Document Dv40Mxz9yOERGoB3NbQG7KgbQ

DownloadSearchable PDFRandom document
FINAL REPORT PROJECT: HLTH-12 1 DIETARY LEAD AND CALCIUM: EFFECTS ON BLOOD PRESSURE AND RENAL NEOPLASIA IN THE RAT By John D. Bogden Sheldon B, Gertner Francis W. Kemp Robbie McLeod Kay S. Bruening Haingsub R. Chung UMDNJ-New Jersey Medical School Newark, New Jersey Confidential July, 1990 N 27038 DUP040007077 J. Bpgden arid S.B. Girther: Lisd<-Calcium Interactions and Blood Pressure. NSF Industry/University Cooperative for Research in Hazardous and Toxic Substances. Report on Research Efforts, June 19,1990. The enclosed manuscript is the product of our research for last year. It has been reviewed by the Journal of Nutrition, and this is the revised copy, which we expect will be accepted for publication shortly, in this paper we describe our first chronic experiment with lead and calcium, in which two concentrations of calcium in the diet were used , viz., 0.2% and 4,0%. We found a graded rise in bipod pressure in the low calcium group with increasing levels of lead in the drinking water. The high calcium group had consistently higher blood pressures than the low calcium group, and there did not appear to be any ability of calcium to prevent the hypertensive effects of lead. A very exciting finding was the fact that the high calcium diet appeared to enhance the tumorigenic potential of lead on the kidney. There were, in addition, very significant changes in trace metal concentrations other than lead in several organs. The high calcium diet did not prevent the accumulation of lead in any of the 6 tissues studied, but did cause significant decreases in kidney copper, femur and plasma magnesium, and iron in kidney, liver and testis. Since then, we instituted a larger multifactorial experiment, begun in October, 1989, to examine in more detail the actions of various calcium and lead concentrations in the diet on blood pressure and neoplasia. In this experiment, which will last about 1 year, we are studying the actions of 3 dietary concentrations of calcium <0.1, 0.5, and 2.5%) in combination with 3 levels of lead in the drinking water (0, 50 and 100 ppm). Since we found previously that the 4.0% calcium diet produced considerable toxicity (nephrolithiasis), the present 2.5% calcium Concentration appears to be better tolerated. We are finding in our DUP040007078 preliminary studies on blood pressure that the 0.1% calcium group given the highest concentration of lead (100 ppm) appears to consistently have the highest blood pressures and that there is a tendency toward lower blood pressures as the concentration of calcium in the diet increases. This experiment will continue until September, 1990, so that the absolute values of pressure will not be available until then. At that time we shall examine the tumorigenic actions on the kindey and do trace metal analysis^ We estimate that this component of our study will be completed by February 1991, if sufficient funds are available to us. Without additional funding , we may not be able to satisfactorilly complete this project. DUP040007079 Dietary Lead and Calcium: Effects on Blood Pressure and Renal Neoplasia in the Rat John D. Bogden, Sheldon B. Gertner, Francis W. Kemp, Robbie McLeod, Kay 5. Bruening, and Haingsub R, Chung, Departments of Preventive Medicine and Community Health, Pharmacology and toxicology, and Pathology University of Medicine and Dentistry of New Jersey - New Jersey Medical School, Newark, NJ 07103. Presented at the meeting of the American Society for Pharmacology and Experimental Therapeutics, Salt Lake City, August 15, 1089, and at the UCLA Colloquium on the Inorganic Chemistry/Molecular Biology Interface, Taos, NM, February 28, 1990 Supported in part by the NSF Industry/University Cooperative for Research in Hazardous and Toxic Substances and the American Heart Association - New Jersey Affiliate. Shortened Title: Calcium, Lead, Blood Pressure, & Renal Tumors Correspondence To: Dr. John D. Bogden, Department of Preventive Medicine, UMDNJ - New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103 2 ABSTRACT 1 We examined the potential of increased Ca in the diet to modify the z effects of Pb on tissue metal concentrations, blood pressure, and the incidence 3 of renal tumors. Male Wistar rats (n=48, 5 weeks old) were randomly assigned 4 to one of 6 treatment groups. They were fed a low (0,2%) or high (4.0%) Ca 5 diet for 31 weeks and given 0, 1,0 or 100 ppm Pb in drinking water. In the low 6 Ca groups, increasing concentrations