BIOMEDICAL RESPONSES OF RATS TO CHRONIC EXPOSURE TO DIETARY CADMIUM FED IN AD LIBITUM AND EQUALIZED REGIMES Robert J. Cousins, Katherine S. Squibb, Stuart L. Feldman, Anthony de Bari, Brian L. Silbon Department of Nutrition, Rutgers University—The State University of New Jersey, New Brunswick, New Jersey
Forty 100 g male rats were fed, in groups of eight, either 0, 5, or 25 ppm cadmium in a purified diet for 14 wk. Three groups were fed each of the levels of cadmium on an ad libitum basis. Two other groups were fed either 0 or 5 ppm cadmium in amounts that were equalized to that consumed by the 25 ppm group fed ad libitum. Cadmium ingestion decreased daily diet consumption, weight gain, and terminal body weight. These parameters were not significantly different in rats whose diet consumption was equalized. Packed cell volume and serum iron as well as serum zinc were decreased in the rats fed 25 ppm cadmium. These effects were not related to diet intake. No major differences were observed in serum ceruloplasmin, glucose, protein, leucine aminopeptidase activity, or copper in any of the groups. Blood urea nitrogen and renal leucine aminopeptidase activity were decreased by cadmium ingestion in the rats fed ad libitum only. In contrast, serum alkaline phosphatase activity was elevated by cadmium in the equalized-intake groups only. Cadmium and zinc concentrations were elevated and the iron concentration was decreased in the kidney, liver, and intestinal mucosa of the cadmium-fed rats irrespective of level of diet consumption. The increased uptake of cadmium in these tissues was coincident with the increased content of the cadmium-binding protein, metallothioneln, in the cytosol fraction. The results indicate that some parameters of chronic cadmium toxicity are associated with diet consumption whereas others are not.
INTRODUCTION Cadmium has been shown to be highly toxic to biological systems. In animals, signs of a toxicity may include renal damage and concomitant proteinuria, decreased serum alkaline phosphatase and renal leucine aminopeptidase activity, changes in carbohydrate metabolism, depressed immune response, osteomalacia-like decreases in bone mineralization, and numerous other changes. These have been reviewed in detail by Fribergand associates (1971, 1973, 1975). This work was supported in part by NIH grant ES 00777, awarded by the National Institute of Environmental Health Sciences. This is a Journal Series Paper, New Jersey Agricultural Experiment Station, Rutgers University. Requests for reprints should be sent to Robert J. Cousins, Department of Nutrition, Rutgers University—The State University of New Jersey, New Brunswick, New Jersey 08903.
929 Journal of Toxicology and Environmental Health, 2:929-943,1977 Copyright © 1977 by Hemisphere Publishing Corporation
930
R.J. COUSINS ETAL.
A vast majority of the toxic manifestations of cadmium have been observed when the metal is injected. This route has two major drawbacks when one is trying to evaluate the major signs of toxicity and relate these signs to the potential influence cadmium could have as an environmental pollutant. The first limitation is that it bypasses the interaction of the metal with nutrients and homeostatic mechanisms intrinsic to the gastrointestinal tract. Second, the concentrations of the doses employed are often very high and are administered over long periods of time, and therefore are of questionable relevance. In an attempt to circumvent the problem of unphysiologic doses and to approach more relevant conditions, cadmium has been provided in the diet (Fox et al., 1971; Cousins et al., 1973; Freeland and Cousins, 1973; Pond and Walker, 1975) or drinking water (Itokawa et al., 1974; Washko and Cousins, 1975; Doyle et al., 1975) in many experiments. However, in studies where food consumption was measured, a moderate to severe reduction in intake was observed (Cousins et al., 1973; Itokawa et al., 1974; Pond and Walker, 1975). A reduction in food consumption obviously limits the quantity of nutrients available for metabolic needs, and thus some of the signs of cadmium toxicity may be secondary to suboptimal nutrition. We are cognizant of this fact and have conducted the experiment reported here, where the consumption of diet in cadmium-fed rats was equalized with that in controls and the various parameters measured were compared