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Damage to the liver, kidney, and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland
Toxicology 186 (2003) 1 /10 www.elsevier.com/locate/toxicol
Damage to the liver, kidney, and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland Monika Damek-Poprawa a,*, Katarzyna Sawicka-Kapusta b a
Department of Raw Materials and Processing of Fruit and Vegetables, Faculty of Food Technology, Agriculture University, ul. Podluzna 3, 30-239 Krakow, Poland b Department of Environmental Monitoring, Institute of Environmental Sciences, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland Received 15 April 2002; received in revised form 21 October 2002; accepted 21 October 2002
Abstract The influence of the steelworks in Warsaw and Krakow as well as the zinc smelters in Bukowno and Miasteczko Slaskie on lead, cadmium, zinc and iron concentrations and the structure of selected tissues of yellow-necked mice were analysed. The Borecka Forest was chosen as a control area. The highest concentrations of lead, 172.36 g/g dry weight, and cadmium, 23.58 g/g, were detected in the femurs and kidneys, respectively, of rodents caught in Bukowno. Zinc and iron concentrations ranged over physiological values. No histopathological changes were observed in analysed tissues of all rodents in the control area. Damage occurred in the liver and kidneys of animals from all other sites and in the testes of rodents from Bukowno. Decreased glycogen content, interstitial fibrosis, and increased number of pyknotic nuclei as well as necrosis were seen in hepatocytes. In the kidneys hyperplasia of the tubules, atrophy of glomeruli, interstitial fibrosis and necrosis were observed. Degenerate cells were present in the lumen of seminiferous tubules of animals from the Bukowno area. Even relatively low concentrations of lead and cadmium, like those found in the liver and kidneys of rodents from the neighbourhood of the steelworks, caused histopathological changes. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Yellow-necked mice; Heavy metals; Histopathology
1. Introduction The emission of pollutants to the environment has been considerably reduced during the last
* Corresponding author E-mail address: [email protected] Poprawa).
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decade, although the neighbourhood of industrial plants still remains heavily contaminated (Mochel, 1998; Sawicka-Kapusta et al., 1999; Appleton et al., 2000; Grzesiak and Sieradzki, 2000). Human activity leads to a large dispersion of heavy metals, which, not submitted to biodegradation, circulate in trophic chains and accumulate in living organisms (Merian, 1991). Some metals like zinc and iron are physiologically essential, but they may
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also alter the function of organisms when the exposure dose exceeds a critical threshold, which is species-specific, and can depend on age, sex, reproductive state and physiological condition of animals (Shore and Rattner, 2001). As for lead and cadmium, they do not play any functional role in the organisms. Lead accumulates mainly in bone but the critical organs for lead intoxication are bone marrow, the nervous system and kidneys (Friberg et al., 1986). Chronic exposure to lead causes renal dysfunction (Venugopal and Luckey, 1978; Nolan and Shaikh, 1992), liver damage (Bolognani Fantin et al., 1992), and decreased fertility in males and increased spontaneous abortion in females (Friberg et al., 1986). Cadmium causes damage primarily to kidney, bone and lungs (Domino, 1994). It accumulates mainly in kidney and affects the re-absorption functions of the proximal tubules (Mingard and Diezi, 1992; Waalkes et al., 1992; Roels et al., 1993). Cadmium also alters the calcium metabolism, which leads to osteomalacia (Ogoshi et al., 1992; Kido et al., 1993). Cadmium can damage the liver and testes (Amdur et al., 1991; Liu et al., 1992; Stevens and Lowe, 2000). This metal is also teratogenic and carcinogenic. After cadmium intoxication cancer of the testes and prostate occurs (Venugopal and Luckey, 1978; Friberg et al., 1986; Waalkes and Perantoni, 1988). In the organism, lead and cadmium can also alter the level of other metals like zinc and iron. When cadmium is present in the diet the absorption of iron diminishes. However, cadmium enhances zinc absorption from the gastrointestinal tract (Friberg et al., 1986; Kabata-Pendias and Pendias, 1999). Rodents are sensitive and accurate monitors of exposure to environmental pollution by heavy metals. The yellow-necked mouse Apodemus flavicollis (Melchior, 1834) belongs to the most numerous rodent species in Polish deciduous forests. The animals are easily caught, have a small migration area and a relatively short life span (Pucek, 1984). Moreover, the pattern of heavy metal distribution and levels of heavy metals in their tissues are similar to those found in humans (Wesenberg et al., 1979). In research, rodents frequently serve as mammalian surrogate
for humans (Shore and Rattner, 2001). This makes rodents ideal for monitoring environmental pollution as well as for evaluating the exposure risk for people living in a contaminated area (O’Brien et al., 1993). Previous studies on histopathological changes in tissues were carried out mostly in a laboratory on rodents fed on a diet containing very high amounts of lead and/or cadmium. Damages to the structure of the internal organs occurred only through substantial concentrations of these metals in the tissues of rodents (Swiergosz et al., 1998; Shore and Rattner, 2001). Analyses of the tissues of rodents trapped in the wild are scarce (Hunter et al., 1982). The aim of this study was to assess the impact of industrial pollution on yellow-necked mice. Concentrations of lead, cadmium, zinc and iron in the liver, kidneys, testes and femurs of rodents were determined. Moreover histological and histochemical analyses were done in order to determine heavy metal influence on tissues of yellow-necked mice living in the wild.
