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Histopathological changes in the liver, kidneys, and testes of bank voles environmentally exposed to heavy metal emissions from the steelworks and zinc smelter in Poland
ARTICLE IN PRESS
Environmental Research 96 (2004) 72–78
Histopathological changes in the liver, kidneys, and testes of bank voles environmentally exposed to heavy metal emissions from the steelworks and zinc smelter in Poland Monika Damek-Poprawaa,* and Katarzyna Sawicka-Kapustab b
a Faculty of Food Technology, Agricultural University, ul. Balicka 122, 30-149 Krakow, Poland Department of Environmental Monitoring, Institute of Environmental Sciences, Jagiellonian University, ul. R. Ingardena 6, 30-060 Krakow, Poland
Received 14 May 2003; received in revised form 30 January 2004; accepted 3 February 2004
Abstract Bank voles were trapped in the neighborhood of the Sendzimir steelworks in Krakow and the ZGH Boleslaw zinc smelter in Bukowno. The Borecka forest in the north of Poland served as a control area. Lead, cadmium, zinc, and iron concentrations were analyzed in the liver, kidneys, testes, and femur bones of the bank voles. Typically, high levels of lead and cadmium were found in the bones and kidneys, respectively. In the femurs of the rodents from Bukowno, 109.26 mg g 1 dry weight of lead was detected. The kidneys of these animals had accumulated 32.98 mg g 1 cadmium. Concentrations of zinc and iron in the tissues were at physiological levels. No damage was found in the tissues of the bank voles from the Borecka forest or in the testes of animals from other areas. Histopathological changes in the kidneys of the rodents from Krakow as well as changes to the liver and kidneys of the animals from Bukowno were demonstrated. r 2004 Elsevier Inc. All rights reserved. Keywords: Bank voles; Heavy metals; Histopathology
1. Introduction Most data concerning the influence of lead, cadmium, zinc, and iron on the structure and function of organisms have been derived from laboratory experiments (Waalkes and Perantoni, 1988; S´wiergosz et al., 1998; Shore and Rattner, 2001). However, since laboratory experiments do not reflect changeable conditions in natural ecosystems and do not provide information about the state of the environment, field studies are indispensable (Talmage and Walton, 1991; Shore and Douben, 1994a, b). Bank voles, Clethrionomys glareolus (Schreber, 1780) living in the wild were the objects of the analyses. The chosen species of rodents fulfills the requirements for being a monitor of environmental contamination (O’Brien et al., 1993; Shore and Rattner, 2001); they *Corresponding author. Present address: Department of Biochemistry, SDM, University of Pennsylvania, 240 S 40th St. Room 520 Levy Research, Philadelphia, PA 19104-6030, USA. E-mail address: [email protected] (M. Damek-Poprawa). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.02.003
occur commonly in high population densities, are shortlived, have a restricted migration area, and are easy to catch (Pucek, 1984). Histological analyses combined with data on the body burden of heavy metals provide valuable information about the influence of heavy metals on the condition of living organisms (Spellerberg, 1991; O’Brien et al., 1993). Of the heavy metals considered in this study, zinc and iron are indispensable to ensure the normal development and function of the organism, whereas lead and cadmium are toxic elements (Merian, 1991; Shore and Rattner, 2001). The level of zinc and iron present in tissues depends on the age, gender, and physiological state of an individual, and homeostatic mechanisms ensure the regulation of the concentration of these metals around the metabolic optimum (Venugopal and Luckey, 1978; Friberg et al., 1986; Sawicka-Kapusta, 1995). Lead accumulates mostly in bones (Friberg et al., 1986; Cooke et al., 1990). However, damage caused by lead poisoning occurs first in bone marrow, the nervous system, and the kidneys (Friberg et al., 1986). Lead poisoning causes renal dysfunction, liver cirrhosis,
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damage to the central nervous system, and anemia (Sheffield et al., 2001). Absorbed cadmium is deposited mostly in the kidneys and the liver, whereas the kidneys, lungs, and bones are target organs for this metal (Hunter et al., 1989; Shore and Rattner, 2001). Cadmium intoxication leads to renal, hepatic, testicular, and prostate dysfunction, osteomalacia, hypertension, as well as growth retardation (Venugopal and Luckey, 1978). It also causes a disturbance of the central nervous system, poor lactation, and reductions in hematocrit value (Friberg et al., 1986). The high accumulation of cadmium in the liver and kidneys entails an increase of the concentration of zinc in these organs (Friberg et al., 1986; Yang et al., 2000). In addition, the presence of cadmium in the diet reduces the intestinal absorption of iron (Friberg et al., 1986; Kabata-Pendias and Pendias, 1999). The aim of this study was to determine the influence of lead, cadmium, zinc, and iron on the histological structure of the liver, kidneys, and testes of bank voles environmentally exposed to heavy metals. Concentrations of lead, cadmium, zinc, and iron in the liver, kidneys, testes, and bones of the animals were assessed, and microscope slides were prepared.