of Pb produced graded increases in mean 7 blood pressure. Rats receiving 4,0% Ca had higher mean blood pressures than 8 the animals fed the 0,2% Ca diet. The 4.0% Ca diet also caused renal and 9 urinary bladder stones to develop in some rats. The high Ca diet did not 10 prevent dose-dependent increases in tissue Pb accumulation, but caused 11 significant decreases in kidney copper, femur magnesium, and iron in kidney, 12 liver and testis. Femur magnesium and iron and liver iron concentrations were 13 lowest in rats receiving 4.0% Ca and 100 ppm Pb. Precancerous and cancerous 14 renal lesions occurred to the greatest extent in the rats receiving 100 ppm Pb 15 and the high Ca diet. These results suggest that very high dietary Ca does not 16 protect against Pb-induced increases in blood pressure or Pb accumulation in 17 tissues and may often produce nephroealcinosis. In addition, high dietary Ca 18 in the presence of Pb may increase the incidence of renal tumors. Key Words: lead, calcium, blood pressure, nephrocalcinosis, kidney tumors, trace metals i DUP040007081 3 INTRODUCTION 1 Two independent types of evidence suggest that there may be an 2 association between low level lead exposure and mild essential hypertension. 3 First, in several studies low level exposure to lead in drinking water produced 4 Increases in blood pressure in the rat or the dog (1-7). This has not been a 5 consistent finding since, in other studies as reviewed by Victery (7), lead 6 exposure did not result in increases in blood pressure. Second, several ' 7 epidemiologic studies have demonstrated a significant association between blood 8 lead concentrations and blood pressure, while other studies have found no 9 association between these two variables (8-12). The reasons for the 10 contrasting results of the various experimental and epidemiological studies are 11 not known. One possible explanation is that dietary calcium may directly 12 influence blood pressure and/or alter the effects of lead on bipod pressure, 13 and this was not considered in the design of prior studies. 14 Harlan, et al (8) studied about 3000 men and women, and found 15 significantly higher blood lead concentrations in subjects who had elevated 16 diastolic blood pressures than in those with diastolic pressures of less than 17 90 mm Hg, No data on lead exposure were presented other than blood levels. 18 However in the subjects they studied, higher blood lead and blood pressure were 19 associated with a significantly lower dietary calcium intake. These data .20 suggest the importance of evaluating lead-calcium-blood pressure interactions. 21 There is both experimental and epidemiologic evidence that increases in 22__dietary calcium may favorably influence blood pressure in laboratory animals 23 and man (13-19). Furthermore, it has been reported that diets deficient in 24 calcium can augment the toxic effects of lead and promote increased lead 25 absorption and decreased excretion (18,19). In addition, several recent 26 articles suggest that the effects of lead on blood pressure are related to 27 lead-induced alterations in intracellular calcium metabolism (20-22). Thus, 28 lead/calcium interactions may in part account for the differing results of 29 studies of the relationship of each of these metals to blood pressure. DUP040007082 4 1 In addition to effects on blood pressure, lead can induce the 2 development of kidney tumors in the rat. These tumors are often bilateral and 3 multiple, being either adenomas or adenocarcinomas of the renal cortex (23). 4 The adenomas can be solid, papillary, tubular, or mixed, and can occur in both 5 male and female rats.. Lead dose and duration of exposure, as well as lead 6 accumulation in the kidney, influence the development of renal tumors, with / 7 higher doses resulting in a greater incidence of tumors in a shorter period of 8 time (23). Because of the central role of the kidney jn the control of blood 9 pressure, studies of lead effects on blood pressure should include 10 histopathologic evaluation of the kidneys, including examination for renal 11 neoplasms. 