- Email: [email protected]
Protection against chronic cadmium toxicity by caloric restriction
Toxicology 133 (1999) 93 – 103
Protection against chronic cadmium toxicity by caloric restriction Zahir A. Shaikh *, Scott A. Jordan, Weifeng Tang Department of Biomedical Sciences, Uni6ersity of Rhode Island, Kingston, RI 02881, USA Received 23 September 1998; accepted 8 January 1999
Abstract Exposure to cadmium (Cd) can result in nephrotoxicity and osteotoxicity. Because Cd-induced nephrotoxicity involves oxidative stress and caloric restriction decreases oxidative stress, we examined whether reduced caloric intake will protect against Cd-induced nephrotoxicity. In addition, the protection against the osteotoxicity was also examined. Male and female Sprague–Dawley rats were provided drinking water containing 100 mg Cd/l. Since fluid intake relative to the body weight was higher in females as compared to the males, the Cd concentration in their water was reduced to 80 mg/l after 3 months and 65 mg/l after 6.5 months. During the 27 month exposure period the males and females consumed a total of about 5 g Cd/kg body weight. Food was restricted to 20 g/day after the first 3 months. During the unrestricted food intake period Cd exposure reduced the bone density in females by 23%, with a partial recovery and stabilization during the caloric restriction phase. Hepatic and renal Cd accumulation and corresponding metallothionein (MT) levels were very similar in both sexes. The reported critical Cd concentration for nephrotoxicity was reached by 9 months. Renal MT levels were maximum at this time. Despite a 1.5-fold increase in renal Cd concentration over the next 18 months, there was no significant increase in renal MT levels. In spite of high renal Cd levels and lack of availability of sufficient MT, there was no sign of nephrotoxicity, as measured by urinary protein and glucose excretion. It is concluded that caloric restriction prevents Cd-induced nephrotoxicity and also appears to control the osteotoxicity of Cd. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium; Metallothionein; Chronic exposure; Caloric restriction; Bone density; Nephrotoxicity; Osteotoxicity
1. Introduction * Corresponding author. Tel.: +1-401-8745036; fax: +1401-8745048. E-mail address: [email protected] (Z.A. Shaikh)
Upon chronic cadmium (Cd) exposure the absorbed Cd is deposited primarily in the liver, where it induces and binds to metallothionein
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 1 2 - 8
94
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
(MT) (Shaikh and Lucis, 1972a,b). Over time, the hepatic CdMT is slowly released into the circulation (Shaikh and Hirayama 1979; Tohyama and Shaikh, 1981). The protein is filtered from the blood by the renal glomeruli and is subsequently reabsorbed by the renal proximal tubular cells where it is degraded (Cherian and Shaikh, 1975; Foulkes, 1978). Cd ions released from CdMT degradation bind to pre-existing renal MT and to newly synthesized MT (Cherian, 1978; Squibb and Fowler, 1984). When the amount of Cd reaching the renal cortex exceeds the binding capability of the endogenous MT, the non-MT-bound Cd is believed to cause nephrotoxicity (Nomiyama and Nomiyama, 1986; Goyer et al., 1989), presumably by generating free radicals (Hassoun and Stohs, 1996). We have shown that oxidative stress plays an important role in chronic Cd-induced nephrotoxicity (Shaikh et al., 1999a). Cd-induced nephrotoxicity is manifested by lowmolecular weight proteinuria, glucosuria, aminoaciduria, and phosphaturia (Piscator, 1986). It is generally agreed that under chronic exposure conditions the critical concentration for causing nephrotoxicity is about 200 mg Cd/g renal cortex (Nomiyama and Nomiyama, 1982; Goyer et al., 1989). In female rats exposed to 100 mg Cd/l drinking water, urinary protein and b2-microglobin excretion, and urinary lactate dehydrogenase activity increase after 8 – 10 months, indicating renal dysfunction (Bernard et al., 1992). Chronic Cd exposure has been linked to Itai – Itai disease, which involves not only renal dysfunction but also, osteomalacia and osteoporosis (Tsuchiya, 1976; Nogawa, 1981). Cd can induce osteotoxicity in animals under a variety of experimental conditions. There are several reports that conclude that osteotoxicity may occur by direct actions of Cd on bone development and mineralization and that it is independent of the renal effects of Cd (Yoshiki et al., 1975; Wang and Bhattacharyya, 1993). The biochemical mechanism of Cd-induced osteotoxicity remains unknown. Chronic studies often employ some form of caloric restriction to delay health problems associated with accelerated aging as a result of obesity (McCay et al., 1935; Weindruch et al., 1986). Age-related nephropathy is either markedly re-
duced in severity or delayed by limiting the amount of food consumed by the animals (Mitch, 1991). There are several mechanisms by which caloric restriction can influence this process. For example, it can modulate hormonal status and the related feedback responses (Abdo et al., 1991; WalbergRankin et al., 1992; Sprangers and Piacsek, 1997). It can also decrease the metabolic rate and production of waste metabolites, and can slow chemicalinduced loss of renal function (Whiting et al., 1988). Furthermore, caloric restriction increases renal glutathione level and reduces renal oxidative stress (Cadenas et al., 1994). Controlling caloric intake is currently the best modulator of oxidative stress (Yu et al., 1982; Yu, 1990). We reported recently that depletion of glutathione enhances chronic Cd-induced nephrotoxicity and co-treatment with antioxidants offers protection (Shaikh et al., 1999a). It was of interest, therefore, whether caloric restriction would have a beneficial effect on chronic Cd toxicity.
2. Materials and methods
2.1. Chemicals Ultrapure CdCl2 (99.9%) was obtained from Alfa ÆSAR (Ward Hill, MA). All other chemicals were of the highest purity commercially available from either Sigma Chemical (St Louis, MO) or Fisher Scientific (Springfield, NJ).
2.2. Animals and treatment Male and female Sprague–Dawley 5 week old rats, weighing 110–130 g, were obtained from Charles River (Wilmington, MA). The animals were housed 6/group in stainless steel cages for the first 3 months and were fed rat chow (Purina Mills, Richmond, IN) ad libitum. Thereafter, the animals were housed individually in stainless steel cages and the food was restricted to 20 g/day. The control rats were provided deionized water, while the Cd-treated animals were given deionized water containing CdCl2 at a concentration of 100 mg Cd/l. Body weight, food, and water consumption were recorded on a weekly basis. Due to differ-
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
95
ences in liquid consumption, relative to body weight, the Cd concentration in the drinking water for the females was reduced to 80 mg/l after 3 months and to 65 mg/l after 6.5 months. Urine was collected over ice in plastic metabolic cages (Nalgene, Rochester, NY) for 24 h prior to euthanizing the animals. Renal proximal tubular function was monitored by urinary protein and glucose measurements. At the beginning of the study 6 untreated males and 6 untreated females were killed by exsanguination under ether anesthesia. Additionally, at 1, 2, 3, 6, 9, 12, 15, and 18 months four groups (5 – 6 animals each), consisting of untreated and Cd-exposed males and females, were killed. The animals began to die because of old age by 21 months, and of the original 12 animals/group 8 Cd-exposed males and 7 Cd-exposed females survived at 21 months; there were no untreated controls for this time point. The survivors at 27 months, out of the original 12 animals/group, consisted of 8 control males, 5 control females, 6 Cd-exposed males, and 9 Cd-exposed females. Upon euthanasia, liver, kidneys, and femurs were removed from each animal. The liver and kidneys were saved at −85°C for analysis of Cd and MT and the femurs were processed for bone density measurement.
Prior to measuring bone density, most of the soft tissue was removed from both femurs with a scalpel and the remaining upon treatment with a 9:1 mixture of 25% KOH and glycerin for 4 days. The bones were soaked for 2 more days in deionized water to remove blood, dried at 88°C for 3 days, and weighed. Bone volume was measured in ethyl alcohol after first soaking the dried bones in alcohol for 2 days. For MT analysis, the tissues were homogenized (10%, w/v) in 125 mM borate NaOH buffer, pH 8.6, containing 0.1% Tween and 0.04% bovine serum albumin. The homogenate was centrifuged at 10 000× g for 10 min, heated at 80°C for 10 min and centrifuged again at 10 000× g for 10 min. MT was analyzed in the supernatant by a radioimmunoassay (Shaikh et al., 1999c).
2.3. Analytic methods
The control as well as Cd-treated rats were given free access to food during the first 3 months. During this time both control and Cdtreated animals grew rapidly (Fig. 1). The males gained about 84% more weight than the females. From this point on the food intake was restricted. Further weight gain during the next 3 months was minimized by the dietary restriction. Over the course of the study the males lost some weight while the females gained some weight such that at 27 months the sex differences in body weight among the control and Cd-treated animals were reduced to 10 and 13%, respectively.