to those in other cadmium-fed groups that were fed ad libitum, to evaluate the role of diet intake in this toxicity syndrome. METHODS Forty male Sprague-Dawley rats weighing 100-120 g were randomly divided into five groups of eight animals each. They were housed individually in hanging, stainless steel, wire-bottom cages and were exposed to 12 hr of incandescent light (continuous, starting at 7:00 A.M.) and 12 hr of darkness daily. All animals in the five groups were fed a purified diet containing 0.6% calcium (Washko and Cousins, 1975) and resindeionized H2O throughout the equalization and comparison period. The rats were fed this diet, which contained no detectable cadmium, for a 6 day equalization period. During the 14 wk comparison period that followed the equalization period, the 0.6% calcium purified diet (referred to as 0 ppm Cd), without added cadmium, was fed to two of the groups. Cadmium as CdCI2 was added, via the mineral mix, to the basal purified diet to achieve diets containing 5 and 25 mg Cd2+/kg (referred to as 5 and 25 ppm Cd, respectively). These cadmium-containing diets were fed to two and one of the groups, respectively. One group fed each level of cadmium (0, 5, and 25 ppm) was fed ad libitum. The other two groups, one fed 0 ppm
BIOCHEMICAL RESPONSES OF RATS TO CADMIUM
931
Cd and one fed 5 ppm Cd, were given an amount of diet equal to that consumed by the 25 ppm Cd group. This later group consumed the least of all three ad libitum groups. In order to accomplish this equalization of intake, each morning the amount of diet consumed by each replicate animal fed 25 ppm Cd in the previous 18 hr period was recorded. That amount of diet was then provided, for each of the corresponding replicate animals fed either the 0 or 5 ppm Cd diet, for the following 18 hr feeding period. Since the amount of diet fed to these two groups was equalized to that of the 25 ppm Cd group they are referred to as 0 ppm Cd-Eq and 5 ppm Cd-Eq, respectively. The total daily diet consumption was measured for each animal in each group. Body weights were measured biweekly. A t the end of the 14 wk comparison period, the packed cell volume (PCV) was measured using heparinized whole blood and the rats were killed by decapitation. The femur, intestinal mucosa, kidney, liver, and spleen were quickly excised, sampled as required, and frozen at —20°C. The lumen of each intestine was perfused with 50 ml of 0.1 M Tris-HCI (pH 8.6) immediately upon being excised. Mucosa was scraped from the serosal layers of the entire length of intestine with a glass slide. The tissues were analyzed for various minerals, including cadmium, copper, iron, and zinc, by atomic absorption spectrophotometry (AAS). The tissue samples (mucosa, right kidney, one lobe of liver, and total spleen) were first dried for 18 hr at 105°C, then ashed for 48 hr at 450°C in an electric furnace. The ash was dissolved in 7 N HNO 3 and diluted, as required, with deionized H 2 O. Serum samples were diluted directly with deionized H 2 O for measurement of copper, iron, and zinc concentration by AAS. Other serum constituents were analyzed by the following methods: urea nitrogen (Folin and Svedberg, 1930), protein (Lowry et al., 1951), alkaline phosphatase (Bessey et al., 1946), leucine aminopeptidase (Goldberg and Rutenberg, 1958), and glucose (Somogyi, 1945). Serum ceruloplasmin was determined spectrophotometrically by measuring oxidase activity with p-phenylenediamine as substrate (Tietz, 1970). For determination of renal leucine aminopeptidase (rLAP), a 33% homogenate of kidney was prepared with a glass-Teflon homogenizer in 10 m/W Tris-HCI (pH 8.6). The homogenate was centrifuged at 40,000 X g for 1 hr at 4°C. The rLAP activity was measured on an aliquot of the 40,000 X g supernatant that was diluted with deionized H 2 O, using a method based on that of Goldberg and Rutenberg (1958) which measures the liberation of j3-naphthylamine from L-leucyl-j3-naphthylamide. The total right femur from each rat was cleaned of adhering tissue, dried at 105°C for 18 hr, then extracted for 24 hr with a 2:1 mixture of chloroform-.methanol in a Soxhlet extractor. The bones were air-dried and ashed for 24 hr at 600°C. Soluble intestinal mucosal, liver, and kidney proteins were fractionated by gel filtration chromatography. A pooled liver and kidney sample for
932
R.J. COUSINS ETAL.
