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Histopathological evidence of vitamin C protection against Cd-nephrotoxicity in Guinea pigs
Exp Toxic Patho11994; 46: 11-14 Gustav Fischer Verlag Jena Institute of Preventive and Clinical Medicine!), Bratislava, Slovak Republic Institute of Pathological Anatomy 2), Medical Faculty of Komenius University, Bratislava, Slovak Republic
Histopathological evidence of vitamin C protection against Cd-nephrotoxicity in Guinea pigs ANNA NAGYOVAl), STEFAN GALBAVy 2) and EMIL GINTER l ) With 3 figures and 2 tables Received: April 13, 1993; Accepted: April 30, 1993 Address for correspondence: ANNA NAGYOVA, Ph. D., Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovak Republic; Fax: +42-7/373906. Key words: Vitamin C; Nephrotoxicity; Cadmium, renal changes; Ascorbic acid.
Summary The critical organ of chronic cadmium (Cd) exposure are kidneys. Long-term exposure leads to Cd accumulation in kidneys and causes renal tubular dysfunctions. Cd-induced renal changes are irreversible, so the primary prevention by different nutrients can play an important role. In this study, male guinea pigs which, like humans, do not synthetize ascorbic acid (AA) received low (2 mg/animal/day) or high (100 mg/animal/day) dosage of AA and cadmium (1 mg Cd/animal/day) in drinking water for 12 weeks. Characteristic finding in renal morphology of Cd-intoxicated guinea pigs was dilatation of interstitial veins with apparent paravenous lymphatic infiltrates. Histomorphological changes were more evident in Cd-intoxicated guinea pigs with low AA intake. High AA intake apparently reduced in Cd-intoxicated guinea pigs the extent of renal damage. Cadmium significantly increased serum creatinine and urea levels in the group of guinea pigs with low AA intake but not in guinea pigs receiving high concentration of AA. The results showed that AA can be effective in the protection of Cdinduced nephrotoxicity.
Introduction Cadmium is a typical cumulative xenobiotic with extremely long biological half-life in the liver and kidneys of humans, about 10-30 years (FRIBERG 1979). Kidneys are considered the critical organ of long-term Cd-exposure, which could be partly explained by the transport of Cd-metallothionein into the kidney (NORBERG 1989). The concentration of Cd in the blood and kidneys, Cd distribution and its binding to intracellular metallothionein take place in cadmium nephrotoxicity. FRIBERG'S et al. (1974) threshold level of Cd in the kidneys when renal lesions can be detected in man is 130 Ilg Cd/g kidney. Cd-
induced renal changes are responsible for renal tubular dysfunctions manifested by decreased reabsorptive capacity of the tubules with the consequent low molecular weight proteinouria (PISCATOR 1962). Histopathological evidence for renal toxicity indicating tubular derangement in experimental animals has been published by several authors (BONNELL 1960; STOWE 1972; KOTSONIS and KLAASSEN 1978). Since the renal changes caused by Cd exposure are irreversible, the primary prevention may play an important role. Optimal intake of different nutrients like minerals (GROTEN et al. 1991) and vitamins in the diet can favourably affect the Cd-toxicity. AA has been shown to prevent in experimental animals the signs of Cd-toxicity such as anemia, growth retardation (Fox et al. 1971) and liver damage (SUZUKI and YOSHIDA 1978). However, the data on protective effect of AA are available mostly from experiments with rats, mice or Japanese quails. We used guinea pigs which, like man, do not synthetize AA, to assess the effects of different AA intake as related to Cd-induced nephrotoxicity.