12 The major objective of the current investigation was to study the 13 effects of 2 dietary calcium concentrations, one below normal and the other 14 substantially above normal, in combination with several levels of lead exposure 15 on both blood pressure and the development of renal tumors in the rat. METHODS 16 Forty-eight male weanling Wistar rats (50-75g) (Charles River; No. 17 Wilmington, MA) were housed individually in stainless steel cages and allowed 18 to adapt to the laboratory environment for one week. Twelve hour light-dark 19 cycles were maintained. The rats were randomly assigned to one of 6 20 treatment groups, with 8 rats per group. Animals were fed diets containing 21 either 0.2% or 4.0% calcium and were given drinking water containing 0, 1.0, 22 or 100 ppm (ug/ml) Pb. The choice of these calcium and lead concentrations 23 was based on the results of previously reported studies (7,24-27) using 24 identical concentrations to separately assess the effects of Ca or Pb bn blood 25 pressure in the rat. It was anticipated that the lead doses used would produce 26 blood lead concentrations similar to those found in United States residents 27 with low to moderate lead exposures. DUPO4OO07O83 5 1 Diets containing 0,4%-0.5% Ca are required to support maximum 2 calcification during growth, but diets 50% lower in calcium can support maximum 3 weight gain (28), The 0.2% Ca diet was intended to be a diet containing the 4 lowest calcium concentration needed to support maximum weight gain in the rat, 5 but greater than the concentration (< 0.1%) that could produce symptomatic 6 calcium deficiencies (28), The 4,0% Ca diet was the highest concentration shown 7 by other investigators to reduce blood pressure in the rat (27), Use of these 8 concentrations resulted in a 20-fold difference between the high and low 9 calcium diets; this large difference was intended to maximize the ability to * 10 detect effects of diet calcium on lead-induced changes in blood pressure and 11 the incidence of renal tumors. The study of intermediate calcium concentrations 12 was reserved for future investigations. The phosphorus and vitamin D contents 13 of both diets were not varied to prevent the introduction of other variables 14 that could complicate interpretation of the study, results, 15 Introduction of the modified calcium diets was done one week after 16 receiving the rats; distilled water containing sodium acetate trihydrate (0 17 ug/ml Pb) (J.T. Baker, PhilTipsburg, N.J.) or lead acetate trihydrate (1.0 and 18 100 ug/ml Pb) (Fisher Scientific, Fair Lawn, N.J.) was also introduced at the 19 same time. The control groups received sodium acetate in the drinking water at 20 an acetate concentration equal to that of the groups receiving 100 ug/ml Pb, 21 Drinking water bottles were fitted with silicone stoppers (VWR, Scientific, San 22 Francisco, CA), 23 Rats were fed a modified AIN-76 diet (Table 1), Calcium carbonate 24 (CaD3 ) was used to prepare the 2 different dietary levels of calcium. The 3 25 diets contained no calcium phosphate. Diets were prepared by Research Diets, 26 Inc., New Brunswick, NJ. Analysis in triplicate of the diets showed a mean 27 calcium concentration of 0.23% for the low calcium diet and 4.04% for the high 28 calcium diet. DUP040007084 6 1 Drinking water consumption was monitored throughout the study. Rats 2 were weighed every two weeks. 3 At the end of 31 weeks, final systolic and diastolic blood pressures 4 were measured directly by use of an indwelling catheter inserted in a tail 5 artery that was connected to a Statham-type physiological pressure transducer 6 (Model CP21, Century technology Company, Inglewood, CA). The transducers were 7 initially calibrated using a mercury manometer. The catheter was inserted y ' 8 under ether anesthesia and the rats allowed to recover from anesthesia for two 9 hours prior to blood pressure measurement. Measurements of rat blood pressure ID during recovery from anesthesia demonstrated that waiting 1 1/2 hours was 11 sufficient to prevent any lowering of blood pressure by the anesthesia used. 12 Blood pressures were measured in triplicate and averaged; replicate values 13 were within + $ mm of mercury. The data obtained were used to calculate a 14 mean blood pressure (MBP= diastolic blood pressure + 1/3 (systolic - diastolic 15 blood pressure) for each rat. 16 Following these measurements, approximately 5 m] of blood was withdrawn 17 from the catheter and the rats were killed by decapitation. The heart, 18 kidneys, liver, right hind femur, brain, and testes were removed and saved for 19 trace metal analyses. One kidney from each rat was preserved in 10% buffered 20 formalin (Fisher Scientific, Fair Lawn, N.J.). This kidney was bisected and 21 embedded in paraffin. Five-micrometer-thick sections of the tissue were made 22 and stained by the hematoxylin and eosin (Fisher Scientific, Fair Lawn, N.J.) 23 technique and evaluated by light microscopy. 