds exhibited mild histopathological changes (leukocyte infiltration) (not shown); the similar changes were also found in one of the reference birds. The Spearman analysis showed a positive correlation (P = 0.0060) between the hepatic Cd and apoptosis, and a negative correlation between the hepatic GSH and apoptosis (P = 0.0018) (Table 3).

Table 3 The Spearman analysis of correlation between the cadmium concentration and histopathology, apoptosis and glutathione in the kidneys and liver of free-ranging magpies (n = 19) Comparison

Spearman correlation coefficient (Rs)

P-value

Kidney Cadmium versus histopathology

0.87

0.0000

Cadmium versus apoptosis

0.72

0.0005

Cadmium versus glutathione

-0.66

0.0020

Glutathione versus apoptosis

-0.62

0.0043

0.61

0.0060

Cadmium versus glutathione

-0.69

0.0011

Glutathione versus apoptosis

-0.67

0.0018

Liver Cadmium versus apoptosis

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Discussion The present work demonstrates that histopathological changes typical for Cd toxicity occur in the kidneys of magpies at Cd concentrations less than 20 lg/g. Thus, the birds living in an industrialized area are probably as highly



susceptible to Cd toxicity as are some mammalian species (Beiglbock et al. 2002; Damek-Poprawa and SawickaKapusta 2003; Pereira et al. 2006). The data from the present study indicate further that this sensitivity to Cd toxicity may be associated with inappropriate concentrations of MT and GSH in the kidneys (Table 1). It is commonly assumed that nephrotoxicity can occur when the tissue Cd exceeds the Cd-binding capacity of intracellular MT which appears to be a primary component of native and acquired tolerance to toxic effects of Cd (Klaassen et al. 1999). The non-MT-bound Cd in the kidneys of magpies from a zinc smelter area appeared to be comparable to that producing renal damage in the MT-null mice (Liu et al. 1998b). It is thus conceivable that this fraction of renal Cd could be responsible for kidney lesions occurring in the magpies. In addition, GSH that is also known to provide a protection against Cd toxicity (Chan and Cherian 1992; Nzengue et al. 2008) declined almost by 60% in the kidneys of these birds, perhaps potentiating the toxicity. Although the liver GSH concentration also declined to the same extent as the renal GSH in the birds from a polluted area, the Cd-binding capacity of hepatic MT exceeded the total Cd concentration, probably providing specific protection against Cd toxicity. In agreement, it has been previously demonstrated that an increase in Cd toxicity caused by a decrease of GSH was totally overcome by the presence of high MT content (Chan and Cherian 1992). Thus, it is possible that extensive renal injury observed in the magpies from a zinc smelter area could be associated with too low levels of both MT and GSH. The precise mechanism underlying the reduction of renal MT as well as renal and hepatic GSH in the magpies is uncertain. The reduced Cd-binding capacity of renal MT in the birds from a polluted area may be linked to a suppression of the protein synthesis and/or its metal composition. So far, it has been demonstrated that some organic co-contaminants, e.g. benzo(a)pyrene, bisphenol A as well as some natural plant substances suppress metal inducibility of MT (Miranda et al. 1982; Sogawa et al. 2001; van den Hurk et al. 2000). Thus, it cannot be ruled out that the process is also taking place in the kidneys of free-living magpies, resulting in low level of the protein. An important role in the reduction of Cd-binding capacity of MT may play also copper (Cu), which appeared to have the ability to displace Cd from MT (Day et al. 1981). It has been shown that the renal concentration of copper increases upon Cd exposure (Goyer et al. 1989) and the renal MT contains excessive amount of Cu in comparison with the hepatic MT (Day et al. 1981). Importantly, the concentration of Cu in the kidneys of magpies from the polluted area increased by about 40% as compared with the concentration in the reference birds (data not shown). Therefore, the possibility