Material and methods Chemicals: Cadmium chloride, hematoxylin and eosin were purchased from Merck, Darmstadt (FRG). Ascorbic acid was obtained from Farmakon, Olomouc (Czecho-Slovakia). Other chemicals were commercial p.a. grade. Animals and treatments: Male short-hair, three-coloured guinea pigs (Velaz, Prague) weighing 350-450 g were used in the experiment. After two weeks of feeding with standard laboratory diet with addition of vegetables the animals were randomly divided into four groups. Two groups received ascorbic acid in drinking water in daily doses either 2 mg per animal (low AA group) or 100 mg per animal (high AA Exp Toxic Patho146 (1994) 1
11
Table 1. Histomorphological changes in the kidney of Cd-intoxicated guinea pigs with low and high AA intake. Lesion
lowAA
low AA+Cd
highAA
high AA+C
kidney weight (g) kidney cadmium (mg/g)
3.57 ± 0.21 1,79 ± 0.23"
3.34 ± 0.24 99,59 ± 10,39b
3.21 ± 0.13 1,10 ± 0,08 a
3.17±0.1l 85,29 ± 8,52
oa
2a O"b
3ab 3ab
oa
8b 4b 14b
oa
SC
a"
16b
a"
7c
a
3
a
2
epithelial proximal tubules vacuolization interstitium edema dilatation of veins paravenous lymphatic i nfi ltra tes lymphatic elements in interstitium
oa
The numbers in the table represent the sum of changes per group of 8 animals. a, b, c - different superscripts indicate significant difference between means in horizontal lines (P < 0.05). Amounts of cadmium in the kidneys are from KADRABOV A et al. 1992.
Fig. 1. Photomicrograph of the kidney from a guinea pig with low AA intake, chronically treated with Cd. Massive lymphocytic infiltrate surrounding dilatated veins and spreading into the tissue between tubules. Magnification: x 200, stain: Hand E. 12
Exp Toxic Pathol46 (1994) 1
Fig. 2. Photomicrograph of the kidney from a guinea pig with high AA intake, chronically treated with Cd. Dilatated vein and a slight lymphocytic infiltrate can be seen. Tubules appear to bear no trace of changes. Magnification: x 200, stain: Hand E.
Table 2. Effect of cadmium on serum parameters reflecting kidney damage in guinea pigs with low and high intake of ascorbic acid*. Groups
10wAA
low AA+Cd
high AA
high AA+Cd
Creatinine (flmol/l)
68 ± 3a
95 ±4b
75 ± 4ae
86 ± 5 be
Urea (mmol/l)
10.6 ± 0.6a
13.4 ± 1.1 b
10.3 ± 0.6a
ILl ± 0.7 ab
* Values are means ± SEM from 8 animals. a, b, c - different supersrcipts indicate significant difference between means in horizontallines (P < 0.05).
group). Two Cd-intoxicated groups were given 1 mg per animal per day of cadmium (as cadmium chloride) in drinking water (low A + Cd group; high AA + Cd group). AA-intake in these groups was the same as in the control groups. During the whole experiment the animals received drinking water and vitamin C-free diet ad libitum. The animals were decapitated after 17 -hour fasting on the 12-th week of cadmium administration. The number of guinea pigs in each group was eight. Histology: The kidneys were weighed, fixed in buffered formalin, embedded in paraffin and sections of the kidneys were prepared and stained with hematoxylin and eosin. Biochemical assays: In blood serum creatinine and urea levels were determined using commercial biochemical kits (Biola-test, Lachema, Czecho-Slovakia). Statistical analysis: The results of histopathological examinations were evaluated by the Fisher 2-sided test in table 2x2. Data in tab. 2 and kidney weight and Cd amounts in tab. 1 were statistically analyzed by variance analysis (ANOVA, Statgraphic). The minimal acceptable level of significance was P < 0.05.