24 Organ concentrations of zinc, copper, iron, calcium, and magnesium were 25 determined by previously described techniques using flame atomic absorption 26 spectrophotometry (Perkin-Elmer Model 403, Norwalk, CT) after digestion with 27 nitric (GFS Chemicals, Columbus, OH) and perchloric (GFS Chemicals, Columbus, 28 OH) acids (29). Lead concentrations were determined by a flameless atomic 29 absorption procedure (Perkin-Elmer Model 503 and HGA-2100, Norwalk, CT). 30 Plasma calcium and magnesium were determined by flame atomic absorption after DUP0400Q7085 7 1 dilution with a 0.1% 1anthanum solution. National Bureau of Standards bovine 2 liver (SRM 1577a, Gaithersburg, HD) was used as a quality control sample for 3 all tissue metal analyses. Assays of this sample gave results within 7% of 4 certified values. 5 Data were analyzed by analysis of variance (ANOVA). If ANOVA indicated 6 that there were significant (p < .05) differences among the 6 treatment groups* / 7 then pair-wise comparisons were made using Duncan's test (30). Data on tissue 8 lead concentrations were log-transformed prior to evaluation by ANOVA. 9 In statistical evaluation of the data on tissue metal concentrations, 36 10 ANOVAs were done, to compensate for multiple comparisons, the level of 11 significance at which the null hypotheses were tested was set at p < 0.01 12 instead of p < 0.05 for evaluation of tissue concentrations. RESULTS 13 Growth curves for the 6 treatment groups are shown in Figure 1. Rats 14 fed the 4.0% calcium diet appeared to grow more slowly than animals consuming 15 the 0.2% Ca diet, but this difference was significant only for the group of 16 rats fed the 4.0% Ca diet and 100 ppm lead in the drinking water. Daily 17 drinking water consumption for rats fed the low calcium diet was 45+7 ml, 37+3 18 ml, and 40+5 ml, respectively, for rats receiving 0, 1.0 or 100 ppm lead. 19 Consumption for rats fed the high calcium diet was 40+5 ml, 46+3 ml, 43+5 ml. 20 These values do not differ significantly among the 6 treatment groups; thus, 21 lead consumption was comparable in corresponding treatment groups fed the low 22 and high calcium diets. 23 None of the 24 rats fed the 0.2% calcium diet died prior to the 24 termination of the study at 31 weeks, but 10 of the 24 rats consuming the 4.0% 25 calcium diet did. These included 4 rats from the 0 ppm Pb group, 3 from the 1 26 ppm Pb group, and 3 from the 100 ppm Pb group. Autopsies of these ten rats 27 showed kidney and/or bladder stones. DUP040007086 8 Blood Pressure 1 Mean blood pressures (MBP) at the termination of the study are shown in 2 Figure 2,, For rats fed the low calcium diet, the control (0 ppm Pb) group had 3 the lowest MBP and the rats consuming 100 ppm Pb in the drinking water had the 4 highest MBP. Only the values for the control and 100 ppm Pb groups were 5 significantly (p < 0.05) different. 6 Rats fed the high calcium diet and 0 or 1.0 ppm Pb had significantly y ' 7 higher MBP than animals fed the low calcium diet. Although rats fed this diet 8 and 100 ppm lead had lower MBP than their counterparts fed the low calcium 9 diet, this difference was not statistically significant. Kidney Histopathology 1.0 The eight rats in the group fed 0.2% calcium and 0 ppm Pb did not have 11 any pathological findings in the kidneys. 12 Of eight rats in the group fed 0.2% calcium and 1 ppm Pb, two showed 13 focal lymphocytic inflammatory cell infiltration of interstitium. 14 In the group fed 0.2% calcium and 100 ppm Pb, 7 of 8 rats showed 15 scattered tubular cell atypia with cellular enlargment, nuclear hypertrophy, 16 hyperchromiasia, coarse chromatin clumping and single or multiple enlarged 17 nucleoli (Figure 3). One rat showed intranuclear inclusions with nuclear 18 vacuolization in convoluted tubules of the kidney. Three rats showed focal 19 lymphocytic inflammatory cell infiltration of interstitium. 