Urinary protein was determined by a dye-binding method (Bradford, 1976) using bovine serum albumin as a standard. Urinary glucose was determined enzymatically using a kit from Sigma. Creatinine was measured by the method of Heinegard and Tiderstrom (1973). Cd in drinking water and in tissues was determined by flame atomic absorption spectrometry. Prior to analysis liver and kidney samples (200 mg) were digested at 100°C for 4 – 5 days with 1 ml 100 parts 70% nitric acid and 4 parts concentrated sulfuric acid. Cd concentration in the renal cortex was estimated by multiplying the Cd concentration in whole kidney by 1.54, based on the reported critical concentration of 200 mg/g renal cortex or 130 mg/g whole kidney (Nomiyama and Nomiyama, 1982; Dudley et al., 1985; Goyer et al., 1989).
2.4. Statistics Statistical analysis was performed by two-sample t-test (PB 0.05) on each set of control and Cd-exposed animals, or male and female animals.
3. Results
3.1. Effect of caloric restriction on body weight
3.2. Sex differences in water consumption The daily water consumption was the same for males and females in both the control and the Cd-treated groups (data not shown). Since the
96
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
females were smaller than the males, their water consumption/kg body weight was significantly higher than that of the males (Fig. 2A). Except at one month, when the water consumption was the same, the Cd-treated females consumed between 18 and 55% more water than the males. The smallest difference in water consumption was at 27 months. Cyclic variations in water consumption with peaks at roughly 1, 15, and 27 months corresponded with the heating season and were apparently as a result of lower humidity in the animal facility during the winter months.
3.3. Cd consumption Because of differences in water consumption, the Cd concentration in the water for females was reduced from 100 mg/l to 80 mg/l at 3 months and further reduced to 65 mg/l after 6.5 months. This adjustment in Cd concentration of the drink-
Fig. 2. Water consumption and cumulative Cd intake in Cd-exposed male and female rats. See legend under Fig. 1 for details of animal treatment and number of animals/group. *Significantly higher than the respective male rats.
ing water equalized the daily Cd intake relative to the body weight and the cumulative Cd consumption, adjusted for body weight, remained very similar in both males and females throughout the study (Fig. 2B). After 27 months the total Cd consumption was 5.239 0.18 g/kg in the males and 4.9590.17 g/kg in the females. Fig. 1. Effect of caloric restriction on body weight of control and Cd-treated rats. Control groups were provided with deionized water. For the first 3 months both Cd-treated males and females received deionized water containing 100 mg Cd/l. Thereafter, the females received 80 mg/l until 6.5 months and 65 mg/l for the remainder of the study. The animals were allowed free access to rat chow for the first 3 months and limited to 20 g/day for the rest of the study. Data plotted are mean 9 SE of 5 – 6 animals/group, except at 21 and 27 months. At 21 months the number of surviving animals was 8 Cd-exposed males and 7 females out of a total of 12 each. Similarly, at 27 months 8/12 control and 6/12 Cd-exposed males, and 5/12 control and 9/12 Cd-exposed females survived. *Significantly higher than the respective female rats (PB 0.05).
3.3.1. Hepatic Cd accumulation and MT le6els No remarkable sex-related differences in hepatic Cd accumulation were observed and the hepatic Cd levels reached a plateau at around 18 months (Fig. 3A). The average Cd concentration in males and females from this point onward was about 160 mg/g. The hepatic MT levels followed a pattern similar to that of Cd accumulation (Fig. 3B). The MT concentration leveled off at about 2.4 mg/g.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
97
3.4. Renal Cd accumulation and MT le6els Only whole kidney Cd concentration was measured in this study. The Cd concentration in the renal cortex was calculated using a multiplication factor of 1.54. The overall pattern of Cd accumulation was very similar in males and females (Fig. 4A). Cd concentration in the kidney increased almost linearly for the first 9 months of the study and reached the reported critical concentration of 130 mg/g (: 200 mg/g renal cortex) for nephrotoxicity under normal dietary conditions. The renal Cd accumulation, like the hepatic Cd accumulation, depicted saturation kinetics and leveled off at approximately 200 mg/g (: 308 mg/g renal cortex) around 18 months. In response to Cd accumulation, MT concentration in the kidneys of both males and females increased during the first 9 months of exposure to
Fig. 4. Renal Cd and MT levels in Cd-exposed male and female rats. Control groups were provided with deionized water. See legend under Fig. 1 for details of animal treatment and number of animals/group. Renal cortex Cd concentration was calculated by multiplying the Cd concentration in whole kidney by 1.54.
about 1.5 mg/g (Fig. 4B). No significant increase in MT levels occurred between 9 and 27 months, even though renal Cd levels increased 1.5-fold during this period.
Fig. 3. Hepatic Cd and MT levels in Cd-exposed male and female rats. See legend under Fig. 1 for details of animal treatment and number of animals/group.
3.4.1. Effect of caloric restriction on Cd nephrotoxicity Urinary protein and glucose levels were measured to detect Cd-induced nephrotoxicity. There were no remarkable treatment or sex-related differences in protein or glucose levels (Fig. 5). In the Cd-exposed animals the protein excretion remained within the normal range during the entire experimental period (Fig. 5A). Similarly, the glucose excretion did not increase even after the renal Cd levels exceeded the critical concentration (Fig. 5B).
98
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
3.5. Effects of caloric restriction on bone density The bone density was used as a measure of osteotoxicity. In both control and Cd-exposed males the bone density increased about 1.8-fold during the first 3 months on unrestricted diet (Fig. 6A). The only significant reduction in bone density in males was noted after 1 month of Cdexposure. In females the bone density increased during the first 3 months by 2.5-fold in controls and 1.9-fold in treated animals (Fig. 6B). At 3 months the femurs of the Cd-treated females were 23% lighter than those of the control females. During the caloric restriction phase an apparent improvement as well as curtailment of further osteotoxicity took place; the difference in bone density of
Fig. 6. Effect of cadmium exposure and caloric restriction on bone density. See legend under Fig. 1 for details of animal treatment and number of animals/group. *Significantly higher than the Cd-exposed rats.
the femurs from the untreated and Cd-exposed females averaged about 12%.
4. Discussion
Fig. 5. Effect of caloric restriction on urinary protein and glucose excretion in control and Cd-exposed rats. See legend under Fig. 1 for details of animal treatment and number of animals/group.
Except for smoking, food is the major source of environmental Cd exposure (Fleischer et al., 1974). Administration of Cd through the drinking water to animals has been used as a model for environmental Cd exposure by a number of investigators (Itokawa et al., 1978; Kotsonis and Klaassen, 1978; Bernard et al., 1981, 1992; Viau et al., 1984). In male Sprague–Dawley and Wistar rats, fed ad libitum and maintained on 100 mg Cd/l drinking water, renal proximal tubular damage was evident as early as 2–4 months after the start of Cd exposure (Itokawa et al., 1978; Kotsonis and Klaassen, 1978). Female Sprague–Daw-
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
ley rats fed ad libitum and exposed to 100 mg Cd/l drinking water, reach the critical renal Cd concentration within 12 months and show significant elevation in urinary parameters of renal dysfunction (Bernard et al., 1992). Interestingly, in an earlier study Bernard’s group (Viau et al., 1984) was unable to demonstrate any significant increase in urinary parameters in female Sprague–Dawley rats exposed to the same concentration of Cd for up to 12 months. A closer examination of the experimental protocol reveals that these investigators restricted the food intake to 12.7 g/rat/day. No rationale for the food restriction, body weight data, or a discussion of why nephrotoxicity was not observed was provided. In the present study renal dysfunction was not detected even after 27 months of maintaining the rats on 100 mg Cd/l drinking water. The lack of renal dysfunction in our study could only be ascribed to caloric restriction. This conclusion is supported by the results reported by Viau et al. (1984). It should be pointed out, however, that the hepatic and renal Cd concentrations in our study were similar to those achieved by Viau et al. (1984) as well as Bernard et al. (1992), and lack of nephrotoxicity in calory-restricted animals was not as a result of lower Cd burden. Sex differences in Cd distribution upon chronic Cd administration have been reported previously. Hepatic Cd concentration after 45 weeks of feeding rats a diet containing 100 mg Cd/kg was 2-fold higher in females than in males (Stonard and Webb, 1976). Similarly, the renal Cd concentration was 2-fold higher in females than in males after exposure to 4 – 17 mg Cd/l drinking water for 10 months (Murthy et al., 1978; Petering et al., 1979). Stonard and Webb (1976) also reported that, when adjusted for body weight, food intake in female rats was about 15% higher than in males. In our study adult female rats were provided the same quantity of food as the males and drank the same amount of water despite weighing less than the age-matched male rats. This means that when female rats are provided unlimited access to Cdcontaining food or drinking water, they consume