Results Cadmium did not apparently change kidney weight in all groups of guinea pigs (tab. 1). Histopathology of kidneys of guinea pigs exposed to cadmium showed dilatation of veins with paravenous predominantly lymphatic infiltrates and interstitium edema as well as epithelial proximal tubule vacuolization. In the kidneys of Cd-intoxicated guinea pigs presence of lymphatic infiltrates in the interstitium was also observed. Histomorphological changes were most apparent in the group of Cd-intoxicated guinea pigs with low AA intake (fig. 1). Cd-intoxicated guinea pigs with high AA intake had evidently reduced extent of renal injury (fig. 2). AA itself showed no influence on kidney morphology (fig. 3). Consistent with his to morphological changes serum creatinine and urea levels were found to be elevated in Cd-intoxicated guinea pigs with low AA intake (by 40 % and 26 % resp.), while in Cd-intoxicated guinea pigs with high AA intake the differences were not significant (tab. 2).
Fig. 3. Photomicrograph of the kidney from a guinea pig with high AA intake. Normal picture of renal morphology. Magnification: x 200, stain: Hand E.
Discussion In the present study, subchronic Cd-treatment of guinea pigs with different AA intake led to significant histopaExp Toxic Pathol46 (1994) 1
13
thological changes in kidneys with characteristic finding of dilatation of interstitial veins and apparent paravenous lymphatic infiltrates which are typical for interstitial nephritis. High AA intake reduced significantly the extent of Cd-induced renal injury. Morphologically observed renal toxicity of Cd was also manifested by the increases in serum creatinine and urea levels which depended on AA intake. Kidneys are the critical organ of chronic Cd-exposure. Interstitial renal fibrosis has been observed in rabbits receiving 160 ppm of Cd in drinking water for 200 days (STOWE et al. 1972). In rats, exposed to aerosols containing 25 and 50 ~g Cd/m3 for 90 days, in a few kidneys signs of swelling of tubuli were observed (PRIGGE 1978). Chronic exposure of rats to Cd at concentrations of 10, 30, 100 ppm in drinking water for 24 weeks resulted in 30- and 100-ppm receiving rats in slight and focal tubular necrosis (KoTsoNIS and KLAASSEN 1978). In this study, the concentration of administered cadmium to guinea pigs was substantially lower (10 mg of Cd per liter of drinking water) and time-exposure was shorter than in the above mentioned studies with rats and rabbits. Concomitantly, the extent of renal damage was not so evident in this study. We did not observe morphological changes reported by other authors, such as renal interstitial fibrosis, tubular atrophy, tubular necrosis or nephrolithiasis in kidney (BONNELL et al. 1960; STOWE et al. 1972; KOTSONIS and KLAASSEN 1978). However, changes like interstitial nephritis which usually precede interstitial fibrosis have been seen in Cd - intoxicated guinea pigs. We used guinea pigs which, like man, are dependent on exogenous AA intake and their sensitivity to the effect of foreign compounds could be different. The long-term addition of AA to cadmium-containing diet prevented in animals signs of cadmium toxicity such as growth retardation, anemia and increase of serum enzymes reflecting liver damage (SUZUKI and YOSHIDA 1978; Fox et al. 1971). The mechanism by which AA counteracts Cd toxicity is not exactly known. AA may interfere in the absorption of Cd from the gastrointestinal tract, stimulate metallothionein synthesis (ONOSAKA et al. 1987) or as an effective antioxidant can protect-SH groups of metallothionein and other proteins against oxidation. More extensive renal injury in Cd-intoxicated guinea pigs with low AA intake could be related to the higher amount of cadmium accumulated in the kidneys ofthese animals (KADRABOVA et al. 1992). These results are potentially important for the prevention of nephrotoxic effects of cadmium in profesionally
14
Exp Toxic Patho146 (1994) 1
exposed workers, where increased AA intake could possibly reduce the toxic effects of cadmium. Acknowledgements: The authors wish to express their appreciation to Dr. S WSOLOVA for statistical analysis of the results and to Dr. M. ZOLDY for stylistic revision of the manuscript.
