EFFECT OF SHORT-TERM ETHANOL ADMINISTRATION ON ...

Alcohol & Alcoholism Vol. 35, No. 5, pp. 439–445, 2000

EFFECT OF SHORT-TERM ETHANOL ADMINISTRATION ON CADMIUM RETENTION AND BIOELEMENT METABOLISM IN RATS CONTINUOUSLY EXPOSED TO CADMIUM · M. M. BRZÓSKA*, J. MONIUSZKO-JAKONIUK, M. JURCZUK, M. GAŁAZYN-SIDORCZUK and J. ROGALSKA Department of Toxicology, Medical University, Mickiewicza 2c str., 15–222 Bialystok, Poland (Received 28 February 2000; in revised form 25 April 2000; accepted 10 May 2000) Abstract — The present study was performed to assess the effect of short-term ethanol administration on cadmium retention and accumulation as well as on bioelement metabolism (zinc, copper, calcium, and magnesium) in rats exposed to an aqueous solution of cadmium chloride for 8 weeks. Intoxication with cadmium led to accumulation of this toxic metal, particularly in the liver and kidney, which was linked to metallothionein synthesis as well as to a disturbance in the metabolism of zinc, copper, and calcium. These effects were dependent on the level of exposure. The administration of ethanol in the final phase of cadmium treatment increased cadmium retention and accumulation in the body with simultaneous elevation in liver and kidney metallothionein concentration. Ethanol alone or with cadmium caused or intensified the cadmium-induced changes in metabolism of zinc and copper. Calcium metabolism disturbed by cadmium was not influenced by ethanol. Neither agents had any effect on magnesium metabolism. We conclude that even short-term ethanol consumption in conditions of exposure to cadmium can increase this heavy metal body burden and lead to more serious disturbances in metabolism of important elements such as zinc and copper. Cadmium- and ethanol-induced changes in the homeostasis of these microelements are probably connected with the ability of both xenobiotics to cause metallothionein induction.

INTRODUCTION

that interactions of cadmium with metabolism and functions of bioelements are one of the mechanisms of this heavy metal toxicity. Long-term ethanol consumption can also interfere with metabolism of these bioelements (Fairweather-Tait et al., 1988; Sharma et al., 1991; Gonzalez-Reimers et al., 1998). It has been shown that repeated ethanol administration influences some effects of cadmium (Flora and Tandon, 1987; Kershaw et al., 1990; Brus et al., 1995). But there are still only few data on the effect of ethanol influence on bioelement metabolism in conditions of exposure to cadmium (Tandon and Tewari, 1987; Hopf et al., 1990; Sharma et al., 1991). Taking the above into consideration, we have focused in the present work on studying the influence of short-term ethanol dosage on cadmium retention and this heavy metal’s influence on zinc, copper, iron, magnesium, and calcium metabolism. Its effect on iron metabolism has been reported in a separate paper (Moniuszko-Jakoniuk et al., 1999).

Recently great attention has been paid not only to the effects of toxic xenobiotics but also to their interactions with one another or with dietary factors. It has been known that uptake, accumulation and toxicity of xenobiotics can be modified (potentiated or reduced) by dietary factors (Grosicki and Doman´ska, 1997; Brzóska and Moniuszko-Jakoniuk, 1998) and stimulants (Flora and Tandon, 1987; Kershaw et al., 1990). Particularly interesting are interactions between xenobiotics to which exposure is often common. Examples of such substances are cadmium and ethanol. Interactions between cadmium and ethanol are an important problem in the field of modern toxicology, as both substances pose a risk to human and animal health (Schioeler, 1991; World Health Organization, 1992; Eight Special Report to the US Congress, 1993). Exposure to cadmium can occur in the workplace and in the environment, because this metal is utilized in a number of industrial practices and is a ubiquitous contaminant of the natural environment and dietary products (World Health Organization, 1992). An important source of general population exposure to this heavy metal is cigarette smoking (Bem et al., 1993; Benedetti et al., 1994). Alcoholism is a serious problem in almost all countries. The excessive consumption of ethanol in the form of alcoholic beverages may be common among some industrial workers exposed to cadmium, including smokers (Schioeler, 1991). Because ethanol increases permeability of biological membranes to cadmium (Pal et al., 1993) it can make alcoholics more susceptible to the effect of this heavy metal, compared to non-alcoholics. One of the effects of continuous cadmium exposure is a disturbance in metabolism and functions of some essential elements, including zinc, copper, iron, calcium, and magnesium (Mahaffey et al., 1981; Chmielnicka and Sowa, 1996; Brzóska et al., 1997a,b, 1998; Jurczuk et al., 1997a,b). It is thought