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may exist that most of metal binding sites on the renal MT could be occupied by Cu, thereby rendering the binding of Cd ions impossible. Still, these possibilities remain to be proven in further studies. The reduction of GSH concentration in the kidneys and liver of magpies may have been associated, at least partly, with Cd intoxication. Similar effect has been shown in rat liver and kidneys (Nigam et al. 1999), in rat liver slices (Chan and Cherian 1992), in oligodendrocytes (Almazan et al. 2000), and in the primary cultures of rat proximal tubular cells (Wang et al. 2009). However, other authors revealed an increase in GSH concentration upon Cd exposure in vivo (Congiu et al. 2000; Shaikh et al. 1999) and in vitro (Nzengue et al. 2008). Still, since the negative correlation between Cd concentration and GSH level in the kidneys and liver of magpies was not very strong (Table 3), probably other organic and inorganic co-contaminants also contributed significantly to the reduction of tissue GSH. It is important to point out that enhanced apoptosis— a controlled form of cell death, occurred in the kidneys and liver of magpies from a zinc smelter area (Tables 1 and 2). Because the apoptotic cells contain high Cd content (Tanimoto et al. 1993), apoptosis is thought to be a mode for elimination of Cd and critically damaged cells from kidneys and liver. Based on the Spearman rank correlation coefficients (Table 3) it may be concluded that Cd appears to be a significant factor inducing apoptosis in the kidneys and liver, although the GSH also may play an important role in the induction of this process. Indeed, it has been previously demonstrated that GSH depletion can sensitize the cells to apoptotic induction (Voehringer 1999). Thus, the reduction of GSH level in the liver and kidneys of magpies makes them probably more sensitive to apoptosis induced by other factors, e.g. Cd which is known to be a potent inducer of this process (Pulido and Parrish 2003). One mechanism by which Cd produces tissue injury is commonly believed to be through generation of reactive oxygen species and lipid peroxidation (Nomiyama and Nomiyama 1998; Shaikh et al. 1999; Thevenod and Friedmann 1999). However, in the present study no increase in lipid peroxidation was observed in the kidneys of magpies exhibiting massive histopathology (Table 1). Thus, it seems unlikely that lipid peroxidation could be responsible for the renal injury. Others have also contested the main role of lipid peroxidation in cell injury induced by Cd and other toxicants (Rush et al. 1985; Stacey et al. 1980; Włostowski et al. 2000). Therefore, another mechanism is probably involved in Cd-induced tissue damage in the magpies; for instance, the loss of ionic control in the proximal tubules due to reduction of ATP synthesis in mitochondria may have been implicated (Nicholson et al. 1983; Cannino et al. 2009).

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A multi-metal analysis of the magpie’s feathers revealed that the birds from the zinc smelter area are intoxicated not only by Cd but also, to some extent, by Pb and Zn (Dmowski 1997). However, we were unable to detect any specific signs of Pb poisoning, e.g. karyomegaly and inclusion bodies (Qu et al. 2002) in the kidneys of these birds. This may suggest that the accumulation of Pb in the kidneys (less than 3 lg/g wet wt; data not shown) was probably too low to elicit the apparent response. In addition, there were no relations between Pb and Zn concentrations, and histopathological changes observed in the kidneys of magpies under study (data not shown). Still, it cannot be ruled out that Pb and other inorganic and organic co-contaminants act additively or synergistically with Cd bringing about the renal injury even at low total Cd concentration. The nephrotoxicity occurring in the free-ranging magpies could also be exacerbated by some ecological factors, e.g. food shortage, bad weather or social stress. However, a role of these factors in the metal-induced nephrotoxicity in the free-living magpies is unknown. It is also unknown whether the observed renal injury impairs the physiological functions of the kidneys and reduces fitness in these birds. This remains to be elucidated in future studies. In conclusion, the data obtained in the present study suggest that nephrotoxicity occurring in the magpies from a zinc smelter area may be linked to Cd intoxication. The apparent sensitivity of the birds to Cd toxicity may be associated with the reduction of renal MT and GSH concentrations.

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