Department of Toxicology, Medical Academy, Mickiewicza 2c str., 15-222 Bialystok and
1 Department of Histology and Embryology, Medical Academy, Kiliskiego 1 str., 15-230 Bialystok, Poland
Received 21 December 2001; in revised form 9 May 2002; accepted 5 July 2002
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ABSTRACT |
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INTRODUCTION |
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Some publications provide data on CdEtOH interactions (Sharma et al., 1991, 1992
; Brus et al., 1995
) but many aspects are still not fully recognized. According to our earlier results short- and long-term EtOH administration affects Cd turnover in rats, and also modifies changes in the metabolism of some essential elements by this heavy metal (Moniuszko-Jakoniuk et al., 1999
, 2001
; Brzóska et al., 2000
, 2002
).
Liver and kidney are important organs of metabolism, detoxification, storage and excretion of xenobiotics and their metabolites, and are especially vulnerable to damage. As the liver is an important target organ of EtOH (Bunout, 1999; Thurman et al., 1999
), and the kidney of Cd toxicity (Kjellström, 1986
; World Health Organization, 1992
; Nordberg et al., 1994
) we have also assessed liver and kidney function and histology.
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MATERIALS AND METHODS |
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Chemicals
All reagents and chemicals were of analytical grade or higher purity. Trace-free nitric acid (Merck, Dormstadt, Germany) and Cd standard solution assigned for atomic absorption spectrometry (Sigma, St Louis, MO, USA) were used for Cd analysis.
Experimental design
The experiment was conducted for 12 weeks. The animals were randomly allocated to four experimental groups of 10 rats each: (1) a control group, which received redistilled water; (2) an EtOH group, which received 10% (w/v) EtOH (POLMOS, Poland); (3) a Cd group, which was exposed to CdCl2 (POCh, Gliwice, Poland) at a concentration of 50 mg Cd/l; (4) a Cd + EtOH group, which received a redistilled water containing 50 mg Cd/l and 10% EtOH. Fluid consumption was measured daily during the whole experiment.
After 12 weeks of treatment all rats were placed separately in glass metabolic cages for 24-h urine collection. After overnight starvation, blood was taken by cardiac puncture, the liver and kidney were removed under ether anaesthesia, washed thoroughly in ice-cold physiological saline [0.9% (w/v) NaCl], and weighed. Whole blood was centrifuged after clotting, and the serum was separated and stored frozen until further analysis.
The study was approved by the Local Ethics Committee for animal experiments in Bialystok, Poland.
Analytical procedures
Cd and EtOH concentrations. Cd concentration in the blood, liver and kidney was determined by atomic absorption spectrometry (Zeiss Jena AAS 30) as described (Brzóska et al., 2000, 2002
). Blood-EtOH concentration was analysed by head-space gas chromatography (Hewlett-Packard, model 5890, series II) according to the manufacturers recommendations.
Alanine aminotransferase (ALAT) and asparate aminotransferase (AspAT) activities in serum. The activities of ALAT (EC 2.6.1.2.) and AspAT (EC 2.6.1.1.) were determined colorimetrically (SEMCO S/E-uv spectrometer) according to standard procedures using commercially available diagnostic laboratory tests (Lachema, Brno, Czech Republic).
Biochemical indicators of renal function. Total protein in serum and urine was determined according to Lowry et al. (1951). Concentrations of creatinine and urea in serum and urine, as well as urinary alkaline phosphatase (ALP, EC 3.1.3.1) activity, were assessed spectrophotometrically (SEMCO S/E-uv spectrometer) using diagnostic laboratory tests (POCh). Creatinine clearance was calculated.
Histopathological studies. Slices of the left liver lobe and left kidney (from seven animals of each group) were fixed in 10% formalin for 24 h, and were embedded in paraffin; 56 µm sections were routinely stained with haematoxylin and eosin (H&E) and assessed in a light microscope (Nikon Eclipse E400). All alterations from the normal structure were registered. The following criteria were used for scoring liver and kidney histology: ++++, a change was very often found in all animals of a group; +++, a change was relatively common in all animals of a group; ++, a change was rare in all animals of a group; +, a change was found in a few animals of a group; ±, a change was sporadic in a group.
Statistical analysis
Statistical analysis of results was performed using the MannWhitney non-parametric U-test. The level of significance was P < 0.05. In order to discern the possible interactions between Cd and EtOH, two-way analysis of variance (ANOVA/ MANOVA) was used. F values having P < 0.05 were considered significant. A linear Pearson correlation was performed for testing relationships between certain parameters. All statistical calculations were done with the STATISTICA 5.0 computer program.