MATERIALS AND METHODS Animals and experimental design The experiment was performed over 8 weeks on 36 locally bred male Wistar rats, of initial body weight 180–200 g, kept under standard laboratory conditions. The animals were randomly allocated to six treatment groups of six rats each: (1) the control group: the animals received redistilled water to drink for the whole course of the experiment; (2) Et group: the rats were given intragastrically (by a stomach tube) a 25% (v/v) aqueous solution of ethanol in a dose of 1.25 g of 96% ethanol/kg body wt/24 h, every 12 h for the final 108 h of the experiment; (3) Cd5 group: this group received as the only drinking fluid an aqueous solution of cadmium chloride (CdCl2) at a concentration of 5 mg/l for 8 weeks; (4) Cd5 + Et group: the rats were administered 5 mg Cd/l of drinking water for 8 weeks as above and ethanol for the last 108 h of cadmium intoxication in the same dose as the Et group above; (5) Cd50 group: the animals received as drinking fluid an aqueous solution

*Author to whom correspondence should be addressed. 439

© 2000 Medical Council on Alcoholism

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M. M. BRZÓSKA et al.

of CdCl2 at a concentration of 50 mg Cd/l for the whole 8-week period of the experiment; (6) Cd50 + Et group: the rats were exposed to cadmium at the same concentration as the Cd50 group and for the last 108 h of the 8-week treatment period they also received (intragastrically) ethanol similarly to the animals from the Et and Cd5 + Et groups. When the rats from the Et, Cd5 + Et, and Cd50 + Et groups received ethanol, the ones from the control, Cd5, and Cd50 groups were administered redistilled water by stomach tube as a control condition. During the whole course of the experiment, the animals were kept under identical conditions and had unlimited access to LSM granulated diet (Fodder Factory, Motycz, Poland) and drinking solutions (redistilled water for the control and Et groups and aqueous solutions of CdCl2 for the remaining groups). Consumption of drinking water was assessed every 24 h during the experiment. Twenty-four hours before the end of the exposure, the rats were weighed and placed in metabolic cages for a 24-h collection of urine and faeces. At that time, the animals were starved and received only their drinking fluids. Next the animals were anaesthetized with ether and the whole blood from the heart was collected (with and without anticoagulant). Various organs, including liver, kidney, spleen, brain, heart, femur, and muscle tissue, were removed. The soft tissues were washed thoroughly in 0.9% (w/v) NaCl (physiological saline). The bones were cleansed of muscle tissue. The prepared tissues were frozen at –20°C until further analysis. After coagulation, whole blood was centrifuged and serum was immediately separated, whereas blood collected in heparinized tubes (assigned

Table 1. Body weight gain, consumption of drinking fluids and cadmium intake

Group Control Et Cd5 Cd5 + Et Cd50 Cd50 + Et

Body weight gain (g/8 weeks/rat)

Mean consumption of drinking fluids (ml/24 h/rat)

Mean Cd intake (µg/24 h/rat)

119.8 ± 10.2 116.4 ± 12.7 100.4 ± 6.1 108.6 ± 8.3 89.3 ± 7.3a 88.4 ± 8.5ad

37.7 ± 1.7 37.0 ± 2.0 34.0 ± 1.9a,c 34.6 ± 1.5a 27.5 ± 1.3a,b 27.4 ± 1.9ad

— — 170.0 ± 9.5c 173.0 ± 7.5 1375.0 ± 65.0b 1370.0 ± 95.0d

Each value is the mean ± SD of six animals. a,b,c,d Values are significantly different compared to the control, Cd5, Cd50, and Cd5 + Et groups, respectively. Et, ethanol-treated group; Cd5 and Cd50, cadmium-treated groups. For other details, see the Materials and methods section.