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The concentrations of lead, cadmium, zinc, and iron were analyzed in the femur bones, the remainder of the liver, the second kidney, and testis of live-trapped animals, as well as in the same tissues dissected from dead-trapped bank voles. These samples were dried to a constant weight at 60 C prior to wet digestion in a mixture of nitric and perchloric acids (4:1) (spectral grade). Afterwards, they were evaporated and doubledistilled water was added up to 10 mL (SawickaKapusta, 1979). Lead and cadmium concentrations were determined in a Perkin-Elmer Analyst 800 graphite furnace atomic absorption spectrophotometer (AAS), and levels of zinc and iron with the use of a flame implementing an AAS 1L-250. To determine whether there is any association between the levels of heavy metals in tissues and the age of the bank vole, regression analysis was used. That no such association was found was probably due to the limited number of animals caught in the field. In consequence, the groups of animals from each site were not divided into age classes for further analyses. The nonparametric Kruskal–Wallis test and the a posteriori test by Sachs were used to determine the significance of differences in the concentrations of heavy metals in every organ of animals from each site (Sokal and Rohlf, 1981; Sachs, 1984).
2. Materials and methods Research was conducted within the neighborhood of the Sendzimir steelworks in Krakow and within the area of the ZGH Boleslaw zinc smelter in Bukowno in the Upper Silesia region. The Borecka forest, an unindustrialized, unpolluted region in the north of Poland (Siuta, 1994; Sawicka-Kapusta et al., 1995), was selected as the control site. The bank voles were trapped during the period between September and October 2000. The catch was carried out on 1-ha plots according to the modified Standard Minimum method (Grodzin´ski et al., 1966: Bejcek, 1988). The age of each animal was determined on the basis of eye lens weight (Kozakiewicz, 1976). Slides for histological analyses were prepared from a section of the liver, one kidney, and one testis of the livetrapped animals. The rodents (six animals from the neighborhood of each industrial plant and four bank voles from the control area) were euthanized by ether before dissection. The samples were fixed in buffered formalin. Following dehydration, the tissues were mounted in paraffin, and 7-mm sections were cut. Staining was done with hematoxylin and eosin, as well as by the Goldner method. The Goldner method was used for dyeing supporting tissues (Litwin, 1995; Romeis, 1948). The periodic acid-Schiff (PAS) histochemical reaction to glycogen (Bancroft, 1967) was also carried out.
3. Results 3.1. Age of bank voles The results of the age determination of bank voles are provided in Table 1. The average age of the animals from the control and Bukowno areas amounted to 3 months, but the age range was wider in the case of the rodents from Bukowno. The bank voles from the neighborhood of the Sendzimir steelworks were 4 months old on average. 3.2. Heavy metal concentrations The average concentrations of lead, cadmium, zinc, and iron in the liver, kidneys, testes, and femurs of the Table 1 The age (months) of bank voles C. glareolus trapped in the Krakow and Bukowno areas and in the Borecka forest Area
na,b
Average ageb
Age range (months)
Borecka forest Krakow Bukowno
11(3) 9(4) 7(3)
3(3) 4(4) 3(5)
1–6 2–9 1–9
a b
n; Number of animals. Data for males is given in parentheses.
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bank voles trapped in the studied areas are presented in Table 2. The highest average level of lead, 109.26 mg g 1, was detected in the femurs of the bank voles caught within the neighborhood of the ZGH Boleslaw in Bukowno. The concentrations of lead in the soft tissues (liver, kidneys, and testes) of these rodents were also higher in comparison to the values found in samples from the other areas. The highest cadmium concentrations were found in the tissues of bank voles from the Bukowno area. The highest level of this metal, 32.98 mg g 1 on average, accumulated in the kidneys. Lower amounts of cadmium were detected in the livers of these rodents, but the values were significantly higher (Po0:05) than those found in the samples from the neighborhood of the steelworks and from the control area. The concentrations of iron in the analyzed organs did not vary significantly from one another with the exception of the average level present in the livers of the bank voles trapped within the Sendzimir steelworks area. In their case, iron concentration amounted to 1079.9 mg g 1. There were significant differences (Po0:05) in zinc levels present in the analyzed tissues. The kidneys, femurs, and testes of the bank voles trapped within the ZGH Boleslaw neighborhood, as well as the testes of the rodents from the Krakow area accumulated more zinc in comparison to the samples from the Borecka forest.
were of a normal shape, and no fibrosis was visible (Fig. 1). Glycogen was evenly distributed in the tissue, and only those cells located near a capsule contained a slightly higher amount of polyose. A similar normal pattern was found in the livers of rodents trapped within the Krakow area. Glycogen distribution was even. However, histopathological changes were found in the livers of five out of the six rodents from Bukowno. In these cases we observed polymorphism of the nuclei, vacuolization of the cells, and degenerate hepatocytes. Interstitial fibrosis and injuries to the epithelium of the blood vessels were also demonstrated (Fig. 2). A vestigial reaction to glycogen was observed close to a capsule, but the reaction faded centripetally. No damage was seen in any specimen of kidneys of bank voles from the control area. The renal cortex and the medulla were both normally developed (Fig. 3). A strong reaction to glycogen was seen in the glomeruli.