99
significantly more Cd/kg body weight than the males and can thus attain a higher body burden than males. In the present study we minimized the sex differences in the amount of Cd ingested/ day by adjusting the concentration of Cd in the drinking water for the females according to their body weights relative to the males. Since the males and females consumed similar total amounts of Cd/kg body weight, their tissue Cd levels were also very similar. While we did not examine hepatic toxicity in this study, it is known that caloric restriction broadly affects hormonal status (Abdo et al., 1991) and protects against chemical-induced hepatic damage (Ramaiah et al., 1998). Caloric restriction could offer protection by decreasing serum progesterone (Holehan and Merry, 1985) and by increasing serum testosterone levels (Snyder et al., 1988), both of which depress Cdinduced hepatotoxicity (Shiraishi et al., 1994; Shimada et al., 1997a,b). Nomiyama and Nomiyama (1993) reported that amelioration of Cd-induced hepatic damage alleviates nephrotoxicity, possibly by depressing CdMT release from the liver. Cd is translocated from the liver to the kidneys as CdMT. Renal damage depends on the rate of transfer of CdMT to the kidneys (Vestergaard and Shaikh, 1994). When the renal Cd reaches the critical concentration, it is hypothesized that the upper limit of MT synthesis is reached and that the non-MT-bound Cd is responsible for causing nephrotoxicity (Nomiyama and Nomiyama, 1986; Goyer et al., 1989). In the present study, the renal cortex Cd concentration reached the critical concentration after 9 months of exposure, and the maximum MT levels were also observed at this time. With continued exposure to Cd, the renal Cd concentration increased, but renal MT levels did not. This indicates that even in the calory-restricted animals the maximum MT synthesizing ability in the kidney is reached when the renal Cd concentration reaches the critical concentration and that excess Cd is free to interact with other ligands. Based on the theory that non-MT-bound Cd is responsible for Cd-induced nephrotoxicity
100
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
(Nomiyama and Nomiyama, 1986; Goyer et al., 1989), severe nephrotoxicity should have been evident in diet-restricted rats but was not observed. The beneficial effects of caloric restrictions on the susceptibility to cancer and to age-related degenerative diseases have been well documented (McCay et al., 1935; Yu et al., 1982; Kim et al., 1996). Caloric restrictions can modulate age-related free radical production (Yu et al., 1982; Kim et al., 1996; Sohal and Weindruch, 1996). Chronic Cd exposure stimulates lipid peroxidation in the liver and kidney, and antioxidants such as Nacetyl cysteine, vitamin E, and glycine depress Cd-induced nephrotoxicity, indicating that oxidative stress plays a critical role in chronic Cd toxicity (Shaikh and Tang, 1999; Shaikh et al., 1999a,b). Caloric restriction may depress the onset of Cd-induced renal damage by increasing renal GSH levels (Cadenas et al., 1994), suppressing the production of reactive oxygen species, increasing catalase and GSH-Px activities, and by maintaining cytosolic ascorbic acid levels (Kim et al., 1996). Besides renal hypertrophy and degenerative changes in tubules, decreased femur weight, narrowing of cortical osseous tissue, and decrease in osteocytes have been reported by Itokawa et al. (1978) in male Wistar rats fed ad libitum a calcium-deficient diet and drinking water containing 50 mg Cd/l for 4 months. Cd-induced bone loss can occur in mice within 72 h of exposure to as little as 25 mg Cd/l drinking water (Bhattacharyya et al., 1988; Wang and Bhattacharyya, 1993). In another study, weanling rats given 10 – 300 mg Cd/kg diet for a period of 3 weeks showed retardation of bone growth and histological lesions resembling osteoporosis, with no associated renal effects (Yoshiki et al., 1975). Both groups of investigators concluded that Cd had a direct effect on bone formation. In the present study bone density was measured to assess Cd-induced bone loss. A significant decrease in bone density occurred in females within 3 months of Cd exposure under normal dietary conditions. Since no renal dysfunction was present, these results suggest that Cd-induced osteotoxicity can indeed occur by direct effect of Cd on bone metabolism. Once the
female rats were placed on reduced-calory diet, the difference in bone density between the control and treated animals was lowered but persisted for the duration of the study. It appears that caloric restriction prevented further deterioration of bone mineralization. Calcium metabolism is regulated by vitamin D (Locker, 1996). In liver, vitamin D is converted to 25-hydroxyvitamin D, which is then metabolized to its active metabolite, 1, 25-dihydroxyvitamin D in renal proximal tubular epithelial cells (Akiba et al., 1980). Tsuritani et al. (1992) found a significant correlation between serum 1, 25-dihydroxyvitamin D level and Cd-induced renal dysfunction in women, suggesting that Cd exposure interferes with vitamin D metabolism in the kidney and contributes to the Cd-induced bone loss. Caloric restriction may protect against Cd-induced bone loss by preventing the age-related progressive decrease in serum 25-hydroxyvitamin D (Kalu et al., 1988). It may also protect by increasing renal GSH concentration (Cadenas et al., 1994), which, together with MT, can sequester excess Cd and thereby reduce the interference of Cd with vitamin D metabolism in the kidney. Epidemiological studies in a Cd-exposed Japanese population estimated that exceeding a maximum total life time dietary intake of about 2 g Cd could result in Cd-induced renal dysfunction (Nogawa et al., 1989, 1992; Kido et al., 1991, 1993; Hochi et al., 1995). The rats in the present study consumed a total of about 5 g Cd/kg during the 27 month period and yet developed no renal dysfunction. It seems possible that the life time total Cd intake limit may be exceeded in individuals whose caloric intake is limited.
5. Conclusion Caloric restriction is beneficial against chronic Cd toxicity as it prevents nephrotoxicity and also appears to control osteotoxicity.
Acknowledgements This work was supported by USPHS grant no.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
ES 03187 from the National Institute of Environmental Health Sciences. Raymond Walsh, Jr., Jane Northup, and Frank Pagliusi provided technical assistance, and Mary Leatham assisted in manuscript preparation.
References Abdo, K.M., Irwin, R., Johnson, J., 1991. Influence of diet restriction on toxicity and hormonal status in chemically treated mice. In: Fishbein, L. (Ed.), Biological Effect of Dietary Restriction. Springer-Verlag, New York, pp. 99– 111. Akiba, T., Endou, H., Koseki, C., Sakai, F., Horiuchi, N., Suda, T., 1980. Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney. Biochem. Biophys. Res. Commun. 94, 313–318. Bernard, A., Lauwerys, R., Gengoux, P., 1981. Characterization of the proteinuria induced by prolonged oral administration of cadmium in female rats. Toxicology 20, 345 – 357. Bernard, A., Lauwerys, R., Amor, A.O., 1992. Loss of glomerular polyanion correlated with albuminuria in experimental cadmium nephropathy. Arch. Toxicol. 66, 272– 278. Bhattacharyya, M.H., Whelton, B.D., Peterson, D.P., et al., 1988. Skeletal changes in multiparous mice fed a nutrientsufficient diet containing cadmium. Toxicology 50, 193– 204. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Cadenas, S., Rojas, C., Perez-Campo, R., Lopez-Torres, M., Barja, G., 1994. Caloric and carbohydrate restriction in the kidney: effects on free radical metabolism. Exp. Gerontol. 29, 77 – 88. Cherian, M.G., 1978. Induction of renal metallothionein synthesis by parenteral cadmium-thionein in rats. Biochem. Pharmacol. 27, 1163 –1166. Cherian, M.G., Shaikh, Z.A., 1975. Metabolism of intravenously injected cadmium-binding protein. Biochem. Biophys. Res. Commun. 65, 863–869. Dudley, R.E., Gammal, L.M., Klaassen, C.D., 1985. Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77, 414–426. Fleischer, M., Sarofim, A.F., Fassett, D.W., et al., 1974. Environmental impact of cadmium: a review by the Panel on Hazardous Trace Substances. Environ. Health Perspect. 7, 253 – 323. Foulkes, E.C., 1978. Renal tubular transport of cadmiummetallothionein. Toxicol. Appl. Pharmacol. 45, 505–512.