2. Material and methods Yellow-necked mice A. flavicollis (Melchior, 1834) were trapped in the area of two steelworks: the ‘Lucchini /Warszawa’ in Warsaw and the ‘Sendzimir Steelworks’ in Krakow as well as within the neighbourhood of two zinc smelters in the Upper Silesia region: ‘ZGH Boleslaw’ in Bukowno and ‘Miasteczko Slaskie’ in Miasteczko Slaskie. Borecka Forest in the north of Poland was chosen as a control area. In the year 2000 the steelworks ‘Lucchini /Warszawa’ emitted 583 kg of lead, 10 kg of cadmium, 3684 kg of zinc, and 6349 kg of iron and copper together. That year the ‘Sendzimir Steelworks’ in Krakow discharged 625 kg of Pb, 23 kg of Cd, and 263 kg of Cu. As for the zinc smelters, 2.2 kg of lead, 0.2 kg of cadmium, and 753 kg of zinc was emitted from the ‘ZGH Boleslaw’ in Bukowno. In 2000, the zinc smelter ‘Miasteczko Slaskie’ discharged 5214 kg of lead, 44 kg of cadmium as well as 7850 kg of zinc (all data on emission were provided by the mentioned steelworks and zinc smelters). The data, available
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from the year 1999, showed that dust fall within the neighbourhood of the steelworks in Warsaw amounted to 70.7 g/m2 (including 24.2 mg/m2 of lead, and 0.627 mg/m2 of cadmium) (Grzesiak and Sieradzki, 2000). That year dust fall in the Krakow area amounted to 100 g/m2 (including 100 mg/m2 of lead, and 2 mg/m2 of cadmium) (Grzesiak and Sieradzki, 2000). In the year 1999, 60 g/m2 of dust (including approximately 100 mg/ m2 of lead, and 34 mg/m2 of cadmium) fell down within the neighbourhood of the ‘Boleslaw’ zinc smelter (Grzesiak and Sieradzki, 2000). As for Miasteczko Slaskie, the average dust fall in 1999 reached 36 g/m2 (90 mg/m2 of lead, and 1.51 mg/ m2 of cadmium in it) (Grzesiak and Sieradzki, 2000). The Borecka Forest is one of the lowest polluted areas in Poland (Siuta, 1994; SawickaKapusta et al., 1995). In the year 2000 the emission of lead, cadmium, and zinc from local sources within the control area was negligible (Grzesiak and Sieradzki, 2001). The trapping was carried out from 24 September to 27 October 2000. In accordance with a modified Standard-Minimum method (Grodzinski et al., 1966; Bejcek, 1988) 242 traps (including 100 livetraps) were placed in each area on the 1 ha plot in a 10/10 m grid. Within the neighbourhood of the steelworks ‘Lucchini /Warszawa’ 18 yellow-necked mice (including 13 alive) were trapped. In the area of the ‘Sendzimir Steelworks’ 22 animals (including eight alive) were caught. As for the zinc smelters, within the neighbourhood of ‘ZGH Boleslaw’ and ‘Miasteczko Slaskie’ 12 (including seven alive) and 20 (nine alive) yellow-necked mice were trapped, respectively. 15 animals (nine alive) were caught in the control area. The rodents caught by live-traps were killed by ether and immediately dissected. A part of the liver, one kidney and one testis were used for slide preparation. The tissues were fixed with buffered formalin, dehydrated with alcohol and xylene and embedded in paraffin wax. Then they were cut into 7 m slices. Next, slides were stained with haematoxylin and eosin as well as by the Goldner method, which was used to demonstrate supporting tissue elements (Litwin, 1995; Romeis, 1948). The PAS (periodic acid */Schiff)
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histochemical reaction (Baucroft, 1967) was also performed. Glycogen, the intracellular storage form of carbohydrates, is PAS positive. Afterwards the slides were examined under a light microscope. The femurs, the rest of the liver, the other kidney and the other testis of live trapped rodents as well as the same tissues dissected from dead trapped animals were analysed for heavy metals. The samples were dried at 60 8C to a constant weight. Afterwards they were wet digested in a mixture of nitric and perchloric acid (4:1) (spectral grade). After evaporating the samples, double distilled water was added until the solution measured 10 ml (Sawicka-Kapusta, 1978). Lead and cadmium concentrations were determined in a Perkin /Elmer AAnalyst 800 graphite furnace atomic absorption spectrophotometer, while zinc and iron levels in a flame using an AAS IL-250. Yellow-necked mice were aged on the basis of eye lens weight according to the method proposed by Nabaglo and Pachinger (1979). The association between the heavy metal concentrations in tissues and the age of animal was checked by a regression analysis. The significance of differences in the heavy metal concentrations in the liver, kidneys, testes and femurs of animals from each site was determined by a non-parametric Kruskal-Wallis test and an a posteriori test by Sachs (Sokal and Rohlf, 1981; Sachs, 1984).
3. Results 3.1. Age of yellow-necked mice The age of the yellow-necked mice caught in the control area and within the neighbourhood of the ‘Lucchini /Warszawa’ steelworks averaged 4 months. Animals from the Krakow area were 3 months of age on average. As for the A. flavicollis from Bukowno, they were 2 months of age on average. Rodents trapped in the neighbourhood of the zinc smelter ‘Miasteczko Slaskie’ were the youngest */at the age of about 1 month.
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3.2. Heavy metal concentrations No association between the age of yellownecked mice and heavy metal concentrations in the tissues of animals was found. Average lead, cadmium, zinc and iron levels in selected tissues are given in the Table 1. The highest lead concentrations, 172.36 and 17.48 g/g, respectively, were detected in femurs of rodents caught in the area of the zinc smelters in Bukowno and in Miasteczko Slaskie. No statistically significant differences between lead levels in the liver of rodents from the control area and from the neighbourhood of the steelworks were found. However, the concentrations of those metals were slightly higher in tissues of yellow-necked mice from Warsaw and Krakow compared with the values in samples from the control area. The highest cadmium levels were found in the kidneys of the yellow-necked mice caught within the neighbourhood of the zinc smelters in Bukowno and Miasteczko Slaskie. There were 23.58 and 6.59 g Cd/g, respectively.
Zinc and iron concentrations in analysed samples ranged among the physiological values. The lowest amounts of zinc were found in samples from the Borecka Forest. The testes of the yellownecked mice from the control area contained on average only 17.4 g Zn/g. As for iron, the statistically significant lowest concentrations were found in liver and kidneys of animals trapped near the ‘ZGH Boleslaw’ zinc smelter. The samples contained on average 134.4 and 110.2 g Fe/g, respectively. 3.3. Histopathology No damage to analysed tissues was observed in the liver, kidneys and testes of all rodents livetrapped in the control area. The normal structure of the liver of the yellownecked mouse is shown in Figs. 1 and 2. Hepatocytes, the bile duct and the portal vein were clearly visible. Glycogen was evenly spread (Fig. 2). The liver of the yellow-necked mice caught within the neighbourhood of the ‘Lucchini /Wars-
Table 1 Average lead, cadmium, zinc and iron concentrations 9/SE (mg/g dry weight) in the selected tissues of the yellow-necked mice A. flavicollis Element Pb
Cd
Zn
Fe
Area Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl.