3.3. Histological analyses No histopathological changes were determined in any of the tissues analyzed from rodents live-trapped within the control area. The livers of bank voles from the Borecka forest had a compact structure, the hepatocytes Table 2 Average lead, cadmium, zinc, and iron concentrations7SE (mg g a
1
dry weight) in the selected tissues of bank voles, C. glareolus
b
Element
Area
n
Pb
Borecka forest Krakow Bukowno
11 9 7
Cd
Borecka forest Krakow Bukowno
11 9 7
0.8070.12 a 1.2670.34a 16.3971.56b
Zn
Borecka forest Krakow Bukowno
11 9 7
95.173.2a 87.776.3a 95.977.9a
Fe
Borecka forest Krakow Bukowno
11 9 7
a b
Fig. 1. The normal structure of the liver of a bank vole from the control area ( 250). BV, blood vessel.
Liver
0.1170.05a 0.4070.18a, b 1.9570.47b
551.4762.1a 1079.97167.4b 680.6797.0a, b
Kidneyb
Femurb
Testisb
0.7470.27a 1.9371.02a 17.8177.18b
0.5170.13a 1.1270.10a 109.26749.64b
0.9670.05a 3.1472.64a 5.6073.17a
3.2170.55a 7.2372.01a 32.9876.14b
0.1170.03a 0.2170.11a 0.9670.39b
0.6370.03a 1.5771.34a 2.1571.05a
80.374.2a 92.775.8a, b 133.279.3b
159.576.6a, b 130.977.4b 179.7711.6a
493.9740.8a 674.37101.4a 465.2743.7a
141.077.9a 163.8724.4a 167.8735.7a
n; Number of animals. Different letters denote statistically significant differences (Po0:05) between sites in heavy metal concentrations in every organ.
14.871.4a 82.9711.9b 87.6716.9b 303.5715.0a 752.17460.6a 455.37207.0a
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Fig. 2. The liver of a bank vole from the Bukowno area ( 250), H, Vacuolized hepatocytes with polymorphic nuclei; BV, blood vessel.
Fig. 4. The medulla of the kidney of a bank vole from the Krakow area ( 250). DT, distal tubule; BV, blood vessel.
Fig. 3. The medulla of the kidney of a bank vole from the control area ( 250). DT, distal tubule.
Fig. 5. The testis of a bank vole from the control area (Goldner; 250). ST, seminiferous tubule.
Proximal and distal tubules, as well as blood vessels, contained a marked smaller amount of glycogen. The walls of the distal tubules had grown thicker, and blood inflow to the organ was demonstrated in four of the six specimens from the Krakow area (Fig. 4). However, the structure of the glomeruli remained intact and the distribution of glycogen was similar to that found in bank voles from the control area. Histopathological damage was also found in the kidneys of five out of the six animals from Bukowno. Atrophy of some glomeruli, adhesion of Bowman’s capsule to the glomerulus, and contraction of proximal tubules were seen. However, no changes to glycogen distribution were observed. There was no damage present in any sample of the testes of the bank voles trapped in the Borecka forest. The seminiferous tubules were normally developed, and the consecutive stages of spermatogenesis were seen (Fig. 5). The testes of young animals (1–3 months old) were strongly PAS positive, whereas the specimens of older males contained a smaller amount of glycogen.