each of the five groups was prepared by combining slices of tissue from each animal to obtain a 5 g composite sample. Homogenates were prepared with a glass-Teflon homogenizer and centrifuged at 40,000 X g for 1/4 hr. The supernatant solution was recentrifuged at 166,500 X g (average) for 1 hr. The postribosomal (cytosol) fraction was applied to columns (1.6 X 40 cm) of Sephadex G-75, which were eluted as described previously to separate the soluble proteins (Cousins et al., 1973; Squibb and Cousins, 1974). Analysis of variance was used to differentiate the sources of variation. The statistical significance of multiple comparisons was determined by Duncan's multiple range test (Hicks, 1965). RESULTS Terminal body weight and rate of gain were significantly influenced by cadmium intake (Table 1) since the 0 ppm-Eq, 5 ppm-Eq, 5 ppm, and 25 ppm groups were not different, while the 0 ppm group—that is, the group fed a diet with no cadmium ad libitum—was significantly different from these groups. This is reconcilable with measurements of diet consumption. The consumptions of the rats in the equalized groups were not significantly different from each other but were significantly different from those of the ad libitum groups. Clearly the reduction of weight gain and terminal weight was secondary to the influence of cadmium on diet consumption. No health problems were noted in any of the groups, nor did any animals die during the duration of the experiment. The constituents of the serum that were measured in this experiment and the PCV values are shown in Table 2. The PCV was significantly depressed in the 25 ppm group compared to all other groups. This indicates that the role of cadmium in iron metabolism is primary and is not related to limited diet consumption. No significant differences were found for TABLE 1. Effect of Dietary Cadmium and Equalized Diet Intake on Body Weight, Daily Gain, and Average Diet Consumption Cadmium intake, ppm
5°
Item Terminal body weight, g Daily weight gain, g/day Average daily diet intake, g/day "Diet consumption ''Diet consumption c Standard error of "'^''Within a row, 0.05).
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Forty 100 g male rats were fed, in groups of eight, either 0, 5, or 25 ppm cadmium in a purified diet for 14 wk. Three groups were fed each of the levels of cadmium on an ad libitum basis. Two other groups were fed either 0 or 5 ppm cadmium in amounts that were equalized to that consumed by the 25 ppm group fed ad libitum. Cadmium ingestion decreased daily diet consumption, weight gain, and terminal body weight. These parameters were not significantly different in rats whose diet consumption was equalized. Packed cell volume and serum iron as well as serum zinc were decreased in the rats fed 25 ppm cadmium. These effects were not related to diet intake. No major differences were observed in serum ceruloplasmin, glucose, protein, leucine aminopeptidase activity, or copper in any of the groups. Blood urea nitrogen and renal leucine aminopeptidase activity were decreased by cadmium ingestion in the rats fed ad libitum only. In contrast, serum alkaline phosphatase activity was elevated by cadmium in the equalized-intake groups only. Cadmium and zinc concentrations were elevated and the iron concentration was decreased in the kidney, liver, and intestinal mucosa of the cadmium-fed rats irrespective of level of diet consumption. The increased uptake of cadmium in these tissues was coincident with the increased content of the cadmium-binding protein, metallothioneln, in the cytosol fraction. The results indicate that some parameters of chronic cadmium toxicity are associated with diet consumption whereas others are not.
INTRODUCTION Cadmium has been shown to be highly toxic to biological systems. In animals, signs of a toxicity may include renal damage and concomitant proteinuria, decreased serum alkaline phosphatase and renal leucine aminopeptidase activity, changes in carbohydrate metabolism, depressed immune response, osteomalacia-like decreases in bone mineralization, and numerous other changes. These have been reviewed in detail by Fribergand associates (1971, 1973, 1975). This work was supported in part by NIH grant ES 00777, awarded by the National Institute of Environmental Health Sciences. This is a Journal Series Paper, New Jersey Agricultural Experiment Station, Rutgers University. Requests for reprints should be sent to Robert J. Cousins, Department of Nutrition, Rutgers University—The State University of New Jersey, New Brunswick, New Jersey 08903.