for cadmium determination) was prepared according to Razniewska and Trzcinka-Ochocka (1995). Chemicals All reagents and chemicals used in this experiment were of analytical grade or higher purity. Analytical procedures Concentrations of cadmium and bioelements in tissues, biological fluids, and faeces. Samples of known weight (slices or whole organs of soft tissues, femur and 24-h faeces) were subjected to dry mineralization in an electric oven according to · Zmudzki (1977). The ash was dissolved in a known volume of 1 N HCl (bone) or HNO3 (soft tissues and faeces). The concentrations of all metals in such preparations, urine, and the levels of bioelements in the serum and cadmium in the blood (after appropriate dilution) were assessed by atomic absorption spectrophotometry (Zeiss Jena, Germany AAS 30) with electrothermal atomization in a graphite cuvette (cadmium) or flame atomization in an air–acetylene burner (bioelements). The cathode lamps of the respective elements were operated under standard conditions using their respective resonance lines: Cd, 228.8 nm; Zn, 213.9 nm; Cu, 324.75 nm; Mg, 285.2 nm; Ca, 422.7 nm. The concentrations of metals were expressed as µg or mg/g of fresh tissue, µg/24-h urine or faeces collection, or µg or mg/100 ml of serum/blood. Metallothionein concentration in the liver and kidney. The levels of metallothionein in homogenates of liver and kidney · were determined by the radiochemical method of Z elazowski and Piotrowski (1977), with the use of a [203Hg] radioisotope (Amersham Life Science Amersham, UK), and are expressed as µmol Hg/g of fresh tissue. Urea concentration in serum. The concentration of urea in serum was assessed using a diagnostic laboratory test (POCh, Gliwice, Poland) and was expressed as mg/100 ml. Protein concentration in urine. The concentration of total protein in urine was determined by the method of Lowry et al. (1951) and expressed as mg/24 h of urine collection. Creatinine clearance. Creatinine clearance was calculated for the assessment of renal function (a critical organ for cadmium). The determinations of creatinine in serum and urine were made according to Jaffe’go’s reaction using a diagnostic laboratory test (POCh, Gliwice, Poland). The creatinine clearance is given in ml/min. Statistics The Mann–Whitney test was used for statistical analysis.

Table 2. Effects of cadmium, ethanol, and their combination on cadmium concentration in blood and tissues Group

Blood (µg/100 ml)

Liver (µg/g)

Control 0.761 ± 0.169 0.029 ± 0.008 Et 0.786 ± 0.111 0.067 ± 0.020a a,c 1.585 ± 0.288 0.626 ± 0.117a,c Cd5 Cd5 + Et 1.447 ± 0.165a 0.846 ± 0.101a,b Cd50 8.697 ± 0.882a,b 8.880 ± 1.395a,b Cd50 + Et 10.512 ± 1.077a,c 13.586 ± 1.581a,c

Kidney (µg/g)

Spleen (µg/g)

Heart (µg/g)

0.064 ± 0.010 0.086 ± 0.013a 1.649 ± 0.132a,c 1.925 ± 0.113a,b 17.354 ± 0.707a,b 18.806 ± 1.049a,c

0.015 ± 0.002 0.020 ± 0.006 0.073 ± 0.008a,c 0.091 ± 0.012a,b 0.887 ± 0.304a,b 1.798 ± 0.394a,c

0.030 ± 0.007 0.030 ± 0.007 0.067 ± 0.007a,c 0.107 ± 0.013a,b 0.333 ± 0.030a,b 0.459 ± 0.041a,c

Each value is the mean ± SD of six animals. Abbreviations are as in Table 1. a,b,c Values are significantly different compared to the control, Cd5, and Cd50 groups, respectively.