4. Discussion Bank voles trapped in the Bukowno area accumulated the highest amounts of lead and cadmium in their tissues. The average level of lead in the femurs was 100 times higher than the value found in samples from the neighborhood of the Sendzimir steelworks. Furthermore, it exceeded by 4200 times the average lead concentration in the bones of the animals from the Borecka forest. The average levels of cadmium in the liver, kidneys, and femurs of the rodents caught in Bukowno were statistically the highest. The kidneys, a critical organ following long-term exposure (Friberg et al., 1986; Sheffield et al., 2001), contained 44 and 10 times more cadmium in comparison with the samples from the Krakow and control areas, respectively. The highest concentrations of lead and cadmium in the tissues of the animals from the neighborhood of the ZGH Boleslaw zinc smelter resulted from considerable environmental pollution in the area. In 1999 the average
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dust fall in Bukowno came to 460 g m 2. Cadmium fall was 34 mg m 2, which exceeded the permissible standard of 10 mg m 2 year 1 by over three times. The permissible level of lead fall of 100 mg m 2 year 1 was not exceeded in 1999. The high natural content of lead, cadmium, and zinc in soil of the Olkusz region, resulting from the occurrence of zinc and lead ores, constituted an additional burden for the organisms living in the Bukowno area (Turzan´ski and Wertz, 2000; Grzesiak and Sieradzki, 2000). As for the area influenced by the Sendzirnir steelworks, the average dust fall in 1999 amounted to 100 g m 2. Cadmium fall was 2 mg m 2. The amount of lead present in the dust did not exceed the permissible standard (Turzan´ski and Wertz, 2000). The zinc and iron concentrations in the analyzed tissues were at physiological levels and, in most cases and did not differ significantly from one another, Homeostatic mechanisms regulating the absorption and excretion thereof by the organism remained intact (Venugopal and Luckey, 1978; Shore and Rattner, 2001). The exceptionally lower amount of zinc present in the testes of bank voles from the Borecka forest could be due to the reproductive status of these animals (Shore and Rattner, 2001). Moreover, in the case of the tissues of the rodents from the Krakow and Bukowno areas, a higher accumulation of cadmium could favor zinc absorption (Friberg et al., 1986; Piasek et al., 1996; Yang et al., 2000). The inhibition of the absorption of iron by high doses of lead and cadmium led to a reduction of iron concentration in the liver and kidneys of rodents from the Bukowno area (Friberg et al., 1986; Piasek et al., 1996; S´wiergosz et al., 1998). A high average of iron concentration, 1079.9 mg g 1, was found only in the livers of the bank voles trapped in the Sendzimir steelworks neighborhood, where iron ore processing is carried out. The highest level of iron compounds was detected in the soil of this neighborhood, in comparison with other regions of the former Krakow Province (now part of the Malopolska Province) (Turzan´ski and Wertz, 1997). The pattern of histopathological changes in the analyzed tissues fully correlated with data obtained in laboratory studies regarding the influence of heavy metals on living organisms (Shore and Rattner, 2001). Lead hepatotoxicity manifests itself in vacuolization of the cells, polymorphism of the nuclei, and a decrease in glycogen content of hepatocytes (Bolognani Fantin et al., 1992; Foulkes, 1996; Pereira et al., 2001). In the kidneys, lead intoxication causes, among other reactions, interstitial fibrosis, as well as both hyperplasia and the gradual atrophy of tubules and glomeruli (Goyer, 1989; Nolan and Shaikh, 1992). Cadmium accumulation leads to histopathological changes in the liver, such as
vacuolization of the cytoplasm of the hepatocytes, fibrosis, polymorphism of nuclei, necrosis of hepatocytes and parenchymal cells, as well as injury to the hepatic endothelial cells (Liu et al., 1992; MotasGuzman et al., 1996; Stevens and Lowe, 2000). Chronic cadmium exposure damages the kidneys. In the first stage, tubular proteinuria, aminoaciduria, glucosuria, and phosphaturia occur (Itokawa et al., 1978; Waalkes et al., 1992). Further adhesion of Bowman’s capsule to the glomerulus and atrophy of the glomeruli are seen. However, in the case of the vacuolization of the hepatocytes, as well as adhesion of Bowman’s capsule to the glomerulus in the kidneys, it was impossible to determine clearly which metal (lead or cadmium) caused a specific lesion. On the one hand, complex laboratory experiments on the influence of lead, cadmium, and lead–cadmium co-treatment on the organism, and on the other hand, the localization of these metals in the analyzed tissues by means of scanning microscopy techniques should be done to clarify the matter. Despite the high concentrations of cadmium present in the tissues of the bank voles, histopathological changes appeared more seldom than in the case of yellow-necked mice trapped in the same areas (DamekPoprawa and Sawicka-Kapusta, 2003). Although 2.1571.05 (SE) mg g 1 of cadmium was detected in the testes of bank voles from Bukowno, no histopathological changes were observed. Damage to the testes of the yellow-necked mice trapped within the same area appeared when the level of cadmium was 0.7070.21 (SE) mg g 1. The higher tolerance of cadmium may result from a higher production of metallothionein, a cysteine-rich heavy metal-binding protein, by the organism (Shore and Rattner, 2001). Higher levels of metallothionein in the tissues of the bank voles, as compared with the yellow-necked mice, were confirmed in parallel studies (Damek-Poprawa, 2002). Histopathological changes in the tissues of bank voles living in the wild occurred at a lower body burden of heavy metal than demonstrated in laboratory experiments. Experimental data indicated that damage to the kidneys of bank voles chronically exposed to cadmium in their diet appeared when the level of cadmium in the tissues analyzed was 10 times higher than that found in samples from the Krakow area (S´wiergosz et al., 1998). The putative cause for greater sensitivity to heavy metals of rodents living in the wild is that an organism is subject to more stress in a field than in laboratory conditions and less energy could be destined for detoxification (Shore and Rattner, 2001). Our research showed that even a relatively low body burden of lead and cadmium can lead to histopathological changes in the tissues of rodents chronically exposed to heavy metals. Since the pattern of heavy metal distribution and their levels in the tissues of rodents are similar to those found in humans
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(Wesenberg et al., 1979), it is conceivable that emissions from the analyzed steelworks and zinc smelter also present a considerable threat to human health.