101
Goyer, R.A., Miller, C.R., Zhu, S.Y., Victery, W., 1989. Non-metallothionein-bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat. Toxicol. Appl. Pharmacol. 101, 232 – 244. Hassoun, E.A., Stohs, S.J., 1996. Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures. Toxicology 112, 219 – 226. Heinegard, D., Tiderstrom, G., 1973. Determination of serum creatinine by a direct colorimetric method. Clin. Chim. Acta. 43, 305 – 310. Hochi, Y., Kido, T., Nogawa, K., Kito, H., Shaikh, Z.A., 1995. Dose-response relationship between total cadmium intake and prevalence of renal dysfunction using general linear models. J. Appl. Toxicol. 15, 109 – 116. Holehan, A.M., Merry, B.J., 1985. The control of puberty in the dietary restricted female rat. Mech. Aging Dev. 32, 179 – 191. Itokawa, Y., Nishino, K., Takashima, M., et al., 1978. Renal and skeletal lesions in experimental cadmium poisoning of rats: histology and renal function. Environ. Res. 15, 206 – 217. Kalu, D.N., Masaro, E.J., Yu, B.P., Hardin, R.R., Hollis, B.W., 1988. Modulation of age-related hyperparathyroidism and senile bone loss in Fischer rats by soy protein and food restriction. Endocrinol. 122, 1847 – 1854. Kido, T., Shaikh, Z.A., Kito, H., Honda, R., Nogawa, K., 1991. Dose-response relationship between dietary cadmium intake and metallothioneinuria in a population from a cadmium-polluted area of Japan. Toxicology 66, 271 – 278. Kido, T., Shaikh, Z.A., Kito, H., Honda, R, Nogawa, K., 1993. Dose-response relationship between total cadmium intake and metallothioneinuria using logistic regression analysis. Toxicology 80, 207 – 215. Kim, J.D., McCarter, R.J., Yu, B.P., 1996. Influence of age, exercise, and dietary restriction on oxidative stress in rats. Aging (Milano) 8, 123 – 129. Kotsonis, F., Klaassen, C.D., 1978. The relationship of metallothionein to the toxicity of cadmium after oral administration to rats. Toxicol. Appl. Pharmacol. 46, 39 – 54. Locker, F.G., 1996. Hormonal regulation of calcium homeostasis. Nurs. Clin. North. Am. 31, 797 – 803. McCay, C.M., Cromwell, M.E., Maynard, L.A., 1935. The effect of retarded growth upon the length of life span and ultimate size. J. Nutr. 10, 63 – 79. Mitch, W.E., 1991. Dietary manipulation and progression of chronic renal failure. Child Nephrol. Urol. 11, 134 – 139. Murthy, L., Rice, D.P., Petering, H.G., 1978. Sex differences with respect to the accumulation of oral cadmium in rats. In: Kirchgessner, M. (Ed.), Trace Element Metabolism in Man and Animals 3. ATW, Weihenstephan, pp. 557 – 560. Nogawa, K., 1981. Itai-Itai disease and follow-up studies. In: Nriagu, J.O. (Ed.), Cadmium in the Environment. Wiley, New York, pp. 1 – 38.
102
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
Nogawa, K., Honda, R., Kido, T., Tsuritani, I., Yamada, Y., Ishizaki, M., Yamaya, H., 1989. A dose-response analysis of cadmium in the general environment with special reference to total cadmium intake limit. Environ. Res. 48, 7–16. Nogawa, K., Kido, T., Shaikh, Z.A., 1992. Dose-response relationship for renal dysfunction in a population environmentally exposed to cadmium. IARC Sci. Publ. 118, 311– 318. Nomiyama, K., Nomiyama, H., 1982. Tissue metallothioneins in rabbits chronically exposed to cadmium, with special reference to the critical concentration of cadmium in the renal cortex. Dev. Toxicol. Environ. Sci. 9, 47–67. Nomiyama, K., Nomiyama, H., 1986. Critical concentration of ’unbound’ cadmium in the rabbit renal cortex. Experientia 42, 149. Nomiyama, K., Nomiyama, H., 1993. Cadmium-induced renal dysfunction was improved by treating hepatic injury with glycyrrhizin. J. Trace Elem. Exp. Med. 6, 171–178. Petering, H.G., Murthy, L., Sorenson, J.R., Levin, L., Stemmer, K.L., 1979. Effect of sex on oral cadmium dose responses in rats: blood pressure and pharmacodynamics. Environ. Res. 20, 289–299. Piscator, M., 1986. The nephropathy of chronic cadmium poisoning. In: Foulkes, E.C. (Ed.), Handbook of Experimental Pharmacology. Springer-Verlag, New York, pp. 180 – 194. Ramaiah, S.K., Soni, M.G., Bucci, T.J., Mehendale, H.M., 1998. Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide. Toxicol. Appl. Pharmacol. 150, 12–21. Shaikh, Z.A., Hirayama, K., 1979. Metallothionein in the extracellular fluids as an index of cadmium toxicity. Environ. Health Perspect. 28, 267–271. Shaikh, Z.A., Lucis, O.J., 1972a. Cadmium and zinc binding in mammalian liver and kidneys. Arch. Environ. Health 24, 419 – 425. Shaikh, Z.A., Lucis, O.J., 1972b. Biological differences in cadmium and zinc turnover. Arch. Environ. Health 24, 410 – 418. Shaikh, Z.A., Tang, W., 1999. Protection against chronic cadmium toxicity by glycine. Toxicology 132, 139–146. Shaikh, Z.A., Vu, T., Zaman, K., 1999a. Oxidative stress as a mechanism of cadmium-induced hepatotoxicity and nephrotoxicity and prevention by antioxidants. Toxicol. Appl. Pharmacol. 154, 256–263. Shaikh, Z.A., Zaman, K., Tang, W., Vu, T., 1999b. Treatment of chronic cadmium nephrotoxicity by N-acetyl cysteine. Toxicol. Lett. 104, 137–142. Shaikh, Z.A., Jordan, S.A., Kido, T., Tang, W., Gross, W.A., Sabbioni, E., 1999c. A sensitive, high capacity radioimmunoassay for metallothionein and its application to urine samples from cadmium-treated rats. Toxicol. Meth. (in press). Shimada, H., Bare, R.M., Hochadel, J.F., Waalkes, M.P., 1997a. Testosterone pretreatment mitigates cadmium toxic-
ity in male C57 mice but not in C3H mice. Toxicology 116, 183 – 191. Shimada, H., Hochadel, J.F., Waalkes, M.P., 1997b. Progesterone pretreatment enhances cellular sensitivity to cadmium despite a marked activation of the metallothionein gene. Toxicol. Appl. Pharmacol. 142, 178 – 185. Shiraishi, N., Barter, R.A., Uno, H., Waalkes, M.P., 1994. Effect of progesterone pretreatment on cadmium toxicity in male Fischer (F344/NCr) and Wistar (WF/NCr) rats. Environ. Health Perspect. 102, 277 – 280. Snyder, D.L., Wostmann, B.S., Pollard, M., 1988. Serum hormones in diet-restricted gnotobiotic and conventional Lobund – Wistar rats. J. Gerontol. 43, B168 – B173. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59 – 63. Sprangers, S.A., Piacsek, B.E., 1997. Chronic underfeeding increases the positive feedback efficacy of estrogen on gonadotropin secretion. Proc. Soc. Exp. Biol. Med. 216, 398 – 403. Squibb, K.S., Fowler, B.A., 1984. Intracellular metabolism and effects of circulating cadmium-metallothionein in the kidney. Environ. Health Perspect. 54, 31 – 35. Stonard, M.D., Webb, M., 1976. Influence of dietary cadmium on the distribution of the essential metals copper, zinc and iron in tissues of the rat. Chem. Biol. Interact. 15, 349 – 363. Tohyama, C., Shaikh, Z.A., 1981. Metallothionein in plasma and urine of cadmium-exposed rats determined by a singleantibody radioimmunoassay. Fundam. Appl. Toxicol. 1, 1 – 7. Tsuchiya, K., 1976. Epidemiological studies on cadmium in the environment in Japan: etiology of Itai – Itai disease. Fed. Proc. 35, 2412 – 2418. Tsuritani, I., Honda, R., Ishizaki, M., Yamada, Y., Kido, T., Nogawa, K., 1992. Impairment of vitamin D metabolism due to environmental cadmium exposure, and possible relevance to sex-related differences in vulnerability to the bone damage. J. Toxicol. Environ. Health 37, 519 – 533. Vestergaard, P., Shaikh, Z.A., 1994. The nephrotoxicity of intravenously administered cadmium-metallothionein: effect of dose, mode of administration, and pre-existing renal cadmium burden. Toxicol. Appl. Pharmacol. 16, 240 – 247. Viau, C., Bernard, A., Lauwerys, R., Maldague, P., 1984. Cadmium, analgesics, and the chronic progressive nephrosis in the female Sprague – Dawley rat. Arch. Toxicol. 55, 247 – 249. Walberg-Rankin, J., Franke, W.D., Gwazdauskas, F.C., 1992. Response of beta-endorphin and estradiol to resistance exercises in females during energy balance and energy restriction. Int. J. Sports Med. 13, 542 – 547. Wang, C., Bhattacharyya, M.H., 1993. Effect of cadmium on bone calcium and 45Ca in non-pregnant mice on a calciumdeficient diet: evidence of direct effect of cadmium on bone. Toxicol. Appl. Pharmacol. 120, 228 – 239. Whiting, P.H., Power, D.A., Petersen, J., Innes, A., Simpson, J.G., Catto, G.R., 1988. The effect of dietary protein restriction on high dose gentamicin nephrotoxicity in rats. Br. J. Exp. Pathol. 69, 35 – 41.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103 Weindruch, R., Walford, R.L., Fligiel, S., Guthrie, D., 1986. The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641 – 654. Yoshiki, S., Yanagisawa, T., Kimura, M., Otaki, N., Suzuki, M., Suda, T., 1975. Bone and kidney lesions in experimental cadmium intoxication. Arch. Environ. Health 30, 559– 562.
.
103
Yu, B.P., 1990. Food restriction research: past and present status. In: Rothstein, M. (Ed.), Review of Biological Research in Aging, vol. 25. Elsevier, Shannon, pp. 337 – 351. Yu, B.P., Masoro, E.J., Murata, I., Bertrand, H.A., Lynd, F.T., 1982. Life span study of SPF Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J. Gerontol. 37, 130 – 141.