Brain (µg/g) 0.054 ± 0.006 0.050 ± 0.012 0.076 ± 0.011a 0.073 ± 0.012a 0.083 ± 0.011a 0.083 ± 0.005a

Muscle tissue (µg/g)

Femur (µg/g)

0.020 ± 0.004 0.017 ± 0.002 0.042 ± 0.006a,c 0.037 ± 0.009a 0.194 ± 0.036a,b 0.215 ± 0.045a

0.141 ± 0.029 0.141 ± 0.033 0.187 ± 0.049a,c 0.210 ± 0.060a 0.443 ± 0.140a,b 0.442 ± 0.125a

CADMIUM, ETHANOL, AND BIOELEMENTS

RESULTS Body weight gain, consumption of drinking solutions, and cadmium intake Administration of 50 mg Cd/l of drinking water for 8 weeks caused retardation in body weight gain, compared to control rats (Table 1). This effect of cadmium was not observed on exposure to 5 mg Cd/l. The administration of ethanol (alone and with cadmium) had no influence on body weight gain. Addition of CdCl2 to water led to a reduction in drinking water consumption compared to the control group. The decrease Table 3. Cadmium excretion with urine and faeces after administration of cadmium, ethanol or both Group

Urine (µg Cd/24 h/rat)

Faeces (µg Cd/24 h/rat)

Control Et Cd5 Cd5 + Et Cd50 Cd50 + Et

0.206 ± 0.034 0.198 ± 0.037 0.806 ± 0.122a,c 0.531 ± 0.061a,b 2.712 ± 0.340a,b 1.995 ± 0.241a,c

1.48 ± 0.35 1.53 ± 0.28 159.10 ± 19.36a,c 130.04 ± 14.30a,b 1239.86 ± 80.13a,b 1020.73 ± 76.65a,c

Each value is the mean ± SD of six animals.a,b,c Values are significantly different compared to the control, Cd5, and Cd50 groups, respectively.

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was higher in rats exposed to 50 mg Cd/l than in those receiving the smaller 5 mg Cd/l dose. The consumption of drinking solutions was not influenced by ethanol administration (Table 1). Cadmium accumulation and excretion in urine and faeces Administration of cadmium resulted in its accumulation in all tissues and organs studied, in a manner dependent on the level of exposure, apart from brain (Table 2). Organs exhibiting the highest cadmium accumulation were kidney and liver, whereas the lowest levels were noted in brain and muscle. Ethanol administered in the last phase of the cadmium treatment exacerbated this heavy metal’s accumulation in liver, kidney, heart and spleen (Table 2) and decreased its urinary and faecal excretion (Table 3), independently of the level of cadmium treatment. In blood, ethanol induced an increase in cadmium concentration only in conditions of the higher level of exposure (Table 2). On the other hand, ethanol administered alone increased cadmium concentration only in the liver and kidney (Table 2). Metallothionein in liver and kidney The concentration of metallothionein in liver and kidney was increased according to the level of cadmium exposure. Ethanol administered alone as well as in combination with cadmium increased or caused a further increase in metallothionein concentration in both these organs (Fig. 1).

Table 4. Effects of cadmium, ethanol, and their combination on zinc concentration in serum and tissues Group Control Et Cd5 Cd5 + Et Cd50 Cd50 + Et

Serum (µg/100 ml)

Liver (µg/g)

Kidney (µg/g)

Spleen (µg/g)

Heart (µg/g)

Brain (µg/g)


Femur (µg/g)

186.52 ± 8.68 185.40 ± 6.15 183.73 ± 7.54c 182.47 ± 8.41 153.56 ± 7.05a,b 147.65 ± 11.98a

34.88 ± 0.95 36.90 ± 1.03a 39.09 ± 1.42a,c 41.15 ± 1.50a,b 41.65 ± 1.80a,b 45.64 ± 1.74a,c

28.10 ± 1.57 30.48 ± 1.58a 33.46 ± 0.82a,c 35.16 ± 1.30a,b 40.37 ± 2.01a,b 42.93 ± 1.63a,c

30.15 ± 2.01 25.52 ± 5.71 28.59 ± 2.93 28.96 ± 5.43 28.99 ± 3.54 31.74 ± 4.77

22.11 ± 4.74 24.07 ± 5.64 23.11 ± 3.50 22.78 ± 3.34 23.20 ± 5.47 21.27 ± 1.86

11.56 ± 1.84 11.82 ± 3.17 11.62 ± 1.92 13.09 ± 1.97 11.79 ± 2.15 13.06 ± 2.11

16.21 ± 1.01 17.84 ± 2.11 15.97 ± 2.61 17.99 ± 3.53 16.97 ± 1.32 14.71 ± 1.60c

193.43 ± 6.26 195.32 ± 6.14 194.58 ± 8.17c 196.41 ± 6.33 172.70 ± 5.74a,b 175.29 ± 9.03c

Each value is the mean ± SD of six animals. a,b,c Values are significantly different compared to the control, Cd5, and Cd50 groups, respectively.