Acknowledgments The research was supported by a grant (No. 6P04G03218) from the Polish Committee for Scientific Research.
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Environmental Research 96 (2004) 72–78
Histopathological changes in the liver, kidneys, and testes of bank voles environmentally exposed to heavy metal emissions from the steelworks and zinc smelter in Poland Monika Damek-Poprawaa,* and Katarzyna Sawicka-Kapustab b
a Faculty of Food Technology, Agricultural University, ul. Balicka 122, 30-149 Krakow, Poland Department of Environmental Monitoring, Institute of Environmental Sciences, Jagiellonian University, ul. R. Ingardena 6, 30-060 Krakow, Poland
Received 14 May 2003; received in revised form 30 January 2004; accepted 3 February 2004
Abstract Bank voles were trapped in the neighborhood of the Sendzimir steelworks in Krakow and the ZGH Boleslaw zinc smelter in Bukowno. The Borecka forest in the north of Poland served as a control area. Lead, cadmium, zinc, and iron concentrations were analyzed in the liver, kidneys, testes, and femur bones of the bank voles. Typically, high levels of lead and cadmium were found in the bones and kidneys, respectively. In the femurs of the rodents from Bukowno, 109.26 mg g 1 dry weight of lead was detected. The kidneys of these animals had accumulated 32.98 mg g 1 cadmium. Concentrations of zinc and iron in the tissues were at physiological levels. No damage was found in the tissues of the bank voles from the Borecka forest or in the testes of animals from other areas. Histopathological changes in the kidneys of the rodents from Krakow as well as changes to the liver and kidneys of the animals from Bukowno were demonstrated. r 2004 Elsevier Inc. All rights reserved. Keywords: Bank voles; Heavy metals; Histopathology
1. Introduction Most data concerning the influence of lead, cadmium, zinc, and iron on the structure and function of organisms have been derived from laboratory experiments (Waalkes and Perantoni, 1988; S´wiergosz et al., 1998; Shore and Rattner, 2001). However, since laboratory experiments do not reflect changeable conditions in natural ecosystems and do not provide information about the state of the environment, field studies are indispensable (Talmage and Walton, 1991; Shore and Douben, 1994a, b). Bank voles, Clethrionomys glareolus (Schreber, 1780) living in the wild were the objects of the analyses. The chosen species of rodents fulfills the requirements for being a monitor of environmental contamination (O’Brien et al., 1993; Shore and Rattner, 2001); they *Corresponding author. Present address: Department of Biochemistry, SDM, University of Pennsylvania, 240 S 40th St. Room 520 Levy Research, Philadelphia, PA 19104-6030, USA. E-mail address: [email protected] (M. Damek-Poprawa). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.02.003
occur commonly in high population densities, are shortlived, have a restricted migration area, and are easy to catch (Pucek, 1984). Histological analyses combined with data on the body burden of heavy metals provide valuable information about the influence of heavy metals on the condition of living organisms (Spellerberg, 1991; O’Brien et al., 1993). Of the heavy metals considered in this study, zinc and iron are indispensable to ensure the normal development and function of the organism, whereas lead and cadmium are toxic elements (Merian, 1991; Shore and Rattner, 2001). The level of zinc and iron present in tissues depends on the age, gender, and physiological state of an individual, and homeostatic mechanisms ensure the regulation of the concentration of these metals around the metabolic optimum (Venugopal and Luckey, 1978; Friberg et al., 1986; Sawicka-Kapusta, 1995). Lead accumulates mostly in bones (Friberg et al., 1986; Cooke et al., 1990). However, damage caused by lead poisoning occurs first in bone marrow, the nervous system, and the kidneys (Friberg et al., 1986). Lead poisoning causes renal dysfunction, liver cirrhosis,
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damage to the central nervous system, and anemia (Sheffield et al., 2001). Absorbed cadmium is deposited mostly in the kidneys and the liver, whereas the kidneys, lungs, and bones are target organs for this metal (Hunter et al., 1989; Shore and Rattner, 2001). Cadmium intoxication leads to renal, hepatic, testicular, and prostate dysfunction, osteomalacia, hypertension, as well as growth retardation (Venugopal and Luckey, 1978). It also causes a disturbance of the central nervous system, poor lactation, and reductions in hematocrit value (Friberg et al., 1986). The high accumulation of cadmium in the liver and kidneys entails an increase of the concentration of zinc in these organs (Friberg et al., 1986; Yang et al., 2000). In addition, the presence of cadmium in the diet reduces the intestinal absorption of iron (Friberg et al., 1986; Kabata-Pendias and Pendias, 1999). The aim of this study was to determine the influence of lead, cadmium, zinc, and iron on the histological structure of the liver, kidneys, and testes of bank voles environmentally exposed to heavy metals. Concentrations of lead, cadmium, zinc, and iron in the liver, kidneys, testes, and bones of the animals were assessed, and microscope slides were prepared.