Protection against chronic cadmium toxicity by caloric restriction Zahir A. Shaikh *, Scott A. Jordan, Weifeng Tang Department of Biomedical Sciences, Uni6ersity of Rhode Island, Kingston, RI 02881, USA Received 23 September 1998; accepted 8 January 1999
Abstract Exposure to cadmium (Cd) can result in nephrotoxicity and osteotoxicity. Because Cd-induced nephrotoxicity involves oxidative stress and caloric restriction decreases oxidative stress, we examined whether reduced caloric intake will protect against Cd-induced nephrotoxicity. In addition, the protection against the osteotoxicity was also examined. Male and female Sprague–Dawley rats were provided drinking water containing 100 mg Cd/l. Since fluid intake relative to the body weight was higher in females as compared to the males, the Cd concentration in their water was reduced to 80 mg/l after 3 months and 65 mg/l after 6.5 months. During the 27 month exposure period the males and females consumed a total of about 5 g Cd/kg body weight. Food was restricted to 20 g/day after the first 3 months. During the unrestricted food intake period Cd exposure reduced the bone density in females by 23%, with a partial recovery and stabilization during the caloric restriction phase. Hepatic and renal Cd accumulation and corresponding metallothionein (MT) levels were very similar in both sexes. The reported critical Cd concentration for nephrotoxicity was reached by 9 months. Renal MT levels were maximum at this time. Despite a 1.5-fold increase in renal Cd concentration over the next 18 months, there was no significant increase in renal MT levels. In spite of high renal Cd levels and lack of availability of sufficient MT, there was no sign of nephrotoxicity, as measured by urinary protein and glucose excretion. It is concluded that caloric restriction prevents Cd-induced nephrotoxicity and also appears to control the osteotoxicity of Cd. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium; Metallothionein; Chronic exposure; Caloric restriction; Bone density; Nephrotoxicity; Osteotoxicity
1. Introduction * Corresponding author. Tel.: +1-401-8745036; fax: +1401-8745048. E-mail address: [email protected] (Z.A. Shaikh)
Upon chronic cadmium (Cd) exposure the absorbed Cd is deposited primarily in the liver, where it induces and binds to metallothionein
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 1 2 - 8
94
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
(MT) (Shaikh and Lucis, 1972a,b). Over time, the hepatic CdMT is slowly released into the circulation (Shaikh and Hirayama 1979; Tohyama and Shaikh, 1981). The protein is filtered from the blood by the renal glomeruli and is subsequently reabsorbed by the renal proximal tubular cells where it is degraded (Cherian and Shaikh, 1975; Foulkes, 1978). Cd ions released from CdMT degradation bind to pre-existing renal MT and to newly synthesized MT (Cherian, 1978; Squibb and Fowler, 1984). When the amount of Cd reaching the renal cortex exceeds the binding capability of the endogenous MT, the non-MT-bound Cd is believed to cause nephrotoxicity (Nomiyama and Nomiyama, 1986; Goyer et al., 1989), presumably by generating free radicals (Hassoun and Stohs, 1996). We have shown that oxidative stress plays an important role in chronic Cd-induced nephrotoxicity (Shaikh et al., 1999a). Cd-induced nephrotoxicity is manifested by lowmolecular weight proteinuria, glucosuria, aminoaciduria, and phosphaturia (Piscator, 1986). It is generally agreed that under chronic exposure conditions the critical concentration for causing nephrotoxicity is about 200 mg Cd/g renal cortex (Nomiyama and Nomiyama, 1982; Goyer et al., 1989). In female rats exposed to 100 mg Cd/l drinking water, urinary protein and b2-microglobin excretion, and urinary lactate dehydrogenase activity increase after 8 – 10 months, indicating renal dysfunction (Bernard et al., 1992). Chronic Cd exposure has been linked to Itai – Itai disease, which involves not only renal dysfunction but also, osteomalacia and osteoporosis (Tsuchiya, 1976; Nogawa, 1981). Cd can induce osteotoxicity in animals under a variety of experimental conditions. There are several reports that conclude that osteotoxicity may occur by direct actions of Cd on bone development and mineralization and that it is independent of the renal effects of Cd (Yoshiki et al., 1975; Wang and Bhattacharyya, 1993). The biochemical mechanism of Cd-induced osteotoxicity remains unknown. Chronic studies often employ some form of caloric restriction to delay health problems associated with accelerated aging as a result of obesity (McCay et al., 1935; Weindruch et al., 1986). Age-related nephropathy is either markedly re-
duced in severity or delayed by limiting the amount of food consumed by the animals (Mitch, 1991). There are several mechanisms by which caloric restriction can influence this process. For example, it can modulate hormonal status and the related feedback responses (Abdo et al., 1991; WalbergRankin et al., 1992; Sprangers and Piacsek, 1997). It can also decrease the metabolic rate and production of waste metabolites, and can slow chemicalinduced loss of renal function (Whiting et al., 1988). Furthermore, caloric restriction increases renal glutathione level and reduces renal oxidative stress (Cadenas et al., 1994). Controlling caloric intake is currently the best modulator of oxidative stress (Yu et al., 1982; Yu, 1990). We reported recently that depletion of glutathione enhances chronic Cd-induced nephrotoxicity and co-treatment with antioxidants offers protection (Shaikh et al., 1999a). It was of interest, therefore, whether caloric restriction would have a beneficial effect on chronic Cd toxicity.
2. Materials and methods
2.1. Chemicals Ultrapure CdCl2 (99.9%) was obtained from Alfa ÆSAR (Ward Hill, MA). All other chemicals were of the highest purity commercially available from either Sigma Chemical (St Louis, MO) or Fisher Scientific (Springfield, NJ).
2.2. Animals and treatment Male and female Sprague–Dawley 5 week old rats, weighing 110–130 g, were obtained from Charles River (Wilmington, MA). The animals were housed 6/group in stainless steel cages for the first 3 months and were fed rat chow (Purina Mills, Richmond, IN) ad libitum. Thereafter, the animals were housed individually in stainless steel cages and the food was restricted to 20 g/day. The control rats were provided deionized water, while the Cd-treated animals were given deionized water containing CdCl2 at a concentration of 100 mg Cd/l. Body weight, food, and water consumption were recorded on a weekly basis. Due to differ-
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
95
ences in liquid consumption, relative to body weight, the Cd concentration in the drinking water for the females was reduced to 80 mg/l after 3 months and to 65 mg/l after 6.5 months. Urine was collected over ice in plastic metabolic cages (Nalgene, Rochester, NY) for 24 h prior to euthanizing the animals. Renal proximal tubular function was monitored by urinary protein and glucose measurements. At the beginning of the study 6 untreated males and 6 untreated females were killed by exsanguination under ether anesthesia. Additionally, at 1, 2, 3, 6, 9, 12, 15, and 18 months four groups (5 – 6 animals each), consisting of untreated and Cd-exposed males and females, were killed. The animals began to die because of old age by 21 months, and of the original 12 animals/group 8 Cd-exposed males and 7 Cd-exposed females survived at 21 months; there were no untreated controls for this time point. The survivors at 27 months, out of the original 12 animals/group, consisted of 8 control males, 5 control females, 6 Cd-exposed males, and 9 Cd-exposed females. Upon euthanasia, liver, kidneys, and femurs were removed from each animal. The liver and kidneys were saved at −85°C for analysis of Cd and MT and the femurs were processed for bone density measurement.
Prior to measuring bone density, most of the soft tissue was removed from both femurs with a scalpel and the remaining upon treatment with a 9:1 mixture of 25% KOH and glycerin for 4 days. The bones were soaked for 2 more days in deionized water to remove blood, dried at 88°C for 3 days, and weighed. Bone volume was measured in ethyl alcohol after first soaking the dried bones in alcohol for 2 days. For MT analysis, the tissues were homogenized (10%, w/v) in 125 mM borate NaOH buffer, pH 8.6, containing 0.1% Tween and 0.04% bovine serum albumin. The homogenate was centrifuged at 10 000× g for 10 min, heated at 80°C for 10 min and centrifuged again at 10 000× g for 10 min. MT was analyzed in the supernatant by a radioimmunoassay (Shaikh et al., 1999c).
2.3. Analytic methods
The control as well as Cd-treated rats were given free access to food during the first 3 months. During this time both control and Cdtreated animals grew rapidly (Fig. 1). The males gained about 84% more weight than the females. From this point on the food intake was restricted. Further weight gain during the next 3 months was minimized by the dietary restriction. Over the course of the study the males lost some weight while the females gained some weight such that at 27 months the sex differences in body weight among the control and Cd-treated animals were reduced to 10 and 13%, respectively.