Fig. 1. Metallothionein concentration in the liver and kidney after administration of cadmium, ethanol or both.

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Concentration of bioelements in serum and tissues and their urinary excretion Zinc. Cadmium led to an increase in zinc concentration in the liver and kidney in a dose-dependent manner (Table 4). In both serum and femur, the concentration of zinc was reduced in rats exposed to the higher level of cadmium. The concentration of zinc in the spleen, heart, brain, muscle tissue (Table 4) and its urinary excretion (Table 8) were not changed by any treatment. Administration of ethanol alone and in conjunction with cadmium increased zinc concentration in the liver and kidney, but had no effect on its level in other tissues or biological fluids.

Copper. In the liver of rats exposed to both levels of cadmium, the concentration of copper was increased, compared to the control group (Table 5). In the kidney and spleen, copper concentration was also increased, but only at the highest level of cadmium. The concentration of copper in the serum, heart, brain, muscle tissue, and femur was not influenced by cadmium treatment (Table 5). Administration of ethanol alone caused an increase in copper concentration in the spleen. Ethanol independently of whether it was administered alone or in combination with cadmium led to a marked decrease in copper concentration in the femur (Table 5). Ethanol administered with 5 mg Cd/l increased copper concentration in muscle tissue,

Table 5. Effects of cadmium, ethanol, and their combination on copper concentration in serum and tissues Group Control Et Cd5 Cd5 + Et Cd50 Cd50 + Et

Serum (µg/100 ml)

Liver (µg/g)

Kidney (µg/g)

169.26 ± 16.94 176.01 ± 12.78 159.05 ± 5.99 164.19 ± 7.69 153.50 ± 12.64 173.31 ± 12.61c

4.555 ± 0.647 4.912 ± 0.915 6.818 ± 1.520a 6.138 ± 0.700a 7.308 ± 1.514a 6.027 ± 0.952a

5.733 ± 0.721 4.582 ± 1.353 6.450 ± 0.360 6.482 ± 0.678 7.702 ± 1.366a 7.550 ± 1.208a

Spleen (µg/g) 1.908 ± 0.616 2.967 ± 0.501a 3.000 ± 1.012 2.735 ± 0.735 2.983 ± 0.585a 3.527 ± 1.290a

Heart (µg/g) 4.983 ± 0.664 5.028 ± 1.194 4.245 ± 0.973 5.227 ± 1.060 4.955 ± 1.015 4.720 ± 0.784

Brain (µg/g) 2.313 ± 0.528 2.075 ± 0.705 2.165 ± 0.711 2.833 ± 0.449 2.632 ± 0.805 1.920 ± 0.513


Femur (µg/g)

1.400 ± 0.329 20.508 ± 3.347 1.313 ± 0.314 5.222 ± 1.737a 1.615 ± 0.354 21.542 ± 5.055 1.933 ± 0.440a 5.245 ± 1.017a,b 1.782 ± 0.385 22.717 ± 4.602 1.473 ± 0.302 4.353 ± 1.304a,c

Each value is the mean ± SD of six animals. a,b,c Values are significantly different compared to the control, Cd5, and Cd50 groups, respectively. Table 6. Effects of cadmium, ethanol, and their combination on magnesium concentration in serum and tissues Group

Serum (mg/100 ml)

Liver (µg/g)


3.31 ± 0.46 3.55 ± 0.21 3.62 ± 0.58 3.37 ± 0.64 3.89 ± 0.60 3.35 ± 0.41

256.32 ± 35.87 256.95 ± 33.68 279.23 ± 45.45 278.02 ± 32.45 281.75 ± 23.61 275.85 ± 34.87

Kidney (µg/g) 201.98 ± 33.67 208.38 ± 24.03 234.18 ± 28.63 233.80 ± 29.95 239.25 ± 33.27 240.33 ± 22.68

