Department of Toxicology, Medical University, Mickiewicza 2c str., 15222 Bialystok, Poland
Received 28 February 2000; in revised form 25 April 2000; accepted 10 May 2000
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ABSTRACT |
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INTRODUCTION |
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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 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).
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MATERIALS AND METHODS |
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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 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 mudzki (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 airacetylene 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 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 MannWhitney test was used for statistical analysis.
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RESULTS |
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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
).
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DISCUSSION |
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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 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 metallo-thionein 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 metalmetallothionein 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 ethanol-induced 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 Doma
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 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 impoverishment of micro- and macrobioelements, and vitamins (Fairweather-Tait et al., 1988; Sharma et al., 1991
; Gonzalez-Reimers et al., 1998
). In this study the duration of ethanol administration was very short and as ethanol has no influence on dietary consumption, it is unlikely to have influenced the gastrointestinal absorption of zinc and copper.
More studies are, however, needed to explain the mechanism of the ethanol-induced disturbances in the metabolism of microelements in rats exposed to cadmium.
This study assessed the effect of short-term ethanol consumption on cadmium retention and this cadmium-induced changes in bioelement metabolism. Alcoholics are frequently heavy smokers and thus interaction of cadmium and ethanol with bioelements may be important clinically. Accordingly, we have started investigation of these potential interactions in conditions of long-term exposure to cadmium and ethanol in an animal model.
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FOOTNOTES |
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