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The concentrations of lead, cadmium, zinc, and iron were analyzed in the femur bones, the remainder of the liver, the second kidney, and testis of live-trapped animals, as well as in the same tissues dissected from dead-trapped bank voles. These samples were dried to a constant weight at 60 C prior to wet digestion in a mixture of nitric and perchloric acids (4:1) (spectral grade). Afterwards, they were evaporated and doubledistilled water was added up to 10 mL (SawickaKapusta, 1979). Lead and cadmium concentrations were determined in a Perkin-Elmer Analyst 800 graphite furnace atomic absorption spectrophotometer (AAS), and levels of zinc and iron with the use of a flame implementing an AAS 1L-250. To determine whether there is any association between the levels of heavy metals in tissues and the age of the bank vole, regression analysis was used. That no such association was found was probably due to the limited number of animals caught in the field. In consequence, the groups of animals from each site were not divided into age classes for further analyses. The nonparametric Kruskal–Wallis test and the a posteriori test by Sachs were used to determine the significance of differences in the concentrations of heavy metals in every organ of animals from each site (Sokal and Rohlf, 1981; Sachs, 1984).
2. Materials and methods Research was conducted within the neighborhood of the Sendzimir steelworks in Krakow and within the area of the ZGH Boleslaw zinc smelter in Bukowno in the Upper Silesia region. The Borecka forest, an unindustrialized, unpolluted region in the north of Poland (Siuta, 1994; Sawicka-Kapusta et al., 1995), was selected as the control site. The bank voles were trapped during the period between September and October 2000. The catch was carried out on 1-ha plots according to the modified Standard Minimum method (Grodzin´ski et al., 1966: Bejcek, 1988). The age of each animal was determined on the basis of eye lens weight (Kozakiewicz, 1976). Slides for histological analyses were prepared from a section of the liver, one kidney, and one testis of the livetrapped animals. The rodents (six animals from the neighborhood of each industrial plant and four bank voles from the control area) were euthanized by ether before dissection. The samples were fixed in buffered formalin. Following dehydration, the tissues were mounted in paraffin, and 7-mm sections were cut. Staining was done with hematoxylin and eosin, as well as by the Goldner method. The Goldner method was used for dyeing supporting tissues (Litwin, 1995; Romeis, 1948). The periodic acid-Schiff (PAS) histochemical reaction to glycogen (Bancroft, 1967) was also carried out.
3. Results 3.1. Age of bank voles The results of the age determination of bank voles are provided in Table 1. The average age of the animals from the control and Bukowno areas amounted to 3 months, but the age range was wider in the case of the rodents from Bukowno. The bank voles from the neighborhood of the Sendzimir steelworks were 4 months old on average. 3.2. Heavy metal concentrations The average concentrations of lead, cadmium, zinc, and iron in the liver, kidneys, testes, and femurs of the Table 1 The age (months) of bank voles C. glareolus trapped in the Krakow and Bukowno areas and in the Borecka forest Area
na,b
Average ageb
Age range (months)
Borecka forest Krakow Bukowno
11(3) 9(4) 7(3)
3(3) 4(4) 3(5)
1–6 2–9 1–9
a b
n; Number of animals. Data for males is given in parentheses.
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bank voles trapped in the studied areas are presented in Table 2. The highest average level of lead, 109.26 mg g 1, was detected in the femurs of the bank voles caught within the neighborhood of the ZGH Boleslaw in Bukowno. The concentrations of lead in the soft tissues (liver, kidneys, and testes) of these rodents were also higher in comparison to the values found in samples from the other areas. The highest cadmium concentrations were found in the tissues of bank voles from the Bukowno area. The highest level of this metal, 32.98 mg g 1 on average, accumulated in the kidneys. Lower amounts of cadmium were detected in the livers of these rodents, but the values were significantly higher (Po0:05) than those found in the samples from the neighborhood of the steelworks and from the control area. The concentrations of iron in the analyzed organs did not vary significantly from one another with the exception of the average level present in the livers of the bank voles trapped within the Sendzimir steelworks area. In their case, iron concentration amounted to 1079.9 mg g 1. There were significant differences (Po0:05) in zinc levels present in the analyzed tissues. The kidneys, femurs, and testes of the bank voles trapped within the ZGH Boleslaw neighborhood, as well as the testes of the rodents from the Krakow area accumulated more zinc in comparison to the samples from the Borecka forest.
were of a normal shape, and no fibrosis was visible (Fig. 1). Glycogen was evenly distributed in the tissue, and only those cells located near a capsule contained a slightly higher amount of polyose. A similar normal pattern was found in the livers of rodents trapped within the Krakow area. Glycogen distribution was even. However, histopathological changes were found in the livers of five out of the six rodents from Bukowno. In these cases we observed polymorphism of the nuclei, vacuolization of the cells, and degenerate hepatocytes. Interstitial fibrosis and injuries to the epithelium of the blood vessels were also demonstrated (Fig. 2). A vestigial reaction to glycogen was observed close to a capsule, but the reaction faded centripetally. No damage was seen in any specimen of kidneys of bank voles from the control area. The renal cortex and the medulla were both normally developed (Fig. 3). A strong reaction to glycogen was seen in the glomeruli.