Spleen (µg/g) 257.78 ± 49.89 247.83 ± 23.36 251.42 ± 52.49 235.32 ± 40.17 250.23 ± 36.35 271.18 ± 32.42

Heart (µg/g)

Brain (µg/g)


Femur (µg/g)

225.53 ± 28.02 233.37 ± 22.33 225.27 ± 40.02 246.85 ± 21.15 232.02 ± 62.52 234.23 ± 13.32

128.60 ± 23.91 129.82 ± 21.66 131.90 ± 27.94 143.02 ± 8.88 130.45 ± 20.17 107.60 ± 22.44

309.23 ± 22.34 289.33 ± 31.34 305.98 ± 56.35 312.13 ± 40.31 301.32 ± 31.66 307.95 ± 33.08

4.147 ± 0.304 4.224 ± 0.459 4.076 ± 0.685 4.083 ± 0.433 3.670 ± 0.476 3.755 ± 0.754

Each value is the mean ± SD of six animals. Table 7. Effects of cadmium, ethanol, and their combination on calcium concentration in serum and tissues Group

Serum (mg/100 ml)


9.21 ± 0.22 9.26 ± 0.19 8.84 ± 0.21a,c 8.82 ± 0.17a 8.51 ± 0.23a,b 8.58 ± 0.24a

Liver (µg/g)

Kidney (µg/g)

Spleen (µg/g)

Heart (µg/g)

51.95 ± 6.98 48.85 ± 9.23 54.03 ± 7.85 49.99 ± 8.69 54.44 ± 5.81 57.12 ± 7.61

65.04 ± 5.21 63.99 ± 6.13 68.70 ± 5.99 71.23 ± 7.25 66.59 ± 6.83 69.12 ± 4.32

54.60 ± 3.60 51.84 ± 9.05 52.96 ± 4.70 56.27 ± 5.96 58.79 ± 4.44 58.23 ± 7.31

51.08 ± 8.02 52.65 ± 7.23 46.80 ± 7.39 49.26 ± 6.64 48.73 ± 9.97 44.50 ± 7.76

Brain (µg/g) 42.54 ± 9.02 40.19 ± 7.12 44.33 ± 10.13 41.68 ± 4.12 46.00 ± 9.01 42.84 ± 10.15


Femur (µg/g)

13.79 ± 1.91 13.56 ± 2.03 13.54 ± 1.78 13.44 ± 1.72 14.15 ± 2.07 14.08 ± 2.11

192.45 ± 5.08 189.57 ± 6.11 178.25 ± 5.12a,c 177.95 ± 6.33a 163.24 ± 4.87a,b 161.97 ± 4.99a

Each value is the mean ± SD of six animals. a,b,c Values are significantly different compared to the control, Cd5, and Cd50 groups, respectively. Table 8. Effects of cadmium, ethanol, and their combination on bioelement urinary excretion Group Control Et Cd5 Cd5 + Et Cd50 Cd50 + Et

Zinc (µg/24 h)

Copper (µg/24 h)

Magnesium (µg/24 h)

6.335 ± 2.037 5.886 ± 2.540 5.381 ± 1.665 5.328 ± 1.036 5.739 ± 0.966 5.662 ± 0.929

3.252 ± 0.898 2.971 ± 1.072 2.405 ± 0.949 2.479 ± 0.367 3.182 ± 0.847 2.545 ± 0.404

2.880 ± 0.554 2.683 ± 0.535 2.844 ± 0.731 2.293 ± 0.783 3.193 ± 0.814 2.778 ± 0.543

Calcium (µg/24 h) 0.811 ± 0.142 0.796 ± 0.110 1.396 ± 0.163ac 1.415 ± 0.180a 1.964 ± 0.213ab 1.896 ± 0.209a


CADMIUM, ETHANOL, AND BIOELEMENTS Table 9. Biochemical parameters in serum and urine after administration of cadmium, ethanol or both

Group

Serum urea (mg/100 ml)

Total protein in urine (mg/24 h)

Creatinine clearance (ml/min)


27.25 ± 2.48 27.93 ± 3.71 26.23 ± 3.10 25.76 ± 2.98 25.47 ± 4.14 26.17 ± 3.15

31.88 ± 4.88 36.38 ± 6.02a 42.21 ± 4.64a,c 40.27 ± 3.18a 64.95 ± 9.11a,b 58.66 ± 9.71a