N 15 18 22 12 20 15 18 22 12 20 15 18 22 12 20 15 18 22 12 20
Liver
Kidney a
0.119/0.02 0.189/0.06a 0.229/0.04a 17.619/3.30b 0.819/0.10 c 0.169/0.03a 0.349/0.10a 0.259/0.08a 8.669/1.54b 4.009/0.84b 68.19/3.3a 117.79/7.1b 123.69/11.3b 99.99/3.3b 115.29/10.4b 449.49/35.7a 402.79/43.4a 536.49/54.5a 134.49/23.3b 492.69/49.7a
Femur a
0.479/0.07 0.449/0.22a 1.439/0.22b 93.219/20.40c 2.519/0.26d 0.559/0.08a 1.199/0.45a 1.169/0.38a 23.589/4.82b 6.599/1.32b 55.49/2.9a 81.59/4.1b 72.69/5.2a,b 73.39/6.1a,b 89.69/5.7b 394.49/19.8a 275.79/16.3b 362.39/21.4a,b 110.29/23.4c 382.89/29.3a,b
Testis a
0.229/0.04 0.829/0.11b 0.879/0.07b 172.369/14.16c 17.489/2.89d 0.029/0.01a 0.059/0.01a 0.039/0.02a 0.439/0.12b 0.109/0.03b 108.89/3.4a 141.09/7.5a,b 128.09/6.1a,b 166.39/7.6b 166.59/13.3b 150.09/11.4a 107.69/6.2a 128.89/10.9a 153.09/9.9a 142.59/14.5a
0.879/0.27a,b 0.149/0.09a 1.049/0.63a,b 6.969/2.36b 5.139/0.96b 0.579/0.15a 0.509/0.10a 0.349/0.21a 0.709/0.21a 1.939/0.60a 17.49/5.1a 97.69/20.7b 88.89/11.2b 64.39/10.3b 91.09/16.5b 329.59/62.7a 263.89/50.4a 241.29/107.4a 137.29/22.5a 617.09/160.5a
N , number of animals; Miasteczko Sl., Miasteczko Slaskie. a,b,c,d Different letters denote statistically significant differences (P B/0.05) in heavy metal concentrations in each organ between sites.
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Fig. 1. The normal structure of the liver of the yellow-necked mouse from the control area (Goldner; /250), B, bile duct; PV, hepatic portal vein. Fig. 2. The normal structure of the liver of the yellow-necked mouse from the control area (PAS; /250), Glycogen stained violet. BV, blood vessel. Fig. 3. The liver of the yellow-necked mouse from the neighbourhood of the ‘Sendzimir Steelworks’ in Krakow (PAS; /250), Glycogen stained violet. BV, blood vessel.
zawa’ steelworks demonstrated fibroses. Glycogen was not evenly distributed. Histopathological changes in tissues were found in seven of the eight rodents live trapped in the Krakow area. Liver cells appeared more round and swollen than the specimens from the control areas and vacuoles were observed in the cytoplasm. Nuclear heterochromatin was increased. Hepatocytes seemed to be neoplasticaly transformed. Glycogen in the liver was not evenly distributed (Fig. 3). Histopathological changes in the liver were also visible in seven of the nine yellow-necked mice
from the Miasteczko Slaskie area. Progressive interstitial fibrosis and fatty changes were seen. As for yellow-necked mice from the Bukowno area, histopathological changes in the liver included an increased number of pyknotic nuclei, necrotic hepatocytes and parenchymal cell, interstitial fibrosis as well as injuries to the epithelium of the blood vessels (Fig. 4). The renal cortex and the medulla of all yellownecked mice caught in the control area were normally developed (Figs. 5 and 6). Basement membranes and the brush borders of the kidney were PAS positive (Fig. 6).
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As for the kidneys of the animals trapped within the neighbourhood of the ‘Lucchini /Warszawa’ steelworks, cytosolic inclusion bodies were found in the renal tubular epithelial cells (Fig. 7). Swelling of both the glomeruli and proximal tubules in the kidney and adhesion of the Bowman’s capsule to the glomerulus were also demonstrated in seven of the eight specimens from the Krakow area. Light chains of immunoglobulin were demonstrated in the kidneys of seven of the nine animals caught within the Miasteczko Slaskie area. Nine yellow-necked mice (five females and four males) live-trapped within the neighbourhood of the ‘ZGH Boleslaw’ zinc smelter had damage to the kidneys. Hyperplasia of the tubules, atrophy of glomeruli, interstitial fibrosis and necrosis were seen (Fig. 8). The testes of animals trapped in the Borecka Forest as well as in the Warsaw, Krakow and Miasteczko Slaskie areas show no histopathological changes (Fig. 9). As for the testes of the yellow-necked mice from the neighbourhood of the zinc smelter in Bukowno, degenerating cells were present in the lumen of seminiferous tubules. The decrease in the diameter of the tubules and the increase in the intertubular space were also observed (Fig. 10).
4. Discussion In spite of proecological technical advances, metallurgical industry remains one of the main sources of heavy metal pollution in the environment. A large amount of iron, but also of lead, zinc, and cadmium is discharged during steel production. Zinc ore processing is a source of emission mainly of zinc, lead, and cadmium (Shore and Rattner, 2001). The highest lead and cadmium concentrations in analysed tissues were found in animals trapped within the neighbourhood of the zinc smelters. Lead concentrations were highest in femurs. Cadmium showed typically high levels (Hunter et al., 1982; Shore and Rattner, 2001) in kidneys. Lead and cadmium levels in samples from the Warsaw and Krakow areas were similar to values for the Borecka Forest. Only lead concentrations in the femurs of the yellow-necked mice from the Warsaw area and in kidneys, as well as in the femurs of animals from the neighbourhood of Krakow were statistically higher. Higher zinc concentrations in the tissues of the yellow-necked mice caught within the neighbourhood of industrial plants probably result from a higher cadmium accumulation, which favours zinc absorption (Friberg et al., 1986; Piasek et al., 1996; Yang et al., 2000). Moreover a reproduction state
Fig. 4. Damage to the liver of the yellow-necked mouse from the neighbourhood of the ‘ZGH Boleslaw’ zinc smelter in Bukowno (Goldner; /250). Arrows indicate fibroses stained green. BV, blood vessel. Fig. 5. The kidney of the yellow-necked mouse from the control area (Goldner; /250), G, glomerulus; PT, proximal tubule.
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Fig. 6. The kidney of the yellow-necked mouse from the control area (PAS; /400), DT, distal tubule.
Fig. 7. The kidney of the yellow-necked mouse trapped in the Warsaw area (PAS; /400), I, inclusion body; DT, distal tubule.
of an individual animal could affect zinc concentrations in the testes (Shore and Rattner, 2001). As for iron, its lower concentration in the liver and kidneys of rodents caught in the Bukowno area, compared with samples from other places can result from the inhibition of iron absorption by high doses of lead and cadmium (Friberg et al., 1986; Piasek et al., 1996; Swiergosz et al., 1998). However, zinc and iron concentrations in the tissues of the animals were at physiological levels (Shore and Rattner, 2001). Homeostatic mechanisms were able to maintain tissue zinc and iron concentration around the metabolic optimum (Venugopal and Luckey, 1978).
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Fig. 8. The kidney of the yellow-necked mouse caught in the Bukowno area (Goldner; /250). Areas of necrosis stained yellow. G, glomerulus; PT, proximal tubule.
Fig. 9. The testis of the yellow-necked mouse from the control area (Goldner; /250), ST, seminiferous tubule; BV, blood vessel.