3.3. Histological analyses No histopathological changes were determined in any of the tissues analyzed from rodents live-trapped within the control area. The livers of bank voles from the Borecka forest had a compact structure, the hepatocytes Table 2 Average lead, cadmium, zinc, and iron concentrations7SE (mg g a
1
dry weight) in the selected tissues of bank voles, C. glareolus
b
Element
Area
n
Pb
Borecka forest Krakow Bukowno
11 9 7
Cd
Borecka forest Krakow Bukowno
11 9 7
0.8070.12 a 1.2670.34a 16.3971.56b
Zn
Borecka forest Krakow Bukowno
11 9 7
95.173.2a 87.776.3a 95.977.9a
Fe
Borecka forest Krakow Bukowno
11 9 7
a b
Fig. 1. The normal structure of the liver of a bank vole from the control area ( 250). BV, blood vessel.
Liver
0.1170.05a 0.4070.18a, b 1.9570.47b
551.4762.1a 1079.97167.4b 680.6797.0a, b
Kidneyb
Femurb
Testisb
0.7470.27a 1.9371.02a 17.8177.18b
0.5170.13a 1.1270.10a 109.26749.64b
0.9670.05a 3.1472.64a 5.6073.17a
3.2170.55a 7.2372.01a 32.9876.14b
0.1170.03a 0.2170.11a 0.9670.39b
0.6370.03a 1.5771.34a 2.1571.05a
80.374.2a 92.775.8a, b 133.279.3b
159.576.6a, b 130.977.4b 179.7711.6a
493.9740.8a 674.37101.4a 465.2743.7a
141.077.9a 163.8724.4a 167.8735.7a
n; Number of animals. Different letters denote statistically significant differences (Po0:05) between sites in heavy metal concentrations in every organ.
14.871.4a 82.9711.9b 87.6716.9b 303.5715.0a 752.17460.6a 455.37207.0a
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Fig. 2. The liver of a bank vole from the Bukowno area ( 250), H, Vacuolized hepatocytes with polymorphic nuclei; BV, blood vessel.
Fig. 4. The medulla of the kidney of a bank vole from the Krakow area ( 250). DT, distal tubule; BV, blood vessel.
Fig. 3. The medulla of the kidney of a bank vole from the control area ( 250). DT, distal tubule.
Fig. 5. The testis of a bank vole from the control area (Goldner; 250). ST, seminiferous tubule.
Proximal and distal tubules, as well as blood vessels, contained a marked smaller amount of glycogen. The walls of the distal tubules had grown thicker, and blood inflow to the organ was demonstrated in four of the six specimens from the Krakow area (Fig. 4). However, the structure of the glomeruli remained intact and the distribution of glycogen was similar to that found in bank voles from the control area. Histopathological damage was also found in the kidneys of five out of the six animals from Bukowno. Atrophy of some glomeruli, adhesion of Bowman’s capsule to the glomerulus, and contraction of proximal tubules were seen. However, no changes to glycogen distribution were observed. There was no damage present in any sample of the testes of the bank voles trapped in the Borecka forest. The seminiferous tubules were normally developed, and the consecutive stages of spermatogenesis were seen (Fig. 5). The testes of young animals (1–3 months old) were strongly PAS positive, whereas the specimens of older males contained a smaller amount of glycogen.