3.489 ± 0.625 4.013 ± 0.892 4.139 ± 0.817 3.657 ± 0.385 3.080 ± 0.637 3.281 ± 0.713


but when administered with 50 mg Cd/l, increased the concentration of copper in the serum. Urinary excretion of copper was unchanged by any treatment (Table 8). Magnesium. The concentrations of magnesium in serum and tissues (Table 6) as well as its urinary excretion (Table 8) in rats exposed to cadmium and/or ethanol were in the range of the control values. Calcium. Exposure to cadmium led to a decrease in calcium concentration in the serum and bone (Table 7) as well as to an increase in its urinary excretion (Table 8) in a manner dependent on the level of exposure. In the other tissues of rats intoxicated with cadmium, the concentration of calcium was in the range of control values. Ethanol administered alone or with cadmium had no effect on calcium metabolism (Tables 7 and 8). Serum urea, total protein in urine, and creatinine clearance Excretion of total protein in the 24-h urine collection increased markedly with the level of cadmium exposure (Table 9). Ethanol administered alone and in combination with cadmium had no effect on urinary protein excretion (Table 9). Serum urea concentration and creatinine clearance were also not changed significantly by any treatment (Table 9). DISCUSSION The present work was aimed at studying the influence of short-term ethanol administration on cadmium retention and accumulation as well as on levels of zinc, copper, calcium, and magnesium in rats continuously exposed to cadmium. We have used cadmium exposure levels corresponding to those which occur in humans, especially smokers, exposed both environmentally (5 mg Cd/l in our study) and occupationally (50 mg Cd/l). Making a simple recalculation, a single dose of ethanol administered to rats (0.625 g of 96% v/v ethanol/kg body wt), can correspond to human consumption of about 100 g of 45% vodka (about 1.4 g/kg body wt, converting to 45% alcohol). However, such a calculation cannot be used as rats metabolize ethanol more rapidly and therefore need a higher dose of ethanol than humans for the same toxic effects. Taking this into consideration, the dose of ethanol used in our study corresponds to a human consumption of less than 100 g of 45% vodka. Cadmium intake and therefore its organ contents were low in animals from the control and Et groups. Administration of cadmium resulted in its accumulation in the body in a manner

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dependent on the level of exposure. Cadmium accumulation is strictly connected with metallothionein synthesis. In tissues, such as liver and kidney, in which the production of metallothionein is high, cadmium concentrations are also considerably higher than in other tissues (Hopf et al., 1990; Sharma et al., 1991). Metallothionein is a low molecular weight, thiol-rich, metal-binding protein, whose main biological function is maintaining a balance of zinc and copper (Ebadi, 1991; Chang and Huang, 1996; Kelly et al., 1996). But this protein also plays an important protective role in heavy metal toxicity, including cadmium (Elinder et al., 1987; Liu et al., 1992, 1994), via the formation of metal–metallothionein complexes. The observations made in the present study indicate that ethanol administered even for a short period of time with a simultaneous exposure to cadmium influences the turnover of this heavy metal. A decrease in cadmium faecal excretion in rats simultaneously exposed to both these substances in comparison to the group exposed to cadmium alone, in the absence of differences in cadmium intake, suggests that ethanol increases cadmium absorption from the gastrointestinal tract. It is known that ethanol increases the permeability of biological membranes to various substances, including metals (Pal et al., 1993). As a result, ethanol can also increase cadmium absorption. The increased cadmium absorption and its decreased urinary excretion by ethanol give clear evidence of increased cadmium retention under conditions of simultaneous exposure to both these substances. The increased cadmium concentrations in whole blood and its accumulation in liver, kidney, heart and spleen of rats exposed to cadmium and ethanol, in comparison with those intoxicated with cadmium alone, further support this conclusion. The mechanism of the ethanol-induced increase in cadmium accumulation in soft tissues may be related to the ability of both substances to induce metallothionein synthesis (Sharma et al., 1991, 1992; Ebadi et al., 1992). It has been proposed that ethanol can induce metallothionein synthesis indirectly by increasing the cadmium body burden, as well as by an alteration in zinc and glucocorticoid homeostasis (Bracken and Klaassen, 1987; Sharma et al., 1992). Because the ethanolinduced increase in the liver and kidney metallothionein concentration was associated with simultaneous elevation of zinc and cadmium levels, it is very probable that ethanol induces the synthesis of this protein indirectly via zinc and cadmium. An excessive exposure to cadmium and its accumulation in the organism lead to disturbances in metabolism of essential elements (Sharma et al., 1991; Chmielnicka and Sowa 1996; Brzóska et al., 1997a,b, 1998; Grosicki and Doman´ska 1997; Jurczuk et al., 1997a,b), The interactions between cadmium and bioelements can take place at different stages of their metabolism: absorption from the gastrointestinal tract, distribution in the organism and excretion in urine, as well as at the stage of biological functions of essential elements (Brzóska et al., 1997a,b; Jurczuk et al., 1997 a,b; Brzóska and Moniuszko-Jakoniuk, 1998). We have shown that exposure to cadmium led to redistribution of zinc in the organism. Cadmium-induced changes in tissue zinc concentration have been widely observed (Sharma et al., 1991; Ninomiya et al., 1993; Liu et al., 1994; Tandon et al., 1994; Brzóska et al., 1998; Eybl et al., 1998). It has been recognized that retention of zinc in the liver and kidney