Histological analyses showed that even relatively low concentrations of lead and cadmium caused damages to the tissues of rodents. Only the liver, kidneys and testes of yellow-necked mice from the control area showed no histopathological changes. Damages to tissues were demonstrated in the liver and kidneys of animals from the other sites and in the testes of rodents from the Bukowno area. Lead intoxication causes a vacuolisation of the cytoplasm, the increase in numbers of pyknotic nuclei and the decrease in glycogen content in hepatocytes (Bolognani Fantin et al., 1992;
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Fig. 10. The testis of the yellow-necked mouse from the Bukowno area (Goldner; /250), ST, seminiferous tubule; BV, blood vessel; D, degenerating cell.
Foulkes, 1996; Pereira et al., 2001). Nephrotoxicity of lead manifests itself in alterations in the mitochondrial structure, the formation of inclusion bodies in the tubular epithelial cells, interstitial fibrosis and both hyperplasia and gradual atrophy of tubules and glomeruli (Goyer, 1989; Nolan and Shaikh, 1992). The inclusion bodies occur at an early stage of renal dysfunction. However, the kidneys of individuals chronically exposed to high doses of lead often show fewer or no inclusion bodies (Kisseberth et al., 1984; Beck, 1991; Nolan and Shaikh, 1992). Lead effects on the male reproductive system consist of prostatic hyperplasia, decreased sperm motility and testicular weight (Friberg et al., 1986; Pinon-Lataillade et al., 1993). As for cadmium, this metal also causes damage to the liver that includes vacuolisation of cytoplasm of hepatocytes, fibrosis, and increased numbers of pyknotic nuclei, necrotic 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). However, the most typical feature of chronic cadmium exposure is damage to the kidneys. Tubular proteinuria, aminoaciduria, glucosuria and phosphaturia occur (Itokawa et al., 1978; Waalkes et al., 1992). Damage to blood vessels and the incorporation of cadmium into enzymes explains its toxicity in gonads (Swiergosz et al.,
1998). After the cadmium injection, a decrease in the diameter of the seminiferous tubules was observed (Godowicz and Kakol, 1988). Moreover the long biological half-life of cadmium may well set the stage for neoplastic transformations (Waalkes et al., 1992). Previous data reported mainly on the basis of laboratory studies showed that histopathological changes in tissues of experimental rodents chronically exposed to different dietary cadmium levels were observed when the metal concentration in the kidneys was 100 times higher and in the liver about 50 times higher than those found in specimens from the Warsaw and Krakow areas (Swiergosz et al., 1998). Damage to kidneys of small mammals from the contaminated area near a battery lead reclamation plant occurred only when lead concentrations were about ten times higher than the levels found in samples from the neighbourhood of the ‘Lucchini-Warszawa’ steelworks (Shore and Rattner, 2001). Rodents in the wild turned out to be more sensitive to heavy metals than those from the laboratory experiments. Energy requirements are greater in the field than in the laboratory because of the expense of acquiring food, defending territories, thermoregulation, and providing food for offspring (Shore and Rattner, 2001). Thus in the field presumably less energy could be devoted to detoxification. Our studies showed that the risk of damage to internal organs of wild rodents exists even at relatively low lead and cadmium concentrations in tissues. Since rodents serve as sentinels for humans in toxicological investigations of potential risk of exposure (Shore and Rattner, 2001), it is conceivable that stated heavy metal levels could cause similar histopathological changes in tissues of people living in the examined areas.
Acknowledgements The research was supported by a grant (no: 1138/P04/2000/18) from the Polish Committee for Scientific Research.
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calcium and phosphorus in environmental cadmium-exposed subjects with renal dysfunction. J. Appl. Toxicol. 13 (1), 43 /47. Kisseberth, W.C., Sundberg, J.P., Nyboer, R.W., Reynolds, J.D., Kasten, S.C., Beasley, V.R., 1984. Industrial lead contamination of an Illinois wildlife refuge and indigenous small mammals. JAVMA 185 (11), 1309 /1313. Litwin, J.A., 1995. Podstawy technik mikroskopowych. CM UJ, Krakow. Liu, J., Kershaw, W.C., Liu, Y.P., Klaassen, C.P., 1992. Cadmium-induced hepatic endothelial cell injury in inbred strains of mice. Toxicology 75, 51 /62. Merian, E. (Ed.), Metals and Their Compounds in the Environment. Occurrence, Analysis, and Biological Relevance. VCH, Weinheim 1991. Mingard, F., Diezi, J., 1992. Cadmium uptake by Brush border membrane vesicles from the rabbit renal external cortex. J. Trace Elem. Electrolytes Health Dis. 6, 111 /115. Mochel, E., 1998. Ocena zanieczyszczenia metalami ciezkimi lasow Legnicko-Glogowskiego i Olkuskiego Okregu Rudnego na podstawie koncentracji metali ciezkich w tkankach nornicy rudej (Clethrionomys glareolus , Schreber 1780). M.Sc. thesis, ZEZ, Jagiellonian University. Motas-Guzman, M., Garcia-Fernandez, A.J., Gomez-Zapata, M., Luna, A., Romero, D., Sanchez-Garcia, J.A., 1996. Effects of dietary chronic cadmium and lead exposure: structural and ultrastructural changes. Toxicol. Lett. 88 (S1), 60. Nabaglo, L., Pachinger, K., 1979. Eye lens as an age indicator in yellow-necked mice. Acta Theriol. 24 (11), 118 /122. Nolan, C.V., Shaikh, Z.A., 1992. Lead nephrotoxicity and associated disorders: biochemical mechanisms. Toxicology 73, 127 /146. O’Brien, D.J., Kaneene, J.B., Poppenga, R.H., 1993. The use of mammals as sentinels for human exposure to toxic contaminants in the environment. Environ. Health Perspect. 99, 351 /368. Ogoshi, K., Yukuo, N., Moriyama, T., 1992. Decrease in bone strength of cadmium-treated young and old rats. Arch. Toxicol. 66, 315 /320. Pereira, R., Pereira, M.L., Ribeiro, R., Goncalves, F., 2001. Wildlife animals as sentinels of threats to human health due to environmental exposure to heavy metals. In: Abstract book. 11th A. Meet. SETAC Eur. 6 /10 May, Soc. Environ. Toxicol. Chem., Madrid, p. 108. Piasek, M., Schonwald, N., Blanusa, M., Kostial, K., 1996. Effects of prenatal and postnatal exposure to cadmium on element concentrations in rats. Toxicol. Lett. 88 (S1), 57 / 58. Pinon-Lataillade, G., Thoreux-Manlay, A., Coffigny, H., Monchaux, G., Masse, R., Soufir, J.-C., 1993. Effect of ingestion and inhalation of lead on the reproductive system and fertility of adult male rats and their progeny. Hum. Exp.Toxicol. 12, 165 /172. Pucek, Z., 1984. Klucz do oznaczania ssakow Polski. PWN, Warsaw.