Urinary protein was determined by a dye-binding method (Bradford, 1976) using bovine serum albumin as a standard. Urinary glucose was determined enzymatically using a kit from Sigma. Creatinine was measured by the method of Heinegard and Tiderstrom (1973). Cd in drinking water and in tissues was determined by flame atomic absorption spectrometry. Prior to analysis liver and kidney samples (200 mg) were digested at 100°C for 4 – 5 days with 1 ml 100 parts 70% nitric acid and 4 parts concentrated sulfuric acid. Cd concentration in the renal cortex was estimated by multiplying the Cd concentration in whole kidney by 1.54, based on the reported critical concentration of 200 mg/g renal cortex or 130 mg/g whole kidney (Nomiyama and Nomiyama, 1982; Dudley et al., 1985; Goyer et al., 1989).
2.4. Statistics Statistical analysis was performed by two-sample t-test (PB 0.05) on each set of control and Cd-exposed animals, or male and female animals.
3. Results
3.1. Effect of caloric restriction on body weight
3.2. Sex differences in water consumption The daily water consumption was the same for males and females in both the control and the Cd-treated groups (data not shown). Since the
96
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
females were smaller than the males, their water consumption/kg body weight was significantly higher than that of the males (Fig. 2A). Except at one month, when the water consumption was the same, the Cd-treated females consumed between 18 and 55% more water than the males. The smallest difference in water consumption was at 27 months. Cyclic variations in water consumption with peaks at roughly 1, 15, and 27 months corresponded with the heating season and were apparently as a result of lower humidity in the animal facility during the winter months.
3.3. Cd consumption Because of differences in water consumption, the Cd concentration in the water for females was reduced from 100 mg/l to 80 mg/l at 3 months and further reduced to 65 mg/l after 6.5 months. This adjustment in Cd concentration of the drink-
Fig. 2. Water consumption and cumulative Cd intake in Cd-exposed male and female rats. See legend under Fig. 1 for details of animal treatment and number of animals/group. *Significantly higher than the respective male rats.
ing water equalized the daily Cd intake relative to the body weight and the cumulative Cd consumption, adjusted for body weight, remained very similar in both males and females throughout the study (Fig. 2B). After 27 months the total Cd consumption was 5.239 0.18 g/kg in the males and 4.9590.17 g/kg in the females. Fig. 1. Effect of caloric restriction on body weight of control and Cd-treated rats. Control groups were provided with deionized water. For the first 3 months both Cd-treated males and females received deionized water containing 100 mg Cd/l. Thereafter, the females received 80 mg/l until 6.5 months and 65 mg/l for the remainder of the study. The animals were allowed free access to rat chow for the first 3 months and limited to 20 g/day for the rest of the study. Data plotted are mean 9 SE of 5 – 6 animals/group, except at 21 and 27 months. At 21 months the number of surviving animals was 8 Cd-exposed males and 7 females out of a total of 12 each. Similarly, at 27 months 8/12 control and 6/12 Cd-exposed males, and 5/12 control and 9/12 Cd-exposed females survived. *Significantly higher than the respective female rats (PB 0.05).
3.3.1. Hepatic Cd accumulation and MT le6els No remarkable sex-related differences in hepatic Cd accumulation were observed and the hepatic Cd levels reached a plateau at around 18 months (Fig. 3A). The average Cd concentration in males and females from this point onward was about 160 mg/g. The hepatic MT levels followed a pattern similar to that of Cd accumulation (Fig. 3B). The MT concentration leveled off at about 2.4 mg/g.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
97
3.4. Renal Cd accumulation and MT le6els Only whole kidney Cd concentration was measured in this study. The Cd concentration in the renal cortex was calculated using a multiplication factor of 1.54. The overall pattern of Cd accumulation was very similar in males and females (Fig. 4A). Cd concentration in the kidney increased almost linearly for the first 9 months of the study and reached the reported critical concentration of 130 mg/g (: 200 mg/g renal cortex) for nephrotoxicity under normal dietary conditions. The renal Cd accumulation, like the hepatic Cd accumulation, depicted saturation kinetics and leveled off at approximately 200 mg/g (: 308 mg/g renal cortex) around 18 months. In response to Cd accumulation, MT concentration in the kidneys of both males and females increased during the first 9 months of exposure to
Fig. 4. Renal Cd and MT levels in Cd-exposed male and female rats. Control groups were provided with deionized water. See legend under Fig. 1 for details of animal treatment and number of animals/group. Renal cortex Cd concentration was calculated by multiplying the Cd concentration in whole kidney by 1.54.
about 1.5 mg/g (Fig. 4B). No significant increase in MT levels occurred between 9 and 27 months, even though renal Cd levels increased 1.5-fold during this period.
Fig. 3. Hepatic Cd and MT levels in Cd-exposed male and female rats. See legend under Fig. 1 for details of animal treatment and number of animals/group.
3.4.1. Effect of caloric restriction on Cd nephrotoxicity Urinary protein and glucose levels were measured to detect Cd-induced nephrotoxicity. There were no remarkable treatment or sex-related differences in protein or glucose levels (Fig. 5). In the Cd-exposed animals the protein excretion remained within the normal range during the entire experimental period (Fig. 5A). Similarly, the glucose excretion did not increase even after the renal Cd levels exceeded the critical concentration (Fig. 5B).
98
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
3.5. Effects of caloric restriction on bone density The bone density was used as a measure of osteotoxicity. In both control and Cd-exposed males the bone density increased about 1.8-fold during the first 3 months on unrestricted diet (Fig. 6A). The only significant reduction in bone density in males was noted after 1 month of Cdexposure. In females the bone density increased during the first 3 months by 2.5-fold in controls and 1.9-fold in treated animals (Fig. 6B). At 3 months the femurs of the Cd-treated females were 23% lighter than those of the control females. During the caloric restriction phase an apparent improvement as well as curtailment of further osteotoxicity took place; the difference in bone density of
Fig. 6. Effect of cadmium exposure and caloric restriction on bone density. See legend under Fig. 1 for details of animal treatment and number of animals/group. *Significantly higher than the Cd-exposed rats.
the femurs from the untreated and Cd-exposed females averaged about 12%.
4. Discussion
Fig. 5. Effect of caloric restriction on urinary protein and glucose excretion in control and Cd-exposed rats. See legend under Fig. 1 for details of animal treatment and number of animals/group.
Except for smoking, food is the major source of environmental Cd exposure (Fleischer et al., 1974). Administration of Cd through the drinking water to animals has been used as a model for environmental Cd exposure by a number of investigators (Itokawa et al., 1978; Kotsonis and Klaassen, 1978; Bernard et al., 1981, 1992; Viau et al., 1984). In male Sprague–Dawley and Wistar rats, fed ad libitum and maintained on 100 mg Cd/l drinking water, renal proximal tubular damage was evident as early as 2–4 months after the start of Cd exposure (Itokawa et al., 1978; Kotsonis and Klaassen, 1978). Female Sprague–Daw-
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
ley rats fed ad libitum and exposed to 100 mg Cd/l drinking water, reach the critical renal Cd concentration within 12 months and show significant elevation in urinary parameters of renal dysfunction (Bernard et al., 1992). Interestingly, in an earlier study Bernard’s group (Viau et al., 1984) was unable to demonstrate any significant increase in urinary parameters in female Sprague–Dawley rats exposed to the same concentration of Cd for up to 12 months. A closer examination of the experimental protocol reveals that these investigators restricted the food intake to 12.7 g/rat/day. No rationale for the food restriction, body weight data, or a discussion of why nephrotoxicity was not observed was provided. In the present study renal dysfunction was not detected even after 27 months of maintaining the rats on 100 mg Cd/l drinking water. The lack of renal dysfunction in our study could only be ascribed to caloric restriction. This conclusion is supported by the results reported by Viau et al. (1984). It should be pointed out, however, that the hepatic and renal Cd concentrations in our study were similar to those achieved by Viau et al. (1984) as well as Bernard et al. (1992), and lack of nephrotoxicity in calory-restricted animals was not as a result of lower Cd burden. Sex differences in Cd distribution upon chronic Cd administration have been reported previously. Hepatic Cd concentration after 45 weeks of feeding rats a diet containing 100 mg Cd/kg was 2-fold higher in females than in males (Stonard and Webb, 1976). Similarly, the renal Cd concentration was 2-fold higher in females than in males after exposure to 4 – 17 mg Cd/l drinking water for 10 months (Murthy et al., 1978; Petering et al., 1979). Stonard and Webb (1976) also reported that, when adjusted for body weight, food intake in female rats was about 15% higher than in males. In our study adult female rats were provided the same quantity of food as the males and drank the same amount of water despite weighing less than the age-matched male rats. This means that when female rats are provided unlimited access to Cdcontaining food or drinking water, they consume