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RESULTS |
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Blood-EtOH concentration
The concentration of EtOH in the blood of rats which were not treated with EtOH (the control and Cd groups) was within the low physiological range (Fig. 5). In the animals drinking 10% EtOH alone, its concentration was significantly higher (P < 0.001), but the joint presence of Cd suppressed this increase (Fig. 5
).
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Biochemical indicators of renal function
Both Cd and EtOH exposure affected some biochemical markers of kidney function. As shown in Table 2, the intensity of these changes were dependent on whether Cd and EtOH were administered separately or in combination. The creatinine clearance was unaffected by Cd or EtOH alone, but their co-administration decreased it by 29% (P < 0.05) versus control and by 25% (P < 0.05) versus the Cd-treated group. The total protein concentration in urine was not influenced by either treatment alone. However, urinary protein excretion in the co-exposed rats was higher (P < 0.05) than in those receiving EtOH or Cd separately (by 11 and 14%, respectively). An increase in serum urea (by 23%, P < 0.01), and a decrease in serum total protein (by 27%, P < 0.05) accompanied by a decrease in urinary urea (by 32%, P < 0.001), and an increase in urinary ALP activity (2.6-fold, P < 0.001) were observed followed EtOH administration. Exposure to Cd alone decreased the urinary urea level (by 36%, P < 0.001), increased the urinary ALP activity (4.5-fold, P < 0.001) and the serum urea concentration (by 16%, P < 0.001), but had no effect on the total protein concentration in serum and urine. In co-exposed animals, serum protein concentration was unchanged, whereas serum urea was increased (by 24%, P < 0.05) vs controls. Furthermore, urinary excretion of urea was markedly reduced (2.1-fold, P < 0.001) while ALP activity was increased (2.4-fold, P < 0.001). In this group, the changes in urinary urea were more, while those in ALP were less pronounced than in the Cd-exposed group.
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Liver and kidney histopathology
The liver of control rats showed a normal structure (Fig. 7), which was influenced by the administration of Cd and/or EtOH (Table 3
and Figs 810
). Following exposure to EtOH alone (Fig. 8
), the trabecular structure of the lobules was slightly or distinctly blurred. The cytoplasm of hepatocytes of zone 2 and 3, contained empty vacuole-like spaces, and were enlarged. Some sinusoids were overfilled with erythrocytes and the walls of most sinusoids showed numerous Kupffer cells. Locally, mononuclear cell infiltrates were observed, most frequently in the hepatocytes of zone 1. In a few animals of this group, an increased density of nuclear chromatin and a very compact nuclear structure were noted (zones 2 and 3). Sporadically, single necrotic cells were evident in zone 1. After exposure to Cd alone (Fig. 9
), the trabecular liver structure was more seriously affected than after EtOH administration (Fig. 8
). Cd-induced degenerative changes were evident in numerous hepatocytes of zones 2 and 3; the cells were enlarged and had light and foamy cytoplasm filled with vacuoles. The walls of the sinusoids in both zones showed numerous Kupffer cells. In a few zone 1 hepatocytes, necrotic changes were evident; a small, pycnotic cellular nucleus with condensed chromatin, lack of nucleolus and strongly acidophilic cytoplasm were observed. Mononuclear cell infiltrates were also noted in zone 1 hepatocytes. In rats co-exposed to Cd and EtOH (Fig. 10
), the trabecular structure of the lobules was blurred. The cytoplasm of some hepatocytes was light, enlarged and contained vacuoles (less numerous than after Cd alone). Numerous Kupffer cells were found in the sinusoid walls. These changes were observed mainly in the hepatocytes of zone 3. Mononuclear cell infiltrates were evident in zone 1. Moreover, increased density of nuclear chromatin and a very compact nuclear structure (zones 2 and 3) were rarely noted in all rats of this group. In a few animals, necrosis of single cells was evident (zone 1).
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DISCUSSION |
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We evaluated liver function by measuring plasma ALAT and AspAT activities. As parameters of kidney function, creatinine, total protein and urea concentrations in serum and urine as well as urinary ALP activity, were determined, and creatinine clearance was also calculated. The structure of both organs was assessed on the basis of histopathological analyses.