20 Of four rats examined from the group receiving 4.0% calcium and 0 ppm 21 Pb, one rat showed a stone in the caliceopelvic system with atypical 22 transitional cell hyperplasia and dilatation of the tubular lumen. Three rats 23 revealed focal mild lymphocytic inflammatory cell infiltration of interstitium, 24 Of the five rats examined from the group fed 4.0% calcium and 1 ppm Pb, 25 one rat showed a stone in the caliceopelvic system along with papillary 26 transitional cell carcinoma of the pelvis. Three rats showed focal lymphocytic 27 inflaiwnatory cell infiltration of interstitium. DUP040007087 9 2 Of five rats examined from the group consuming 4.0% calcium and 100 ppm 2 Pb, four rats showed a stone in the dilated caliceopelvic system. Three rats 3 had transitional cell hyperplasia and two had invasive transitional cell 4 carcinoma of the pelvis (Figure 4}. Two rats with stones showed dilatation of 5 convoluted tubules. Tubular epithelial atypia with enlarged cells and nuclei, 6 hyperchromasia, and single or multiple enlarged nucleoli were seen in three 7 rats having a stone. Convoluted tubules were dilated in two rats. In three /' 8 rats, there were focal lymphocytic inflammatory cell infiltrations and in one 9 rat there was polymorphonuclear leukocytic infiltration of interstitium, 10 interstitial fibrosis, and edema with renal and perinephric abscess formation. Tissue Metal Concentrations 11 Metal concentrations for blood, whole brain, kidney, liver, femur, 12 heart, and testis are provided in Tables 2-8, respectively. 13 Concentrations of lead in whole blood, heart, and brain were 14 significantly increased in rats consuming 100 ppm but not 1,0 ppm lead. 15 Testicular lead concentrations were significantly increased only in rats 16 receiving 100 ppm Pb and the high calcium diet. For kidney, liver, and femur, 17 Pb concentrations were significantly increased in rats receiving 1,0 ppm Pb in 18 the drinking water and were further increased in animals consuming 100 ppm Pb 19 in their water. 20 Plasma magnesium concentrations were reduced by the high calcium diet. 21 Plasma calcium was significantly elevated only in the group of rats receiving 22 4.0% Ca and 100 ppm Pb. The high calcium diet resulted in large increases in 23 the calcium content of the kidney, but did not significantly increase calcium 24 concentrations in any other organ. Kidney concentrations of copper and iron, 25 but not magnesium and zinc, were substantially reduced by the high calcium 26 diet. 27 Concentrations of iron in testis and liver were also significantly 28 decreased by the high calcium diet, the latter especially in animals consuming DUP040007088 10 1 100 ppm Pb ip the drinking water, feeding the high calcium diet also resulted 2 in decreased femur magnesium and iron concentrations, particularly in the group 3 of animals receiving 100 ppm lead. DISCUSSION Blood Pressure y' 4 the results of this study are in agreement with prior investigations 5 (1-7') that demonstrate that chronic low-dose exposure to lead can increase 6 blood pressure in the rat. However, the results do not support prior studies 7 that show that high dietary calcium concentrations (up to 4.0%) reduce blood 8 pressure in the rat. In contrast, the rats fed the 4.0% calcium diet and 0 or 9 1.0 ppm Pb in the present study had higher MBP than rats consuming a low10 normal (0.2%) calcium diet and the same Pb concentrations. Only the rats 11 consuming the 4.0% calcium diet and 100 ppm Pb had lower MBP than their 12 counterparts on the low Ca diet, but this difference was not statistically 13 significant and is based on blood pressure in only three rats in the 4,0% 14 Ca/100 ppm Pb group. 15 The higher mean blood pressures in the rats consuming 4.0% calcium may 16 have developed as a result of the nephrocalctnosis observed in these animals. 17 Alternatively, other factors may be responsible for the differences between 18 these results and those of other studies, including differences in duration of 19 the study, other dietary components besides calcium, strain of fats studied, or 20 whether the blood, pressure was measured indirectly or directly, as in the 21 current study. Nephrocaleinosis 22 The development of nephrocalcinosis in rats is dependent upon the 23 dietary ratio of calcium to phosphorus as well as the magnesium content of the 24 diet (31,32,33). Hoek et al, (31) have shown that increases in dietary calcium 25 from 0,13 to 0.50% and phosphorus from 0,20 to 0.40% increased the Ca and DUP040007089 11 1 phosphorus content of the kidney and the extent of nephrocaleinosis, However, 2 further increases in dietary Ca to 0.75% actually reduced the degree of 3 nephrocaleinosis . The phosphorus content of-all diets in our study was held 4 constant at 0.4%, and the magnesium content was 0,05%; at these levels, an 5 increase in dietary calcium from 0.2% to 4.0% led to the development of marked 6 nephrocaleinosis. However, from these results it cannot be determined if the 7 nephrocaleinosis was the result of the high diet Ca concentration, its ratio to 8 phosphorus or magnesium, or the combined influence of these factors, 9 Greger et al. (32) have shown that feeding dibasic calcium phosphate to,, ID rats results in 20 fold higher kidney calcium concentrations when compared to 11 other calcium salts. For this reason, we used calcium carbonate instead of 12 calciuin phosphate in our diets. Nevertheless, the 4.0% calcium diet did 13 produce substantial nephrocaleinosis. 