929 Journal of Toxicology and Environmental Health, 2:929-943,1977 Copyright © 1977 by Hemisphere Publishing Corporation
930
R.J. COUSINS ETAL.
A vast majority of the toxic manifestations of cadmium have been observed when the metal is injected. This route has two major drawbacks when one is trying to evaluate the major signs of toxicity and relate these signs to the potential influence cadmium could have as an environmental pollutant. The first limitation is that it bypasses the interaction of the metal with nutrients and homeostatic mechanisms intrinsic to the gastrointestinal tract. Second, the concentrations of the doses employed are often very high and are administered over long periods of time, and therefore are of questionable relevance. In an attempt to circumvent the problem of unphysiologic doses and to approach more relevant conditions, cadmium has been provided in the diet (Fox et al., 1971; Cousins et al., 1973; Freeland and Cousins, 1973; Pond and Walker, 1975) or drinking water (Itokawa et al., 1974; Washko and Cousins, 1975; Doyle et al., 1975) in many experiments. However, in studies where food consumption was measured, a moderate to severe reduction in intake was observed (Cousins et al., 1973; Itokawa et al., 1974; Pond and Walker, 1975). A reduction in food consumption obviously limits the quantity of nutrients available for metabolic needs, and thus some of the signs of cadmium toxicity may be secondary to suboptimal nutrition. We are cognizant of this fact and have conducted the experiment reported here, where the consumption of diet in cadmium-fed rats was equalized with that in controls and the various parameters measured were compared to those in other cadmium-fed groups that were fed ad libitum, to evaluate the role of diet intake in this toxicity syndrome. METHODS Forty male Sprague-Dawley rats weighing 100-120 g were randomly divided into five groups of eight animals each. They were housed individually in hanging, stainless steel, wire-bottom cages and were exposed to 12 hr of incandescent light (continuous, starting at 7:00 A.M.) and 12 hr of darkness daily. All animals in the five groups were fed a purified diet containing 0.6% calcium (Washko and Cousins, 1975) and resindeionized H2O throughout the equalization and comparison period. The rats were fed this diet, which contained no detectable cadmium, for a 6 day equalization period. During the 14 wk comparison period that followed the equalization period, the 0.6% calcium purified diet (referred to as 0 ppm Cd), without added cadmium, was fed to two of the groups. Cadmium as CdCI2 was added, via the mineral mix, to the basal purified diet to achieve diets containing 5 and 25 mg Cd2+/kg (referred to as 5 and 25 ppm Cd, respectively). These cadmium-containing diets were fed to two and one of the groups, respectively. One group fed each level of cadmium (0, 5, and 25 ppm) was fed ad libitum. The other two groups, one fed 0 ppm
BIOCHEMICAL RESPONSES OF RATS TO CADMIUM
931
Cd and one fed 5 ppm Cd, were given an amount of diet equal to that consumed by the 25 ppm Cd group. This later group consumed the least of all three ad libitum groups. In order to accomplish this equalization of intake, each morning the amount of diet consumed by each replicate animal fed 25 ppm Cd in the previous 18 hr period was recorded. That amount of diet was then provided, for each of the corresponding replicate animals fed either the 0 or 5 ppm Cd diet, for the following 18 hr feeding period. Since the amount of diet fed to these two groups was equalized to that of the 25 ppm Cd group they are referred to as 0 ppm Cd-Eq and 5 ppm Cd-Eq, respectively. The total daily diet consumption was measured for each animal in each group. Body weights were measured biweekly. A t the end of the 14 wk comparison period, the packed cell volume (PCV) was measured using heparinized whole blood and the rats were killed by decapitation. The femur, intestinal mucosa, kidney, liver, and spleen were quickly excised, sampled as required, and frozen at —20°C. The lumen of each intestine was perfused with 50 ml of 0.1 M Tris-HCI (pH 