The level of Cd treatment used in this study corresponds to human (especially smokers) occupational exposure to this heavy metal, or environmental exposure in heavily contaminated areas (World Health Organization, 1992). The level of intoxication with EtOH may be tantamount to its misuse in man (Wis'niewska-Knypl and Wro
ska-Nofer, 1994).
Since the relative liver and kidney weights did not change in the co-exposed rats, the decrease in their weights reflects a retardation in body weight gain, which is a consequence of reduced food (Brzóska et al., 2002) and water intake, and of CdEtOH interaction. Other authors also reported the unfavourable effect of co-exposure to Cd and EtOH on body weight gain (Tandon and Tewari, 1987
; Gupta and Gill, 2000
).
As animals receiving Cd and EtOH simultaneously develop a stronger aversion to drinking than those intoxicated separately, so they ingest less Cd and EtOH. The difference of intake is noteworthy and has to be taken into account in interpretation of the present results.
Cd accumulation in the liver and kidney of rats exposed to this metal alone as well as in combination with EtOH resulted in serious changes in the histology and function of these two organs. Similar or more advanced changes in liver and kidney histology and function under Cd influence, have been reported by others (Aughey et al., 1984; Kjellström, 1986
; Mitsumori et al., 1998
). Aughey et al. (1984)
noted early pathological changes in rat kidney already after 6 weeks of administration of 50 mg Cd/l in drinking water. After 12 weeks, they revealed signs of tubular necrosis, interstitial fibrosis and glomerular epithelial cell hypertrophy in small areas of the kidney cortex. Pathological changes in kidney ultrastructure (injured brush-border microvilli and swollen mitochondria in the proximal convoluted tubular cells) were observed when Cd concentration in this organ exceeded 10 µg/g and they became more pronounced as concentration increased. At a Cd level of about 30 µg/g, necrotic changes were observed (Aughey et al., 1984
). In our experiment, Cd concentration in kidney ranged from about 20 to 30 µg/g, depending on whether Cd was administered alone or in combination with EtOH. The results of this study and of other investigations (Aughey et al., 1984
) show that the critical Cd concentration in the kidney cortex is lower than 200 µg/g (the kidney cortex/whole kidney ratio of Cd concentration is about 1.25). Such high Cd concentrations in the kidney cortex were measured in rats fed with diet containing 200 mg Cd/kg for 24 months (Mitsumori et al., 1998
).
Increased serum transaminase activities were observed in our study following Cd and EtOH co-administration and similar changes have been reported by other authors (Tandon and Tewari, 1987; Thurman et al., 1999
).
Morphological observations, together with functional tests, show that Cd and EtOH, administered separately and especially in combination, lead to liver and kidney injury, thus posing a serious risk for health. The changes observed in these organs of co-exposed rats can be a result of an independent effect of Cd and EtOH and also of their interaction. Since EtOH alone also had affected the liver and kidney, on the basis of this study it is difficult to make any definite assessment as to whether EtOH influenced Cd toxicity, and if so, to what extent. However, such an effect of EtOH is very likely, and can be linked to changes in Cd body burden. In this work, we measured the Cd concentrations only in the liver and kidney, but in a previous study a profound effect of EtOH on Cd turnover was reported in the same experimental model (Brzóska et al., 2002). We have noted that in the Cd + EtOH group the whole Cd pool in the internal organs was at the same level as in those receiving Cd alone, in spite of its lower intake. In the absence of the modifying effect of EtOH, the concentrations and content of Cd in the co-exposed animals should be lower, compared to the Cd-only exposed ones. Thus, our results clearly show that EtOH influences Cd turnover (increases gastrointestinal absorption and retention of absorbed metal), making the organism more susceptible to its accumulation.
Due to the different intakes of Cd and EtOH during their co-administration, than after their separate dosages, we cannot correctly interpret the interactive effects of the two substances on the liver and kidney. Nevertheless, our findings allow us to conclude that EtOH increases Cd nephrotoxicity, although the present results give no clear evidence of enhanced Cd hepatotoxicity. However, it seems likely that, if the consumption of Cd and EtOH were the same in co-exposed and separately exposed animals, the disturbances in liver and kidney function as well as histology, would be more serious in the co-exposed ones. On the basis of the present and previous studies (Brzóska et al., 2000, 2002
), we hypothesize that subjects exposed simultaneously to Cd and EtOH are more vulnerable to Cd accumulation and thus its deleterious health effects, including kidney damage. Further studies are needed to explain Cd EtOH interactions in conditions of long-term co-exposure and their consequences for health.
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FOOTNOTES |
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