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Roels, H., Bernard, A.M., Cardenas, A., Buchet, J.P., Lauwerys, R.R., Hotter, G., Ramis, I., Mutti, A., Franchini, I., Bundschuh, I., Stolte, H., De Broe, M.E., Nuyts, G.D., Taylor, S.A., Price, R.G., 1993. Markers of early changes induced by industrial pollutants. III Application to workers exposed to cadmium. Br. J. Ind. Med. 50, 37 /48. Romeis, B., 1948. Mikroskopische Technik. Leibniz Verlag, Munchen, 348. Sachs, L., 1984. Applied Statistics. A Handbook of Techniques, second ed.. Springer, New York. Sawicka-Kapusta, K., 1978. Ocena zawartosci metali ciezkich w porozach saren lasow slaskich. Arch. Ochr. Srod. 1, 107 / 121. Sawicka-Kapusta, K., Zakrzewska, M., Kowalska, A., Lenda, B., Skrobacz, M., 1995. Heavy metal concentrations in small mammals from Borecka Forest. Arch. Ochr. Srod. 34, 229 /234. Sawicka-Kapusta, K., Zakrzewska, M., Orzechowski, T., 1999. Seasonal changes of metal concentration in small mammals’ populations from different contaminated areas. In: Ketrup, A., Schramm, K.-W. (Eds), Proc. SECOTOX 99. Fifth Eur. Conf. Ecotoxicol. Environ. Safety, March 15 /17, Munich. Shore, R.F., Rattner, B.A. (Eds.), Ecotoxicology of Wild Mammals. Wiley, Chichester, New York, Weinheim 2001. Situa, J. (Ed.), Monitoring Srodowiska. Stacja kompleksowego monitoringu srodowiska Puszcza Borecka. Inst. Ochr. Srod, Warsaw 1994.
Sokal, R.R., Rohlf, F.J., 1981. Biometry, second ed. Freeman, San Francisco. Stevens, A., Lowe, J., 2000. In: Zabel, M. (Ed.), Histologia. PZWL, Warsaw. Swiergosz, R., Zakrzewska, M., Sawicka-Kapusta, K., Bacia, K., Janowska, I., 1998. Accumulation of cadmium in and its effect on bank vole tissues after chronic exposure. Ecotoxicol. Environ. Safety. 41, 130 /136. Venugopal, B., Luckey, T.D., 1978. Metal Toxicity In Mammals. Plenum Press, New York, pp. 185 /195. Waalkes, M.P., Coogan, T.P., Barter, R.A., 1992. Toxicological principles of metal carcinogenesis with special emphasis on cadmium. Critical Rev. Toxicol. 22, 175 /201. Waalkes, M.P., Perantoni, A., 1988. In vitro assessment of target cell specificity in cadmium carcinogenesis: interactions of cadmium and zinc with isolated interstitial cells of the rat testes. In Vitro Cell. Dev. Biol. 24 (6), 558 /565. Wesenberg, G.B.R., Fosse, G., Justessen, N.-P.B., Rasmussen, P., 1979. Lead and cadmium in teeth, bone and kidneys of rats with a standard Pb /Cd supply. Inter. J. Environ. Stud. 14, 223 /230. Yang, M.S., Lai, K.P., Cheng, K.Y., Wong, C.K.C., 2000. Changes in endogenous Zn and Cu distribution in different cytosolic protein fractions in mouse liver after administration of single sublethal dose of CdCl2. Toxicology 154 (1-3), 103 /111.
Damage to the liver, kidney, and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland Monika Damek-Poprawa a,*, Katarzyna Sawicka-Kapusta b a
Department of Raw Materials and Processing of Fruit and Vegetables, Faculty of Food Technology, Agriculture University, ul. Podluzna 3, 30-239 Krakow, Poland b Department of Environmental Monitoring, Institute of Environmental Sciences, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland Received 15 April 2002; received in revised form 21 October 2002; accepted 21 October 2002
Abstract The influence of the steelworks in Warsaw and Krakow as well as the zinc smelters in Bukowno and Miasteczko Slaskie on lead, cadmium, zinc and iron concentrations and the structure of selected tissues of yellow-necked mice were analysed. The Borecka Forest was chosen as a control area. The highest concentrations of lead, 172.36 g/g dry weight, and cadmium, 23.58 g/g, were detected in the femurs and kidneys, respectively, of rodents caught in Bukowno. Zinc and iron concentrations ranged over physiological values. No histopathological changes were observed in analysed tissues of all rodents in the control area. Damage occurred in the liver and kidneys of animals from all other sites and in the testes of rodents from Bukowno. Decreased glycogen content, interstitial fibrosis, and increased number of pyknotic nuclei as well as necrosis were seen in hepatocytes. In the kidneys hyperplasia of the tubules, atrophy of glomeruli, interstitial fibrosis and necrosis were observed. Degenerate cells were present in the lumen of seminiferous tubules of animals from the Bukowno area. Even relatively low concentrations of lead and cadmium, like those found in the liver and kidneys of rodents from the neighbourhood of the steelworks, caused histopathological changes. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Yellow-necked mice; Heavy metals; Histopathology
1. Introduction The emission of pollutants to the environment has been considerably reduced during the last
* Corresponding author E-mail address: [email protected] Poprawa).
(M.