8.6) immediately upon being excised. Mucosa was scraped from the serosal layers of the entire length of intestine with a glass slide. The tissues were analyzed for various minerals, including cadmium, copper, iron, and zinc, by atomic absorption spectrophotometry (AAS). The tissue samples (mucosa, right kidney, one lobe of liver, and total spleen) were first dried for 18 hr at 105°C, then ashed for 48 hr at 450°C in an electric furnace. The ash was dissolved in 7 N HNO 3 and diluted, as required, with deionized H 2 O. Serum samples were diluted directly with deionized H 2 O for measurement of copper, iron, and zinc concentration by AAS. Other serum constituents were analyzed by the following methods: urea nitrogen (Folin and Svedberg, 1930), protein (Lowry et al., 1951), alkaline phosphatase (Bessey et al., 1946), leucine aminopeptidase (Goldberg and Rutenberg, 1958), and glucose (Somogyi, 1945). Serum ceruloplasmin was determined spectrophotometrically by measuring oxidase activity with p-phenylenediamine as substrate (Tietz, 1970). For determination of renal leucine aminopeptidase (rLAP), a 33% homogenate of kidney was prepared with a glass-Teflon homogenizer in 10 m/W Tris-HCI (pH 8.6). The homogenate was centrifuged at 40,000 X g for 1 hr at 4°C. The rLAP activity was measured on an aliquot of the 40,000 X g supernatant that was diluted with deionized H 2 O, using a method based on that of Goldberg and Rutenberg (1958) which measures the liberation of j3-naphthylamine from L-leucyl-j3-naphthylamide. The total right femur from each rat was cleaned of adhering tissue, dried at 105°C for 18 hr, then extracted for 24 hr with a 2:1 mixture of chloroform-.methanol in a Soxhlet extractor. The bones were air-dried and ashed for 24 hr at 600°C. Soluble intestinal mucosal, liver, and kidney proteins were fractionated by gel filtration chromatography. A pooled liver and kidney sample for
932
R.J. COUSINS ETAL.
each of the five groups was prepared by combining slices of tissue from each animal to obtain a 5 g composite sample. Homogenates were prepared with a glass-Teflon homogenizer and centrifuged at 40,000 X g for 1/4 hr. The supernatant solution was recentrifuged at 166,500 X g (average) for 1 hr. The postribosomal (cytosol) fraction was applied to columns (1.6 X 40 cm) of Sephadex G-75, which were eluted as described previously to separate the soluble proteins (Cousins et al., 1973; Squibb and Cousins, 1974). Analysis of variance was used to differentiate the sources of variation. The statistical significance of multiple comparisons was determined by Duncan's multiple range test (Hicks, 1965). RESULTS Terminal body weight and rate of gain were significantly influenced by cadmium intake (Table 1) since the 0 ppm-Eq, 5 ppm-Eq, 5 ppm, and 25 ppm groups were not different, while the 0 ppm group—that is, the group fed a diet with no cadmium ad libitum—was significantly different from these groups. This is reconcilable with measurements of diet consumption. The consumptions of the rats in the equalized groups were not significantly different from each other but were significantly different from those of the ad libitum groups. Clearly the reduction of weight gain and terminal weight was secondary to the influence of cadmium on diet consumption. No health problems were noted in any of the groups, nor did any animals die during the duration of the experiment. The constituents of the serum that were measured in this experiment and the PCV values are shown in Table 2. The PCV was significantly depressed in the 25 ppm group compared to all other groups. This indicates that the role of cadmium in iron metabolism is primary and is not related to limited diet consumption. No significant differences were found for TABLE 1. Effect of Dietary Cadmium and Equalized Diet Intake on Body Weight, Daily Gain, and Average Diet Consumption Cadmium intake, ppm
5°
Item Terminal body weight, g Daily weight gain, g/day Average daily diet intake, g/day "Diet consumption ''Diet consumption c Standard error of "'^''Within a row, 0.05).
401^
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