99
significantly more Cd/kg body weight than the males and can thus attain a higher body burden than males. In the present study we minimized the sex differences in the amount of Cd ingested/ day by adjusting the concentration of Cd in the drinking water for the females according to their body weights relative to the males. Since the males and females consumed similar total amounts of Cd/kg body weight, their tissue Cd levels were also very similar. While we did not examine hepatic toxicity in this study, it is known that caloric restriction broadly affects hormonal status (Abdo et al., 1991) and protects against chemical-induced hepatic damage (Ramaiah et al., 1998). Caloric restriction could offer protection by decreasing serum progesterone (Holehan and Merry, 1985) and by increasing serum testosterone levels (Snyder et al., 1988), both of which depress Cdinduced hepatotoxicity (Shiraishi et al., 1994; Shimada et al., 1997a,b). Nomiyama and Nomiyama (1993) reported that amelioration of Cd-induced hepatic damage alleviates nephrotoxicity, possibly by depressing CdMT release from the liver. Cd is translocated from the liver to the kidneys as CdMT. Renal damage depends on the rate of transfer of CdMT to the kidneys (Vestergaard and Shaikh, 1994). When the renal Cd reaches the critical concentration, it is hypothesized that the upper limit of MT synthesis is reached and that the non-MT-bound Cd is responsible for causing nephrotoxicity (Nomiyama and Nomiyama, 1986; Goyer et al., 1989). In the present study, the renal cortex Cd concentration reached the critical concentration after 9 months of exposure, and the maximum MT levels were also observed at this time. With continued exposure to Cd, the renal Cd concentration increased, but renal MT levels did not. This indicates that even in the calory-restricted animals the maximum MT synthesizing ability in the kidney is reached when the renal Cd concentration reaches the critical concentration and that excess Cd is free to interact with other ligands. Based on the theory that non-MT-bound Cd is responsible for Cd-induced nephrotoxicity
100
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
(Nomiyama and Nomiyama, 1986; Goyer et al., 1989), severe nephrotoxicity should have been evident in diet-restricted rats but was not observed. The beneficial effects of caloric restrictions on the susceptibility to cancer and to age-related degenerative diseases have been well documented (McCay et al., 1935; Yu et al., 1982; Kim et al., 1996). Caloric restrictions can modulate age-related free radical production (Yu et al., 1982; Kim et al., 1996; Sohal and Weindruch, 1996). Chronic Cd exposure stimulates lipid peroxidation in the liver and kidney, and antioxidants such as Nacetyl cysteine, vitamin E, and glycine depress Cd-induced nephrotoxicity, indicating that oxidative stress plays a critical role in chronic Cd toxicity (Shaikh and Tang, 1999; Shaikh et al., 1999a,b). Caloric restriction may depress the onset of Cd-induced renal damage by increasing renal GSH levels (Cadenas et al., 1994), suppressing the production of reactive oxygen species, increasing catalase and GSH-Px activities, and by maintaining cytosolic ascorbic acid levels (Kim et al., 1996). Besides renal hypertrophy and degenerative changes in tubules, decreased femur weight, narrowing of cortical osseous tissue, and decrease in osteocytes have been reported by Itokawa et al. (1978) in male Wistar rats fed ad libitum a calcium-deficient diet and drinking water containing 50 mg Cd/l for 4 months. Cd-induced bone loss can occur in mice within 72 h of exposure to as little as 25 mg Cd/l drinking water (Bhattacharyya et al., 1988; Wang and Bhattacharyya, 1993). In another study, weanling rats given 10 – 300 mg Cd/kg diet for a period of 3 weeks showed retardation of bone growth and histological lesions resembling osteoporosis, with no associated renal effects (Yoshiki et al., 1975). Both groups of investigators concluded that Cd had a direct effect on bone formation. In the present study bone density was measured to assess Cd-induced bone loss. A significant decrease in bone density occurred in females within 3 months of Cd exposure under normal dietary conditions. Since no renal dysfunction was present, these results suggest that Cd-induced osteotoxicity can indeed occur by direct effect of Cd on bone metabolism. Once the
female rats were placed on reduced-calory diet, the difference in bone density between the control and treated animals was lowered but persisted for the duration of the study. It appears that caloric restriction prevented further deterioration of bone mineralization. Calcium metabolism is regulated by vitamin D (Locker, 1996). In liver, vitamin D is converted to 25-hydroxyvitamin D, which is then metabolized to its active metabolite, 1, 25-dihydroxyvitamin D in renal proximal tubular epithelial cells (Akiba et al., 1980). Tsuritani et al. (1992) found a significant correlation between serum 1, 25-dihydroxyvitamin D level and Cd-induced renal dysfunction in women, suggesting that Cd exposure interferes with vitamin D metabolism in the kidney and contributes to the Cd-induced bone loss. Caloric restriction may protect against Cd-induced bone loss by preventing the age-related progressive decrease in serum 25-hydroxyvitamin D (Kalu et al., 1988). It may also protect by increasing renal GSH concentration (Cadenas et al., 1994), which, together with MT, can sequester excess Cd and thereby reduce the interference of Cd with vitamin D metabolism in the kidney. Epidemiological studies in a Cd-exposed Japanese population estimated that exceeding a maximum total life time dietary intake of about 2 g Cd could result in Cd-induced renal dysfunction (Nogawa et al., 1989, 1992; Kido et al., 1991, 1993; Hochi et al., 1995). The rats in the present study consumed a total of about 5 g Cd/kg during the 27 month period and yet developed no renal dysfunction. It seems possible that the life time total Cd intake limit may be exceeded in individuals whose caloric intake is limited.
5. Conclusion Caloric restriction is beneficial against chronic Cd toxicity as it prevents nephrotoxicity and also appears to control osteotoxicity.
Acknowledgements This work was supported by USPHS grant no.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
ES 03187 from the National Institute of Environmental Health Sciences. Raymond Walsh, Jr., Jane Northup, and Frank Pagliusi provided technical assistance, and Mary Leatham assisted in manuscript preparation.
References Abdo, K.M., Irwin, R., Johnson, J., 1991. Influence of diet restriction on toxicity and hormonal status in chemically treated mice. In: Fishbein, L. (Ed.), Biological Effect of Dietary Restriction. Springer-Verlag, New York, pp. 99– 111. Akiba, T., Endou, H., Koseki, C., Sakai, F., Horiuchi, N., Suda, T., 1980. Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney. Biochem. Biophys. Res. Commun. 94, 313–318. Bernard, A., Lauwerys, R., Gengoux, P., 1981. Characterization of the proteinuria induced by prolonged oral administration of cadmium in female rats. Toxicology 20, 345 – 357. Bernard, A., Lauwerys, R., Amor, A.O., 1992. Loss of glomerular polyanion correlated with albuminuria in experimental cadmium nephropathy. Arch. Toxicol. 66, 272– 278. Bhattacharyya, M.H., Whelton, B.D., Peterson, D.P., et al., 1988. Skeletal changes in multiparous mice fed a nutrientsufficient diet containing cadmium. Toxicology 50, 193– 204. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Cadenas, S., Rojas, C., Perez-Campo, R., Lopez-Torres, M., Barja, G., 1994. Caloric and carbohydrate restriction in the kidney: effects on free radical metabolism. Exp. Gerontol. 29, 77 – 88. Cherian, M.G., 1978. Induction of renal metallothionein synthesis by parenteral cadmium-thionein in rats. Biochem. Pharmacol. 27, 1163 –1166. Cherian, M.G., Shaikh, Z.A., 1975. Metabolism of intravenously injected cadmium-binding protein. Biochem. Biophys. Res. Commun. 65, 863–869. Dudley, R.E., Gammal, L.M., Klaassen, C.D., 1985. Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77, 414–426. Fleischer, M., Sarofim, A.F., Fassett, D.W., et al., 1974. Environmental impact of cadmium: a review by the Panel on Hazardous Trace Substances. Environ. Health Perspect. 7, 253 – 323. Foulkes, E.C., 1978. Renal tubular transport of cadmiummetallothionein. Toxicol. Appl. Pharmacol. 45, 505–512.