Damek-
decade, although the neighbourhood of industrial plants still remains heavily contaminated (Mochel, 1998; Sawicka-Kapusta et al., 1999; Appleton et al., 2000; Grzesiak and Sieradzki, 2000). Human activity leads to a large dispersion of heavy metals, which, not submitted to biodegradation, circulate in trophic chains and accumulate in living organisms (Merian, 1991). Some metals like zinc and iron are physiologically essential, but they may
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 5 9 5 - 4
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also alter the function of organisms when the exposure dose exceeds a critical threshold, which is species-specific, and can depend on age, sex, reproductive state and physiological condition of animals (Shore and Rattner, 2001). As for lead and cadmium, they do not play any functional role in the organisms. Lead accumulates mainly in bone but the critical organs for lead intoxication are bone marrow, the nervous system and kidneys (Friberg et al., 1986). Chronic exposure to lead causes renal dysfunction (Venugopal and Luckey, 1978; Nolan and Shaikh, 1992), liver damage (Bolognani Fantin et al., 1992), and decreased fertility in males and increased spontaneous abortion in females (Friberg et al., 1986). Cadmium causes damage primarily to kidney, bone and lungs (Domino, 1994). It accumulates mainly in kidney and affects the re-absorption functions of the proximal tubules (Mingard and Diezi, 1992; Waalkes et al., 1992; Roels et al., 1993). Cadmium also alters the calcium metabolism, which leads to osteomalacia (Ogoshi et al., 1992; Kido et al., 1993). Cadmium can damage the liver and testes (Amdur et al., 1991; Liu et al., 1992; Stevens and Lowe, 2000). This metal is also teratogenic and carcinogenic. After cadmium intoxication cancer of the testes and prostate occurs (Venugopal and Luckey, 1978; Friberg et al., 1986; Waalkes and Perantoni, 1988). In the organism, lead and cadmium can also alter the level of other metals like zinc and iron. When cadmium is present in the diet the absorption of iron diminishes. However, cadmium enhances zinc absorption from the gastrointestinal tract (Friberg et al., 1986; Kabata-Pendias and Pendias, 1999). Rodents are sensitive and accurate monitors of exposure to environmental pollution by heavy metals. The yellow-necked mouse Apodemus flavicollis (Melchior, 1834) belongs to the most numerous rodent species in Polish deciduous forests. The animals are easily caught, have a small migration area and a relatively short life span (Pucek, 1984). Moreover, the pattern of heavy metal distribution and levels of heavy metals in their tissues are similar to those found in humans (Wesenberg et al., 1979). In research, rodents frequently serve as mammalian surrogate
for humans (Shore and Rattner, 2001). This makes rodents ideal for monitoring environmental pollution as well as for evaluating the exposure risk for people living in a contaminated area (O’Brien et al., 1993). Previous studies on histopathological changes in tissues were carried out mostly in a laboratory on rodents fed on a diet containing very high amounts of lead and/or cadmium. Damages to the structure of the internal organs occurred only through substantial concentrations of these metals in the tissues of rodents (Swiergosz et al., 1998; Shore and Rattner, 2001). Analyses of the tissues of rodents trapped in the wild are scarce (Hunter et al., 1982). The aim of this study was to assess the impact of industrial pollution on yellow-necked mice. Concentrations of lead, cadmium, zinc and iron in the liver, kidneys, testes and femurs of rodents were determined. Moreover histological and histochemical analyses were done in order to determine heavy metal influence on tissues of yellow-necked mice living in the wild.
2. Material and methods Yellow-necked mice A. flavicollis (Melchior, 1834) were trapped in the area of two steelworks: the ‘Lucchini /Warszawa’ in Warsaw and the ‘Sendzimir Steelworks’ in Krakow as well as within the neighbourhood of two zinc smelters in the Upper Silesia region: ‘ZGH Boleslaw’ in Bukowno and ‘Miasteczko Slaskie’ in Miasteczko Slaskie. Borecka Forest in the north of Poland was chosen as a control area. In the year 2000 the steelworks ‘Lucchini /Warszawa’ emitted 583 kg of lead, 10 kg of cadmium, 3684 kg of zinc, and 6349 kg of iron and copper together. That year the ‘Sendzimir Steelworks’ in Krakow discharged 625 kg of Pb, 23 kg of Cd, and 263 kg of Cu. As for the zinc smelters, 2.2 kg of lead, 0.2 kg of cadmium, and 753 kg of zinc was emitted from the ‘ZGH Boleslaw’ in Bukowno. In 2000, the zinc smelter ‘Miasteczko Slaskie’ discharged 5214 kg of lead, 44 kg of cadmium as well as 7850 kg of zinc (all data on emission were provided by the mentioned steelworks and zinc smelters). The data, available
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from the year 1999, showed that dust fall within the neighbourhood of the steelworks in Warsaw amounted to 70.7 g/m2 (including 24.2 mg/m2 of lead, and 0.627 mg/m2 of cadmium) (Grzesiak and Sieradzki, 2000). That year dust fall in the Krakow area amounted to 100 g/m2 (including 100 mg/m2 of lead, and 2 mg/m2 of cadmium) (Grzesiak and Sieradzki, 2000). In the year 1999, 60 g/m2 of dust (including approximately 100 mg/ m2 of lead, and 34 mg/m2 of cadmium) fell down within the neighbourhood of the ‘Boleslaw’ zinc smelter (Grzesiak and Sieradzki, 2000). As for Miasteczko Slaskie, the average dust fall in 1999 reached 36 g/m2 (90 mg/m2 of lead, and 1.51 mg/ m2 of cadmium in it) (Grzesiak and Sieradzki, 2000). The Borecka Forest is one of the lowest polluted areas in Poland (Siuta, 1994; SawickaKapusta et al., 1995). In the year 2000 the emission of lead, cadmium, and zinc from local sources within the control area was negligible (Grzesiak and Sieradzki, 2001). The trapping was carried out from 24 September to 27 October 2000. In accordance with a modified Standard-Minimum method (Grodzinski et al., 1966; Bejcek, 1988) 242 traps (including 100 livetraps) were placed in each area on the 1 ha plot in a 10/10 m grid. Within the neighbourhood of the steelworks ‘Lucchini /Warszawa’ 18 yellow-necked mice (including 13 alive) were trapped. In the area of the ‘Sendzimir Steelworks’ 22 animals (including eight alive) were caught. As for the zinc smelters, within the neighbourhood of ‘ZGH Boleslaw’ and ‘Miasteczko Slaskie’ 12 (including seven alive) and 20 (nine alive) yellow-necked mice were trapped, respectively. 15 animals (nine alive) were caught in the control area. The rodents caught by live-traps were killed by ether and immediately dissected. A part of the liver, one kidney and one testis were used for slide preparation. The tissues were fixed with buffered formalin, dehydrated with alcohol and xylene and embedded in paraffin wax. Then they were cut into 7 m slices. Next, slides were stained with haematoxylin and eosin as well as by the Goldner method, which was used to demonstrate supporting tissue elements (Litwin, 1995; Romeis, 1948). The PAS (periodic acid */Schiff)
3
histochemical reaction (Baucroft, 1967) was also performed. Glycogen, the intracellular storage form of carbohydrates, is PAS positive. Afterwards the slides were examined under a light microscope. The femurs, the rest of the liver, the other kidney and the other testis of live trapped rodents as well as the same tissues dissected from dead trapped animals were analysed for heavy metals. The samples were dried at 60 8C to a constant weight. Afterwards they were wet digested in a mixture of nitric and perchloric acid (4:1) (spectral grade). After evaporating the samples, double distilled water was added until the solution measured 10 ml (Sawicka-Kapusta, 1978). Lead and cadmium concentrations were determined in a Perkin /Elmer AAnalyst 800 graphite furnace atomic absorption spectrophotometer, while zinc and iron levels in a flame using an AAS IL-250. Yellow-necked mice were aged on the basis of eye lens weight according to the method proposed by Nabaglo and Pachinger (1979). The association between the heavy metal concentrations in tissues and the age of animal was checked by a regression analysis. The significance of differences in the heavy metal concentrations in the liver, kidneys, testes and femurs of animals from each site was determined by a non-parametric Kruskal-Wallis test and an a posteriori test by Sachs (Sokal and Rohlf, 1981; Sachs, 1984).