References I. BONNELL JA, Ross JH, KING E: Renal lesions in experimental cadmium poisoning. Brit J Industr Med 1960; 17: 69-80. 2. Fox MRS, FRY BE, JR, HARLAND BF, et al.: Effect of ascorbic acid on cadmium toxicity in the young coturnix. J of Nutr 1971; 101: 1295-1306. 3. FRIBERG L, KJELLSTROM T, NORDBERG GF, et al.: in Handbook on the Toxicology of Metals. Biomedical Press, Elsevier, North Holland, Amsterdam - New York Oxford, 1979, p. 355. 4. FRIBERG L, PISCATOR M, NORDBERG GF, et al.: Cadmium in the environment, 2nd ed. Cleveland, Ohio, CRC Press, 1974. 5. GROTEN JP, SINKELDAM EJ, Muys TH, et al.: Interaction of dietary Ca, P, Mg, Mn, Cu, Fe, Zn and Se with the accumulation and oral toxicity of cadmium in rats. Food Chern Toxicol1991; 29: 249-258. 6. KADRABOV AJ, MADARIi': A, GINTER E: The effect of ascorbic acid on cadmium accumulation in guinea pigs tissues. Experientia 1992; 48: 989-991. 7. KOTSONIS FN, KLAASSEN CD: The relationship of metallothionein to the toxicity of cadmium after prolonged oral administration to rats. Toxicol and Appl Pharmacol 1978;46: 39-54. 8. NORDBERG GF: Modulation of metal toxicity by metallothionein. BioI Trace Elem Res 1989; 21: 131-135. 9. ONOSAKA S, KAWAKAMI D, MIN K, et al.: Induced synthesis of metallothionein by ascorbic acid in mouse liver. Toxicology 1987; 43: 251-259. 10. PRIGGE E.: Early signs of oral and inhalative cadmium uptake in rats. Arch of Toxicol1978; 40: 231-247. 11. PISCATOR M: Proteinouria in chronic cadmium poisoning. Arch Environ Health 1962; 5: 55-62. 12. STOWE HD, WILSON M, GOYER RA: Clinical and morphologic effects of oral cadmium toxicity in rabbits. Arch Path 1972; 94: 389-405. 13. SUZUKI T, YOSHIDA A: Long-term effectiveness of dietary iron and ascorbic acid in the prevention and cure of cadmium toxicity in rats. Am J Clin Nutr 1978; 31: 1491-1498.
Histopathological evidence of vitamin C protection against Cd-nephrotoxicity in Guinea pigs ANNA NAGYOVAl), STEFAN GALBAVy 2) and EMIL GINTER l ) With 3 figures and 2 tables Received: April 13, 1993; Accepted: April 30, 1993 Address for correspondence: ANNA NAGYOVA, Ph. D., Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovak Republic; Fax: +42-7/373906. Key words: Vitamin C; Nephrotoxicity; Cadmium, renal changes; Ascorbic acid.
Summary The critical organ of chronic cadmium (Cd) exposure are kidneys. Long-term exposure leads to Cd accumulation in kidneys and causes renal tubular dysfunctions. Cd-induced renal changes are irreversible, so the primary prevention by different nutrients can play an important role. In this study, male guinea pigs which, like humans, do not synthetize ascorbic acid (AA) received low (2 mg/animal/day) or high (100 mg/animal/day) dosage of AA and cadmium (1 mg Cd/animal/day) in drinking water for 12 weeks. Characteristic finding in renal morphology of Cd-intoxicated guinea pigs was dilatation of interstitial veins with apparent paravenous lymphatic infiltrates. Histomorphological changes were more evident in Cd-intoxicated guinea pigs with low AA intake. High AA intake apparently reduced in Cd-intoxicated guinea pigs the extent of renal damage. Cadmium significantly increased serum creatinine and urea levels in the group of guinea pigs with low AA intake but not in guinea pigs receiving high concentration of AA. The results showed that AA can be effective in the protection of Cdinduced nephrotoxicity.