14 Evans and Weaver (34) have found that a diet containing 2.5% calcium as 15 the carbonate produces nephrocaleinosis in spontaneously hypertensive rats 16 consuming diets containing 0.01% or 0.05%, but not 0.75%, magnesium. Blood 17 pressure was lowered by increasing dietary calcium despite the 18 nephrocaleinosis, an observation that puzzled these authors.. The above 19 results suggest that the development of nephrocaleinosis should be monitored 2D in all blood pressure studies in the rat, especially those in which dietary 21 calcium, phosphorus, or magnesium are modifiad. Renal Tumors 22 The occurrence of kidney lesions including renal tumors in rats as a 23 result of lead exposure is well known (23,35,36). ATI rats studied in the 24 group fed 4.0% calcium and 100 ppm Pb had hyperplasia or invasive transitional 25 cell carcinoma, but none of the rats fed 0,2% calcium and TOO ppm Pb exhibited 26 these changes. Thus, the high calcium diet promoted the development of kidney 27 tumors in the rats receiving 100 ppm lead in the drinking water. This result 28 is in agreement with the observations of Kasprzak et al, (37), who found that DUP040007090 n 1 lead-induced renal carcinogenesis was enhanced by increases in dietary calcium 2 despite the fact that higher diet calcium concentrations reduced kidney lead 3 concentrations. These results are also in agreement with the observation that 4 a high calcium diet is more likely to promote chemical carcinogenesis than to 5 inhibit It (38). 6 Renal tumors developed in our rats receiving 100 ppm Pb and 4.0% Ca 7 after 7 months. This period of time is shorter than that reported by other 8 authors (39,40) using diets of normal calcium content even though they used 9 higher doses of lead, Kasprzak et al, (37) did not find renal tumors In their 10 rats until 58 weeks after beginning lead exposure. This may be due to the 11 greater promoting ability of the high Calcium diet that we used, which had a 12 higher calcium content (4.0%) than the approximately 2.4% Ca diet used by 13 Kasprzak and co-workers. Alternatively, the tumors that we observed in 14 transitional cells of the kidney pelvis may develop more quickly than the 15 tubular cell neoplasms observed by Kasprzak et al, (37), The fall in body 16 weight at the end of the study of the rats receiving 4.0% calcium and 100 ppm 17 lead could be related to the renal tumors that developed in this treatment 18 group. 19 Typical rodent diets have high calcium concentrations in comparison to 20 human diets. Little is known about the role of dietary calcium in promoting 21 chemically-induced tumors, but it Is possible that other chemicals besides lead 22 acetate that have been shown to be animal carcinogens may also require 23 promotion by calcium. It may be that prior studies of chemically-induced cancer 24 in rodents overestimate the risk to humans because of promotion by the usually 25 high calcium content of rodent diets. This may have important public health 26 implications, since judgements about the safety of many chemicals are based on 27 carcinogenicity studies in rodents. 28 Hale rats have unique urinary proteins, alpha-2 microglobulins, that 29 may be a factor that predisposes them to the development of kidney tumors (4130 43). Thus, the applicability of these findings to female rats is uncertain. DUP040007091 13 1 Several studies suggest that lead can act synergistically with other 2 Carcinogens, including chromate, 2-acetylaminof1uorene, cadmium, and 3 benz(a)pyrene, to produce renal tumors (23). Other types of tumors may also be 4 produced by these combined exposures. Lead stimulates proliferation of renal 5 tubular epithelium in rats and mice, a finding that may have pre-neoplastic 6 significance (23)* A consistent effect of lead is to cause atypical 7 hyperplasia of kidney tubular epithelium, with nuclei that are enlarged and 8 irregular in shape. The renal tumors that develop may arise from the 9 hyperplastic nodules. However, the effects of high dietary and kidney calcium 10 on this process are not known. There are no reports that lead causes kidney 11 cancer in humans (23). 