3. Results 3.1. Age of yellow-necked mice The age of the yellow-necked mice caught in the control area and within the neighbourhood of the ‘Lucchini /Warszawa’ steelworks averaged 4 months. Animals from the Krakow area were 3 months of age on average. As for the A. flavicollis from Bukowno, they were 2 months of age on average. Rodents trapped in the neighbourhood of the zinc smelter ‘Miasteczko Slaskie’ were the youngest */at the age of about 1 month.
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3.2. Heavy metal concentrations No association between the age of yellownecked mice and heavy metal concentrations in the tissues of animals was found. Average lead, cadmium, zinc and iron levels in selected tissues are given in the Table 1. The highest lead concentrations, 172.36 and 17.48 g/g, respectively, were detected in femurs of rodents caught in the area of the zinc smelters in Bukowno and in Miasteczko Slaskie. No statistically significant differences between lead levels in the liver of rodents from the control area and from the neighbourhood of the steelworks were found. However, the concentrations of those metals were slightly higher in tissues of yellow-necked mice from Warsaw and Krakow compared with the values in samples from the control area. The highest cadmium levels were found in the kidneys of the yellow-necked mice caught within the neighbourhood of the zinc smelters in Bukowno and Miasteczko Slaskie. There were 23.58 and 6.59 g Cd/g, respectively.
Zinc and iron concentrations in analysed samples ranged among the physiological values. The lowest amounts of zinc were found in samples from the Borecka Forest. The testes of the yellownecked mice from the control area contained on average only 17.4 g Zn/g. As for iron, the statistically significant lowest concentrations were found in liver and kidneys of animals trapped near the ‘ZGH Boleslaw’ zinc smelter. The samples contained on average 134.4 and 110.2 g Fe/g, respectively. 3.3. Histopathology No damage to analysed tissues was observed in the liver, kidneys and testes of all rodents livetrapped in the control area. The normal structure of the liver of the yellownecked mouse is shown in Figs. 1 and 2. Hepatocytes, the bile duct and the portal vein were clearly visible. Glycogen was evenly spread (Fig. 2). The liver of the yellow-necked mice caught within the neighbourhood of the ‘Lucchini /Wars-
Table 1 Average lead, cadmium, zinc and iron concentrations 9/SE (mg/g dry weight) in the selected tissues of the yellow-necked mice A. flavicollis Element Pb
Cd
Zn
Fe
Area Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl. Borecka Forest Warsaw Krakow Bukowno Miasteczko Sl.
N 15 18 22 12 20 15 18 22 12 20 15 18 22 12 20 15 18 22 12 20
Liver
Kidney a
0.119/0.02 0.189/0.06a 0.229/0.04a 17.619/3.30b 0.819/0.10 c 0.169/0.03a 0.349/0.10a 0.259/0.08a 8.669/1.54b 4.009/0.84b 68.19/3.3a 117.79/7.1b 123.69/11.3b 99.99/3.3b 115.29/10.4b 449.49/35.7a 402.79/43.4a 536.49/54.5a 134.49/23.3b 492.69/49.7a
Femur a
0.479/0.07 0.449/0.22a 1.439/0.22b 93.219/20.40c 2.519/0.26d 0.559/0.08a 1.199/0.45a 1.169/0.38a 23.589/4.82b 6.599/1.32b 55.49/2.9a 81.59/4.1b 72.69/5.2a,b 73.39/6.1a,b 89.69/5.7b 394.49/19.8a 275.79/16.3b 362.39/21.4a,b 110.29/23.4c 382.89/29.3a,b
Testis a
0.229/0.04 0.829/0.11b 0.879/0.07b 172.369/14.16c 17.489/2.89d 0.029/0.01a 0.059/0.01a 0.039/0.02a 0.439/0.12b 0.109/0.03b 108.89/3.4a 141.09/7.5a,b 128.09/6.1a,b 166.39/7.6b 166.59/13.3b 150.09/11.4a 107.69/6.2a 128.89/10.9a 153.09/9.9a 142.59/14.5a
0.879/0.27a,b 0.149/0.09a 1.049/0.63a,b 6.969/2.36b 5.139/0.96b 0.579/0.15a 0.509/0.10a 0.349/0.21a 0.709/0.21a 1.939/0.60a 17.49/5.1a 97.69/20.7b 88.89/11.2b 64.39/10.3b 91.09/16.5b 329.59/62.7a 263.89/50.4a 241.29/107.4a 137.29/22.5a 617.09/160.5a
N , number of animals; Miasteczko Sl., Miasteczko Slaskie. a,b,c,d Different letters denote statistically significant differences (P B/0.05) in heavy metal concentrations in each organ between sites.
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Fig. 1. The normal structure of the liver of the yellow-necked mouse from the control area (Goldner; /250), B, bile duct; PV, hepatic portal vein. Fig. 2. The normal structure of the liver of the yellow-necked mouse from the control area (PAS; /250), Glycogen stained violet. BV, blood vessel. Fig. 3. The liver of the yellow-necked mouse from the neighbourhood of the ‘Sendzimir Steelworks’ in Krakow (PAS; /250), Glycogen stained violet. BV, blood vessel.
zawa’ steelworks demonstrated fibroses. Glycogen was not evenly distributed. Histopathological changes in tissues were found in seven of the eight rodents live trapped in the Krakow area. Liver cells appeared more round and swollen than the specimens from the control areas and vacuoles were observed in the cytoplasm. Nuclear heterochromatin was increased. Hepatocytes seemed to be neoplasticaly transformed. Glycogen in the liver was not evenly distributed (Fig. 3). Histopathological changes in the liver were also visible in seven of the nine yellow-necked mice
from the Miasteczko Slaskie area. Progressive interstitial fibrosis and fatty changes were seen. As for yellow-necked mice from the Bukowno area, histopathological changes in the liver included an increased number of pyknotic nuclei, necrotic hepatocytes and parenchymal cell, interstitial fibrosis as well as injuries to the epithelium of the blood vessels (Fig. 4). The renal cortex and the medulla of all yellownecked mice caught in the control area were normally developed (Figs. 5 and 6). Basement membranes and the brush borders of the kidney were PAS positive (Fig. 6).
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As for the kidneys of the animals trapped within the neighbourhood of the ‘Lucchini /Warszawa’ steelworks, cytosolic inclusion bodies were found in the renal tubular epithelial cells (Fig. 7). Swelling of both the glomeruli and proximal tubules in the kidney and adhesion of the Bowman’s capsule to the glomerulus were also demonstrated in seven of the eight specimens from the Krakow area. Light chains of immunoglobulin were demonstrated in the kidneys of seven of the nine animals caught within the Miasteczko Slaskie area. Nine yellow-necked mice (five females and four males) live-trapped within the neighbourhood of the ‘ZGH Boleslaw’ zinc smelter had damage to the kidneys. Hyperplasia of the tubules, atrophy of glomeruli, interstitial fibrosis and necrosis were seen (Fig. 8). The testes of animals trapped in the Borecka Forest as well as in the Warsaw, Krakow and Miasteczko Slaskie areas show no histopathological changes (Fig. 9). As for the testes of the yellow-necked mice from the neighbourhood of the zinc smelter in Bukowno, degenerating cells were present in the lumen of seminiferous tubules. The decrease in the diameter of the tubules and the increase in the intertubular space were also observed (Fig. 10).