Introduction Cadmium is a typical cumulative xenobiotic with extremely long biological half-life in the liver and kidneys of humans, about 10-30 years (FRIBERG 1979). Kidneys are considered the critical organ of long-term Cd-exposure, which could be partly explained by the transport of Cd-metallothionein into the kidney (NORBERG 1989). The concentration of Cd in the blood and kidneys, Cd distribution and its binding to intracellular metallothionein take place in cadmium nephrotoxicity. FRIBERG'S et al. (1974) threshold level of Cd in the kidneys when renal lesions can be detected in man is 130 Ilg Cd/g kidney. Cd-
induced renal changes are responsible for renal tubular dysfunctions manifested by decreased reabsorptive capacity of the tubules with the consequent low molecular weight proteinouria (PISCATOR 1962). Histopathological evidence for renal toxicity indicating tubular derangement in experimental animals has been published by several authors (BONNELL 1960; STOWE 1972; KOTSONIS and KLAASSEN 1978). Since the renal changes caused by Cd exposure are irreversible, the primary prevention may play an important role. Optimal intake of different nutrients like minerals (GROTEN et al. 1991) and vitamins in the diet can favourably affect the Cd-toxicity. AA has been shown to prevent in experimental animals the signs of Cd-toxicity such as anemia, growth retardation (Fox et al. 1971) and liver damage (SUZUKI and YOSHIDA 1978). However, the data on protective effect of AA are available mostly from experiments with rats, mice or Japanese quails. We used guinea pigs which, like man, do not synthetize AA, to assess the effects of different AA intake as related to Cd-induced nephrotoxicity.
Material and methods Chemicals: Cadmium chloride, hematoxylin and eosin were purchased from Merck, Darmstadt (FRG). Ascorbic acid was obtained from Farmakon, Olomouc (Czecho-Slovakia). Other chemicals were commercial p.a. grade. Animals and treatments: Male short-hair, three-coloured guinea pigs (Velaz, Prague) weighing 350-450 g were used in the experiment. After two weeks of feeding with standard laboratory diet with addition of vegetables the animals were randomly divided into four groups. Two groups received ascorbic acid in drinking water in daily doses either 2 mg per animal (low AA group) or 100 mg per animal (high AA Exp Toxic Patho146 (1994) 1
11
Table 1. Histomorphological changes in the kidney of Cd-intoxicated guinea pigs with low and high AA intake. Lesion
lowAA
low AA+Cd
highAA
high AA+C
kidney weight (g) kidney cadmium (mg/g)
3.57 ± 0.21 1,79 ± 0.23"
3.34 ± 0.24 99,59 ± 10,39b
3.21 ± 0.13 1,10 ± 0,08 a
3.17±0.1l 85,29 ± 8,52
oa
2a O"b
3ab 3ab
oa
8b 4b 14b
oa
SC
a"
16b
a"
7c
a
3
a
2
epithelial proximal tubules vacuolization interstitium edema dilatation of veins paravenous lymphatic i nfi ltra tes lymphatic elements in interstitium
oa
The numbers in the table represent the sum of changes per group of 8 animals. a, b, c - different superscripts indicate significant difference between means in horizontal lines (P < 0.05). Amounts of cadmium in the kidneys are from KADRABOV A et al. 1992.
Fig. 1. Photomicrograph of the kidney from a guinea pig with low AA intake, chronically treated with Cd. Massive lymphocytic infiltrate surrounding dilatated veins and spreading into the tissue between tubules. Magnification: x 200, stain: Hand E. 12
Exp Toxic Pathol46 (1994) 1
Fig. 2. Photomicrograph of the kidney from a guinea pig with high AA intake, chronically treated with Cd. Dilatated vein and a slight lymphocytic infiltrate can be seen. Tubules appear to bear no trace of changes. Magnification: x 200, stain: Hand E.