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in conditions of exposure to cadmium is a secondary effect of its accumulation and metallothionein synthesis in these organs. The decrease in zinc concentration in serum and femur noted in rats exposed to the higher level of cadmium was caused, at least partly, by increased zinc retention in the liver and kidney. The probability of the above mechanism is suggested by the fact that an increase in zinc concentration in the liver and kidney is the first change in zinc metabolism noted in conditions of exposure to cadmium. We have shown that ethanol administered in the last phase of cadmium exposure potentiates this heavy metal-induced retention of zinc in these organs. The other metal with metabolism connected to metallothionein is copper. As noted in the case of zinc, cadmium also increased copper concentration in the liver or in the liver and kidney dependent on the level of exposure. Changes in copper metabolism in rats exposed to cadmium has been reported by other authors (Mahaffey et al., 1981; Sharma et al., 1991; Chmielnicka and Sowa, 1996; Saito, 1996). The changes observed in copper concentration in some tissues after ethanol treatment demonstrate the disturbed distribution of this bioelement. Apart from zinc and copper, important metabolic roles are played by magnesium and calcium. The effects of cadmium on calcium metabolism and probable mechanisms of such effects have been reported previously by us (Brzóska et al., 1997a; Brzóska and Moniuszko-Jakoniuk, 1998). Our results in this respect are in agreement with other works (Mahaffey et al., 1981; Ogoshi et al., 1992). We have shown that cadmium at exposure levels leading to clear disturbances in zinc, copper, iron, and calcium metabolism has no effect on magnesium metabolism. On this basis, it can be concluded that magnesium is the most stable of bioelements under conditions of cadmium intoxication. We have shown that ethanol administered even in a low dose and for such a short period, as in our experiment, can cause clear changes in the metabolism of zinc, copper and, as we reported previously, iron (Moniuszko-Jakoniuk et al., 1999). Moreover, ethanol used simultaneously with cadmium can potentiate some cadmium-induced changes in metabolism of microelements and cause changes which have not been observed in conditions of exposure to each of these substances alone. However, ethanol has no effect on the metabolism of macroelements, such as calcium and magnesium. Ethanol-induced disturbances in microelement metabolism in rats exposed to cadmium may be a result of direct as well as indirect actions of ethanol, such as increased cadmium body burden in conditions of simultaneous exposure to both these xenobiotics. The increased level of cadmium further disturbs the homeostasis of bioelements. Changes in bioelement metabolism can also result from cadmium and/or ethanol influence on nutritional status. We have noted (unpublished data) that administration of 50 mg Cd/l of drinking water leads to reduction in diet consumption. Indeed, in the present study, in rats exposed to this level of cadmium decreased body weight gain was noted compared to controls. One should therefore take into consideration diminished intake of these elements in this type of study. It is known that long-term excessive consumption of ethanol can lead to anorexia and malabsorption as well as to increased urinary loss of many essential substances and, as a consequence, to

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