4. Discussion In spite of proecological technical advances, metallurgical industry remains one of the main sources of heavy metal pollution in the environment. A large amount of iron, but also of lead, zinc, and cadmium is discharged during steel production. Zinc ore processing is a source of emission mainly of zinc, lead, and cadmium (Shore and Rattner, 2001). The highest lead and cadmium concentrations in analysed tissues were found in animals trapped within the neighbourhood of the zinc smelters. Lead concentrations were highest in femurs. Cadmium showed typically high levels (Hunter et al., 1982; Shore and Rattner, 2001) in kidneys. Lead and cadmium levels in samples from the Warsaw and Krakow areas were similar to values for the Borecka Forest. Only lead concentrations in the femurs of the yellow-necked mice from the Warsaw area and in kidneys, as well as in the femurs of animals from the neighbourhood of Krakow were statistically higher. Higher zinc concentrations in the tissues of the yellow-necked mice caught within the neighbourhood of industrial plants probably result from a higher cadmium accumulation, which favours zinc absorption (Friberg et al., 1986; Piasek et al., 1996; Yang et al., 2000). Moreover a reproduction state
Fig. 4. Damage to the liver of the yellow-necked mouse from the neighbourhood of the ‘ZGH Boleslaw’ zinc smelter in Bukowno (Goldner; /250). Arrows indicate fibroses stained green. BV, blood vessel. Fig. 5. The kidney of the yellow-necked mouse from the control area (Goldner; /250), G, glomerulus; PT, proximal tubule.
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Fig. 6. The kidney of the yellow-necked mouse from the control area (PAS; /400), DT, distal tubule.
Fig. 7. The kidney of the yellow-necked mouse trapped in the Warsaw area (PAS; /400), I, inclusion body; DT, distal tubule.
of an individual animal could affect zinc concentrations in the testes (Shore and Rattner, 2001). As for iron, its lower concentration in the liver and kidneys of rodents caught in the Bukowno area, compared with samples from other places can result from the inhibition of iron absorption by high doses of lead and cadmium (Friberg et al., 1986; Piasek et al., 1996; Swiergosz et al., 1998). However, zinc and iron concentrations in the tissues of the animals were at physiological levels (Shore and Rattner, 2001). Homeostatic mechanisms were able to maintain tissue zinc and iron concentration around the metabolic optimum (Venugopal and Luckey, 1978).
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Fig. 8. The kidney of the yellow-necked mouse caught in the Bukowno area (Goldner; /250). Areas of necrosis stained yellow. G, glomerulus; PT, proximal tubule.
Fig. 9. The testis of the yellow-necked mouse from the control area (Goldner; /250), ST, seminiferous tubule; BV, blood vessel.
Histological analyses showed that even relatively low concentrations of lead and cadmium caused damages to the tissues of rodents. Only the liver, kidneys and testes of yellow-necked mice from the control area showed no histopathological changes. Damages to tissues were demonstrated in the liver and kidneys of animals from the other sites and in the testes of rodents from the Bukowno area. Lead intoxication causes a vacuolisation of the cytoplasm, the increase in numbers of pyknotic nuclei and the decrease in glycogen content in hepatocytes (Bolognani Fantin et al., 1992;
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Fig. 10. The testis of the yellow-necked mouse from the Bukowno area (Goldner; /250), ST, seminiferous tubule; BV, blood vessel; D, degenerating cell.
Foulkes, 1996; Pereira et al., 2001). Nephrotoxicity of lead manifests itself in alterations in the mitochondrial structure, the formation of inclusion bodies in the tubular epithelial cells, interstitial fibrosis and both hyperplasia and gradual atrophy of tubules and glomeruli (Goyer, 1989; Nolan and Shaikh, 1992). The inclusion bodies occur at an early stage of renal dysfunction. However, the kidneys of individuals chronically exposed to high doses of lead often show fewer or no inclusion bodies (Kisseberth et al., 1984; Beck, 1991; Nolan and Shaikh, 1992). Lead effects on the male reproductive system consist of prostatic hyperplasia, decreased sperm motility and testicular weight (Friberg et al., 1986; Pinon-Lataillade et al., 1993). As for cadmium, this metal also causes damage to the liver that includes vacuolisation of cytoplasm of hepatocytes, fibrosis, and increased numbers of pyknotic nuclei, necrotic 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). However, the most typical feature of chronic cadmium exposure is damage to the kidneys. Tubular proteinuria, aminoaciduria, glucosuria and phosphaturia occur (Itokawa et al., 1978; Waalkes et al., 1992). Damage to blood vessels and the incorporation of cadmium into enzymes explains its toxicity in gonads (Swiergosz et al.,
1998). After the cadmium injection, a decrease in the diameter of the seminiferous tubules was observed (Godowicz and Kakol, 1988). Moreover the long biological half-life of cadmium may well set the stage for neoplastic transformations (Waalkes et al., 1992). Previous data reported mainly on the basis of laboratory studies showed that histopathological changes in tissues of experimental rodents chronically exposed to different dietary cadmium levels were observed when the metal concentration in the kidneys was 100 times higher and in the liver about 50 times higher than those found in specimens from the Warsaw and Krakow areas (Swiergosz et al., 1998). Damage to kidneys of small mammals from the contaminated area near a battery lead reclamation plant occurred only when lead concentrations were about ten times higher than the levels found in samples from the neighbourhood of the ‘Lucchini-Warszawa’ steelworks (Shore and Rattner, 2001). Rodents in the wild turned out to be more sensitive to heavy metals than those from the laboratory experiments. Energy requirements are greater in the field than in the laboratory because of the expense of acquiring food, defending territories, thermoregulation, and providing food for offspring (Shore and Rattner, 2001). Thus in the field presumably less energy could be devoted to detoxification. Our studies showed that the risk of damage to internal organs of wild rodents exists even at relatively low lead and cadmium concentrations in tissues. Since rodents serve as sentinels for humans in toxicological investigations of potential risk of exposure (Shore and Rattner, 2001), it is conceivable that stated heavy metal levels could cause similar histopathological changes in tissues of people living in the examined areas.
Acknowledgements The research was supported by a grant (no: 1138/P04/2000/18) from the Polish Committee for Scientific Research.
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