Table 2. Effect of cadmium on serum parameters reflecting kidney damage in guinea pigs with low and high intake of ascorbic acid*. Groups
10wAA
low AA+Cd
high AA
high AA+Cd
Creatinine (flmol/l)
68 ± 3a
95 ±4b
75 ± 4ae
86 ± 5 be
Urea (mmol/l)
10.6 ± 0.6a
13.4 ± 1.1 b
10.3 ± 0.6a
ILl ± 0.7 ab
* Values are means ± SEM from 8 animals. a, b, c - different supersrcipts indicate significant difference between means in horizontallines (P < 0.05).
group). Two Cd-intoxicated groups were given 1 mg per animal per day of cadmium (as cadmium chloride) in drinking water (low A + Cd group; high AA + Cd group). AA-intake in these groups was the same as in the control groups. During the whole experiment the animals received drinking water and vitamin C-free diet ad libitum. The animals were decapitated after 17 -hour fasting on the 12-th week of cadmium administration. The number of guinea pigs in each group was eight. Histology: The kidneys were weighed, fixed in buffered formalin, embedded in paraffin and sections of the kidneys were prepared and stained with hematoxylin and eosin. Biochemical assays: In blood serum creatinine and urea levels were determined using commercial biochemical kits (Biola-test, Lachema, Czecho-Slovakia). Statistical analysis: The results of histopathological examinations were evaluated by the Fisher 2-sided test in table 2x2. Data in tab. 2 and kidney weight and Cd amounts in tab. 1 were statistically analyzed by variance analysis (ANOVA, Statgraphic). The minimal acceptable level of significance was P < 0.05.
Results Cadmium did not apparently change kidney weight in all groups of guinea pigs (tab. 1). Histopathology of kidneys of guinea pigs exposed to cadmium showed dilatation of veins with paravenous predominantly lymphatic infiltrates and interstitium edema as well as epithelial proximal tubule vacuolization. In the kidneys of Cd-intoxicated guinea pigs presence of lymphatic infiltrates in the interstitium was also observed. Histomorphological changes were most apparent in the group of Cd-intoxicated guinea pigs with low AA intake (fig. 1). Cd-intoxicated guinea pigs with high AA intake had evidently reduced extent of renal injury (fig. 2). AA itself showed no influence on kidney morphology (fig. 3). Consistent with his to morphological changes serum creatinine and urea levels were found to be elevated in Cd-intoxicated guinea pigs with low AA intake (by 40 % and 26 % resp.), while in Cd-intoxicated guinea pigs with high AA intake the differences were not significant (tab. 2).
Fig. 3. Photomicrograph of the kidney from a guinea pig with high AA intake. Normal picture of renal morphology. Magnification: x 200, stain: Hand E.
Discussion In the present study, subchronic Cd-treatment of guinea pigs with different AA intake led to significant histopaExp Toxic Pathol46 (1994) 1
13
thological changes in kidneys with characteristic finding of dilatation of interstitial veins and apparent paravenous lymphatic infiltrates which are typical for interstitial nephritis. High AA intake reduced significantly the extent of Cd-induced renal injury. Morphologically observed renal toxicity of Cd was also manifested by the increases in serum creatinine and urea levels which depended on AA intake. Kidneys are the critical organ of chronic Cd-exposure. Interstitial renal fibrosis has been observed in rabbits receiving 160 ppm of Cd in drinking water for 200 days (STOWE et al. 1972). In rats, exposed to aerosols containing 25 and 50 ~g Cd/m3 for 90 days, in a few kidneys signs of swelling of tubuli were observed (PRIGGE 1978). Chronic exposure of rats to Cd at concentrations of 10, 30, 100 ppm in drinking water for 24 weeks resulted in 30- and 100-ppm receiving rats in slight and focal tubular necrosis (KoTsoNIS and KLAASSEN 1978). In this study, the