101
Goyer, R.A., Miller, C.R., Zhu, S.Y., Victery, W., 1989. Non-metallothionein-bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat. Toxicol. Appl. Pharmacol. 101, 232 – 244. Hassoun, E.A., Stohs, S.J., 1996. Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures. Toxicology 112, 219 – 226. Heinegard, D., Tiderstrom, G., 1973. Determination of serum creatinine by a direct colorimetric method. Clin. Chim. Acta. 43, 305 – 310. Hochi, Y., Kido, T., Nogawa, K., Kito, H., Shaikh, Z.A., 1995. Dose-response relationship between total cadmium intake and prevalence of renal dysfunction using general linear models. J. Appl. Toxicol. 15, 109 – 116. Holehan, A.M., Merry, B.J., 1985. The control of puberty in the dietary restricted female rat. Mech. Aging Dev. 32, 179 – 191. Itokawa, Y., Nishino, K., Takashima, M., et al., 1978. Renal and skeletal lesions in experimental cadmium poisoning of rats: histology and renal function. Environ. Res. 15, 206 – 217. Kalu, D.N., Masaro, E.J., Yu, B.P., Hardin, R.R., Hollis, B.W., 1988. Modulation of age-related hyperparathyroidism and senile bone loss in Fischer rats by soy protein and food restriction. Endocrinol. 122, 1847 – 1854. Kido, T., Shaikh, Z.A., Kito, H., Honda, R., Nogawa, K., 1991. Dose-response relationship between dietary cadmium intake and metallothioneinuria in a population from a cadmium-polluted area of Japan. Toxicology 66, 271 – 278. Kido, T., Shaikh, Z.A., Kito, H., Honda, R, Nogawa, K., 1993. Dose-response relationship between total cadmium intake and metallothioneinuria using logistic regression analysis. Toxicology 80, 207 – 215. Kim, J.D., McCarter, R.J., Yu, B.P., 1996. Influence of age, exercise, and dietary restriction on oxidative stress in rats. Aging (Milano) 8, 123 – 129. Kotsonis, F., Klaassen, C.D., 1978. The relationship of metallothionein to the toxicity of cadmium after oral administration to rats. Toxicol. Appl. Pharmacol. 46, 39 – 54. Locker, F.G., 1996. Hormonal regulation of calcium homeostasis. Nurs. Clin. North. Am. 31, 797 – 803. McCay, C.M., Cromwell, M.E., Maynard, L.A., 1935. The effect of retarded growth upon the length of life span and ultimate size. J. Nutr. 10, 63 – 79. Mitch, W.E., 1991. Dietary manipulation and progression of chronic renal failure. Child Nephrol. Urol. 11, 134 – 139. Murthy, L., Rice, D.P., Petering, H.G., 1978. Sex differences with respect to the accumulation of oral cadmium in rats. In: Kirchgessner, M. (Ed.), Trace Element Metabolism in Man and Animals 3. ATW, Weihenstephan, pp. 557 – 560. Nogawa, K., 1981. Itai-Itai disease and follow-up studies. In: Nriagu, J.O. (Ed.), Cadmium in the Environment. Wiley, New York, pp. 1 – 38.
102
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103
Nogawa, K., Honda, R., Kido, T., Tsuritani, I., Yamada, Y., Ishizaki, M., Yamaya, H., 1989. A dose-response analysis of cadmium in the general environment with special reference to total cadmium intake limit. Environ. Res. 48, 7–16. Nogawa, K., Kido, T., Shaikh, Z.A., 1992. Dose-response relationship for renal dysfunction in a population environmentally exposed to cadmium. IARC Sci. Publ. 118, 311– 318. Nomiyama, K., Nomiyama, H., 1982. Tissue metallothioneins in rabbits chronically exposed to cadmium, with special reference to the critical concentration of cadmium in the renal cortex. Dev. Toxicol. Environ. Sci. 9, 47–67. Nomiyama, K., Nomiyama, H., 1986. Critical concentration of ’unbound’ cadmium in the rabbit renal cortex. Experientia 42, 149. Nomiyama, K., Nomiyama, H., 1993. Cadmium-induced renal dysfunction was improved by treating hepatic injury with glycyrrhizin. J. Trace Elem. Exp. Med. 6, 171–178. Petering, H.G., Murthy, L., Sorenson, J.R., Levin, L., Stemmer, K.L., 1979. Effect of sex on oral cadmium dose responses in rats: blood pressure and pharmacodynamics. Environ. Res. 20, 289–299. Piscator, M., 1986. The nephropathy of chronic cadmium poisoning. In: Foulkes, E.C. (Ed.), Handbook of Experimental Pharmacology. Springer-Verlag, New York, pp. 180 – 194. Ramaiah, S.K., Soni, M.G., Bucci, T.J., Mehendale, H.M., 1998. Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide. Toxicol. Appl. Pharmacol. 150, 12–21. Shaikh, Z.A., Hirayama, K., 1979. Metallothionein in the extracellular fluids as an index of cadmium toxicity. Environ. Health Perspect. 28, 267–271. Shaikh, Z.A., Lucis, O.J., 1972a. Cadmium and zinc binding in mammalian liver and kidneys. Arch. Environ. Health 24, 419 – 425. Shaikh, Z.A., Lucis, O.J., 1972b. Biological differences in cadmium and zinc turnover. Arch. Environ. Health 24, 410 – 418. Shaikh, Z.A., Tang, W., 1999. Protection against chronic cadmium toxicity by glycine. Toxicology 132, 139–146. Shaikh, Z.A., Vu, T., Zaman, K., 1999a. Oxidative stress as a mechanism of cadmium-induced hepatotoxicity and nephrotoxicity and prevention by antioxidants. Toxicol. Appl. Pharmacol. 154, 256–263. Shaikh, Z.A., Zaman, K., Tang, W., Vu, T., 1999b. Treatment of chronic cadmium nephrotoxicity by N-acetyl cysteine. Toxicol. Lett. 104, 137–142. Shaikh, Z.A., Jordan, S.A., Kido, T., Tang, W., Gross, W.A., Sabbioni, E., 1999c. A sensitive, high capacity radioimmunoassay for metallothionein and its application to urine samples from cadmium-treated rats. Toxicol. Meth. (in press). Shimada, H., Bare, R.M., Hochadel, J.F., Waalkes, M.P., 1997a. Testosterone pretreatment mitigates cadmium toxic-
ity in male C57 mice but not in C3H mice. Toxicology 116, 183 – 191. Shimada, H., Hochadel, J.F., Waalkes, M.P., 1997b. Progesterone pretreatment enhances cellular sensitivity to cadmium despite a marked activation of the metallothionein gene. Toxicol. Appl. Pharmacol. 142, 178 – 185. Shiraishi, N., Barter, R.A., Uno, H., Waalkes, M.P., 1994. Effect of progesterone pretreatment on cadmium toxicity in male Fischer (F344/NCr) and Wistar (WF/NCr) rats. Environ. Health Perspect. 102, 277 – 280. Snyder, D.L., Wostmann, B.S., Pollard, M., 1988. Serum hormones in diet-restricted gnotobiotic and conventional Lobund – Wistar rats. J. Gerontol. 43, B168 – B173. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59 – 63. Sprangers, S.A., Piacsek, B.E., 1997. Chronic underfeeding increases the positive feedback efficacy of estrogen on gonadotropin secretion. Proc. Soc. Exp. Biol. Med. 216, 398 – 403. Squibb, K.S., Fowler, B.A., 1984. Intracellular metabolism and effects of circulating cadmium-metallothionein in the kidney. Environ. Health Perspect. 54, 31 – 35. Stonard, M.D., Webb, M., 1976. Influence of dietary cadmium on the distribution of the essential metals copper, zinc and iron in tissues of the rat. Chem. Biol. Interact. 15, 349 – 363. Tohyama, C., Shaikh, Z.A., 1981. Metallothionein in plasma and urine of cadmium-exposed rats determined by a singleantibody radioimmunoassay. Fundam. Appl. Toxicol. 1, 1 – 7. Tsuchiya, K., 1976. Epidemiological studies on cadmium in the environment in Japan: etiology of Itai – Itai disease. Fed. Proc. 35, 2412 – 2418. Tsuritani, I., Honda, R., Ishizaki, M., Yamada, Y., Kido, T., Nogawa, K., 1992. Impairment of vitamin D metabolism due to environmental cadmium exposure, and possible relevance to sex-related differences in vulnerability to the bone damage. J. Toxicol. Environ. Health 37, 519 – 533. Vestergaard, P., Shaikh, Z.A., 1994. The nephrotoxicity of intravenously administered cadmium-metallothionein: effect of dose, mode of administration, and pre-existing renal cadmium burden. Toxicol. Appl. Pharmacol. 16, 240 – 247. Viau, C., Bernard, A., Lauwerys, R., Maldague, P., 1984. Cadmium, analgesics, and the chronic progressive nephrosis in the female Sprague – Dawley rat. Arch. Toxicol. 55, 247 – 249. Walberg-Rankin, J., Franke, W.D., Gwazdauskas, F.C., 1992. Response of beta-endorphin and estradiol to resistance exercises in females during energy balance and energy restriction. Int. J. Sports Med. 13, 542 – 547. Wang, C., Bhattacharyya, M.H., 1993. Effect of cadmium on bone calcium and 45Ca in non-pregnant mice on a calciumdeficient diet: evidence of direct effect of cadmium on bone. Toxicol. Appl. Pharmacol. 120, 228 – 239. Whiting, P.H., Power, D.A., Petersen, J., Innes, A., Simpson, J.G., Catto, G.R., 1988. The effect of dietary protein restriction on high dose gentamicin nephrotoxicity in rats. Br. J. Exp. Pathol. 69, 35 – 41.
Z.A. Shaikh et al. / Toxicology 133 (1999) 93–103 Weindruch, R., Walford, R.L., Fligiel, S., Guthrie, D., 1986. The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641 – 654. Yoshiki, S., Yanagisawa, T., Kimura, M., Otaki, N., Suzuki, M., Suda, T., 1975. Bone and kidney lesions in experimental cadmium intoxication. Arch. Environ. Health 30, 559– 562.
.
103
Yu, B.P., 1990. Food restriction research: past and present status. In: Rothstein, M. (Ed.), Review of Biological Research in Aging, vol. 25. Elsevier, Shannon, pp. 337 – 351. Yu, B.P., Masoro, E.J., Murata, I., Bertrand, H.A., Lynd, F.T., 1982. Life span study of SPF Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J. Gerontol. 37, 130 – 141.