4. Discussion Bank voles trapped in the Bukowno area accumulated the highest amounts of lead and cadmium in their tissues. The average level of lead in the femurs was 100 times higher than the value found in samples from the neighborhood of the Sendzimir steelworks. Furthermore, it exceeded by 4200 times the average lead concentration in the bones of the animals from the Borecka forest. The average levels of cadmium in the liver, kidneys, and femurs of the rodents caught in Bukowno were statistically the highest. The kidneys, a critical organ following long-term exposure (Friberg et al., 1986; Sheffield et al., 2001), contained 44 and 10 times more cadmium in comparison with the samples from the Krakow and control areas, respectively. The highest concentrations of lead and cadmium in the tissues of the animals from the neighborhood of the ZGH Boleslaw zinc smelter resulted from considerable environmental pollution in the area. In 1999 the average
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dust fall in Bukowno came to 460 g m 2. Cadmium fall was 34 mg m 2, which exceeded the permissible standard of 10 mg m 2 year 1 by over three times. The permissible level of lead fall of 100 mg m 2 year 1 was not exceeded in 1999. The high natural content of lead, cadmium, and zinc in soil of the Olkusz region, resulting from the occurrence of zinc and lead ores, constituted an additional burden for the organisms living in the Bukowno area (Turzan´ski and Wertz, 2000; Grzesiak and Sieradzki, 2000). As for the area influenced by the Sendzirnir steelworks, the average dust fall in 1999 amounted to 100 g m 2. Cadmium fall was 2 mg m 2. The amount of lead present in the dust did not exceed the permissible standard (Turzan´ski and Wertz, 2000). The zinc and iron concentrations in the analyzed tissues were at physiological levels and, in most cases and did not differ significantly from one another, Homeostatic mechanisms regulating the absorption and excretion thereof by the organism remained intact (Venugopal and Luckey, 1978; Shore and Rattner, 2001). The exceptionally lower amount of zinc present in the testes of bank voles from the Borecka forest could be due to the reproductive status of these animals (Shore and Rattner, 2001). Moreover, in the case of the tissues of the rodents from the Krakow and Bukowno areas, a higher accumulation of cadmium could favor zinc absorption (Friberg et al., 1986; Piasek et al., 1996; Yang et al., 2000). The inhibition of the absorption of iron by high doses of lead and cadmium led to a reduction of iron concentration in the liver and kidneys of rodents from the Bukowno area (Friberg et al., 1986; Piasek et al., 1996; S´wiergosz et al., 1998). A high average of iron concentration, 1079.9 mg g 1, was found only in the livers of the bank voles trapped in the Sendzimir steelworks neighborhood, where iron ore processing is carried out. The highest level of iron compounds was detected in the soil of this neighborhood, in comparison with other regions of the former Krakow Province (now part of the Malopolska Province) (Turzan´ski and Wertz, 1997). The pattern of histopathological changes in the analyzed tissues fully correlated with data obtained in laboratory studies regarding the influence of heavy metals on living organisms (Shore and Rattner, 2001). Lead hepatotoxicity manifests itself in vacuolization of the cells, polymorphism of the nuclei, and a decrease in glycogen content of hepatocytes (Bolognani Fantin et al., 1992; Foulkes, 1996; Pereira et al., 2001). In the kidneys, lead intoxication causes, among other reactions, interstitial fibrosis, as well as both hyperplasia and the gradual atrophy of tubules and glomeruli (Goyer, 1989; Nolan and Shaikh, 1992). Cadmium accumulation leads to histopathological changes in the liver, such as
vacuolization of the cytoplasm of the hepatocytes, fibrosis, polymorphism of nuclei, necrosis of hepatocytes and parenchymal cells, as well as injury to the hepatic endothelial cells (Liu et al., 1992; MotasGuzman et al., 1996; Stevens and Lowe, 2000). Chronic cadmium exposure damages the kidneys. In the first stage, tubular proteinuria, aminoaciduria, glucosuria, and phosphaturia occur (Itokawa et al., 1978; Waalkes et al., 1992). Further adhesion of Bowman’s capsule to the glomerulus and atrophy of the glomeruli are seen. However, in the case of the vacuolization of the hepatocytes, as well as adhesion of Bowman’s capsule to the glomerulus in the kidneys, it was impossible to determine clearly which metal (lead or cadmium) caused a specific lesion. On the one hand, complex laboratory experiments on the influence of lead, cadmium, and lead–cadmium co-treatment on the organism, and on the other hand, the localization of these metals in the analyzed tissues by means of scanning microscopy techniques should be done to clarify the matter. Despite the high concentrations of cadmium present in the tissues of the bank voles, histopathological changes appeared more seldom than in the case of yellow-necked mice trapped in the same areas (DamekPoprawa and Sawicka-Kapusta, 2003). Although 2.1571.05 (SE) mg g 1 of cadmium was detected in the testes of bank voles from Bukowno, no histopathological changes were observed. Damage to the testes of the yellow-necked mice trapped within the same area appeared when the level of cadmium was 0.7070.21 (SE) mg g 1. The higher tolerance of cadmium may result from a higher production of metallothionein, a cysteine-rich heavy metal-binding protein, by the organism (Shore and Rattner, 2001). Higher levels of metallothionein in the tissues of the bank voles, as compared with the yellow-necked mice, were confirmed in parallel studies (Damek-Poprawa, 2002). Histopathological changes in the tissues of bank voles living in the wild occurred at a lower body burden of heavy metal than demonstrated in laboratory experiments. Experimental data indicated that damage to the kidneys of bank voles chronically exposed to cadmium in their diet appeared when the level of cadmium in the tissues analyzed was 10 times higher than that found in samples from the Krakow area (S´wiergosz et al., 1998). The putative cause for greater sensitivity to heavy metals of rodents living in the wild is that an organism is subject to more stress in a field than in laboratory conditions and less energy could be destined for detoxification (Shore and Rattner, 2001). Our research showed that even a relatively low body burden of lead and cadmium can lead to histopathological changes in the tissues of rodents chronically exposed to heavy metals. Since the pattern of heavy metal distribution and their levels in the tissues of rodents are similar to those found in humans
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(Wesenberg et al., 1979), it is conceivable that emissions from the analyzed steelworks and zinc smelter also present a considerable threat to human health.
Acknowledgments The research was supported by a grant (No. 6P04G03218) from the Polish Committee for Scientific Research.
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