concentration of administered cadmium to guinea pigs was substantially lower (10 mg of Cd per liter of drinking water) and time-exposure was shorter than in the above mentioned studies with rats and rabbits. Concomitantly, the extent of renal damage was not so evident in this study. We did not observe morphological changes reported by other authors, such as renal interstitial fibrosis, tubular atrophy, tubular necrosis or nephrolithiasis in kidney (BONNELL et al. 1960; STOWE et al. 1972; KOTSONIS and KLAASSEN 1978). However, changes like interstitial nephritis which usually precede interstitial fibrosis have been seen in Cd - intoxicated guinea pigs. We used guinea pigs which, like man, are dependent on exogenous AA intake and their sensitivity to the effect of foreign compounds could be different. The long-term addition of AA to cadmium-containing diet prevented in animals signs of cadmium toxicity such as growth retardation, anemia and increase of serum enzymes reflecting liver damage (SUZUKI and YOSHIDA 1978; Fox et al. 1971). The mechanism by which AA counteracts Cd toxicity is not exactly known. AA may interfere in the absorption of Cd from the gastrointestinal tract, stimulate metallothionein synthesis (ONOSAKA et al. 1987) or as an effective antioxidant can protect-SH groups of metallothionein and other proteins against oxidation. More extensive renal injury in Cd-intoxicated guinea pigs with low AA intake could be related to the higher amount of cadmium accumulated in the kidneys ofthese animals (KADRABOVA et al. 1992). These results are potentially important for the prevention of nephrotoxic effects of cadmium in profesionally
14
Exp Toxic Patho146 (1994) 1
exposed workers, where increased AA intake could possibly reduce the toxic effects of cadmium. Acknowledgements: The authors wish to express their appreciation to Dr. S WSOLOVA for statistical analysis of the results and to Dr. M. ZOLDY for stylistic revision of the manuscript.
References I. BONNELL JA, Ross JH, KING E: Renal lesions in experimental cadmium poisoning. Brit J Industr Med 1960; 17: 69-80. 2. Fox MRS, FRY BE, JR, HARLAND BF, et al.: Effect of ascorbic acid on cadmium toxicity in the young coturnix. J of Nutr 1971; 101: 1295-1306. 3. FRIBERG L, KJELLSTROM T, NORDBERG GF, et al.: in Handbook on the Toxicology of Metals. Biomedical Press, Elsevier, North Holland, Amsterdam - New York Oxford, 1979, p. 355. 4. FRIBERG L, PISCATOR M, NORDBERG GF, et al.: Cadmium in the environment, 2nd ed. Cleveland, Ohio, CRC Press, 1974. 5. GROTEN JP, SINKELDAM EJ, Muys TH, et al.: Interaction of dietary Ca, P, Mg, Mn, Cu, Fe, Zn and Se with the accumulation and oral toxicity of cadmium in rats. Food Chern Toxicol1991; 29: 249-258. 6. KADRABOV AJ, MADARIi': A, GINTER E: The effect of ascorbic acid on cadmium accumulation in guinea pigs tissues. Experientia 1992; 48: 989-991. 7. KOTSONIS FN, KLAASSEN CD: The relationship of metallothionein to the toxicity of cadmium after prolonged oral administration to rats. Toxicol and Appl Pharmacol 1978;46: 39-54. 8. NORDBERG GF: Modulation of metal toxicity by metallothionein. BioI Trace Elem Res 1989; 21: 131-135. 9. ONOSAKA S, KAWAKAMI D, MIN K, et al.: Induced synthesis of metallothionein by ascorbic acid in mouse liver. Toxicology 1987; 43: 251-259. 10. PRIGGE E.: Early signs of oral and inhalative cadmium uptake in rats. Arch of Toxicol1978; 40: 231-247. 11. PISCATOR M: Proteinouria in chronic cadmium poisoning. Arch Environ Health 1962; 5: 55-62. 12. STOWE HD, WILSON M, GOYER RA: Clinical and morphologic effects of oral cadmium toxicity in rabbits. Arch Path 1972; 94: 389-405. 13. SUZUKI T, YOSHIDA A: Long-term effectiveness of dietary iron and ascorbic acid in the prevention and cure of cadmium toxicity in rats. Am J Clin Nutr 1978; 31: 1491-1498.