12 Tumors of the renal pelvis have been reported to account for 7-8% of 13 kidney cancers, and most of these are transitional cell tumors (44). There are 14 reports that kidney stones are associated with the development of squamous and 15 possibly transitional cell kidney carcinoma in humans (44-47). It has been 16 suggested that kidney stones irritate the mucosa, transforming the transitional 17 epithelium to squamous epithelium that may progress to leukoplasia and cancer 18 (45). nevertheless, the relevance of our results to human kidney cancer is 19 uncertain. Tissue Metal Concentrations 20 Lead concentrations in whole blood and the 6 organs analyzed were not 21 reduced by the 4,13% calcium diet in comparison to the 0.2% calcium diet. Thus, 22 differences in kidney lead accumulation cannot explain the tumpr enhancing 23 effects of the high Ca diet. These results support prior studies that suggest 24 that high dietary calcium does not protect against organ lead accumulation when 25 compared to normal or low-normal diet calcium concentrations (48). However, we 26 cannot exclude the possibility that such protection might have been observed if 27 we had studied other dietary calcium concentrations, particularly comparison 28 of a low concentration less than 0.1% with higher concentrations, as has been DUP040007092 14 1 reported by other investigators (48,49). Mylroie et al. (50) have found that 2 rats fed semipurified diets are more susceptible to lead toxicity than rats fed 3 stock diets. Our use of semipurified diets of different calcium content may 4 be responsible for differences between our results and those of some other 5 investigators. In particular, use of the AIN-76 diet can enhance the 6 development of nephrocalcinosis in the rat (51); thus our use of a modified 7 AIN-76 diet may have contributed to the development of nephrocalcinosis in the 8 rats fed the 4.0% Ca'diet. 9 The nephrocalcinosis observed in the rats consuming the 4.0% calcium 10 diet was associated with a four-fold increase in kidney calcium. Kidney copper 11 and iron concentrations were reduced two-fold by the high calcium diet, but 12 kidney magnesium and zinc concentrations were not affected. It is possible 13 that calcium accumulation in the nephron resulted in the displacement of iron 14 and copper. However, if this were the reason for the decrease in kidney copper 15 and iron, it is surprising that magnesium and zinc were not similarly 16 decreased. Alternatively, the decreases in kidney iron and copper may be 17 secondary to the previously described pathologic changes that occurred in the 18 kidneys of our rats fed the 4.0% Ca diet. 19 The high calcium diet produced a two-fold decrease in the femur 20 magnesium concentration, as well as decreases in plasma magnesium, but it did 21 not affect the magnesium concentrations of any other organ. The decrease in 22 femur magnesium was more pronounced in rats also consuming lead. Thus, 23 simultaneous exposure to both lead and high dietary calcium appears to be 24 particularly effective in reducing bone magnesium. Rats fed the high calcium 25 diet and 100 ppm Pb also had the lowest femur and liver iron concentrations; 26 this suggests that iron stores can be reduced by this combination of exposures. 27 There were relatively small but statistically significant differences 28 among treatment groups for liver magnesium and zinc. However, it is unlikely 29 that these differences are physiologically important. DUP040Q07093 15 1 Rats fed the high calcium diet and IDO ppm lead had significantly 2 higher plasma calcium concentrations than rats in the other treatment groups. 3 Although based on a small number of observations, this result suggests that 4 the combination of lead exposure and high dietary calcium may compromise 5 homeostatic mechanisms that control plasma calcium concentrations, 6 The rats receiving 1,0 ppm lead in the drinking water had whole blood y 7 lead concentrations lower than those of most United States residents (52,53), 8 yet experienced an increase in mean blood pressure. The rats consuming 100 ppm 9 lead had blood concentrations similar to humans with moderate to high lead 10 exposures. Thus, the concentrations of lead used produced blood lead levels U comparable to these found in man. 12 Both tumor development and increases in blood pressure are associated 2+ 13 with increases in free intracellular calcium (Ca ) (26,54), It may be i 14 that the effects of the high calcium diet on blood pressure and renal tumor 2+ 15 development found in this study are the results of increases in (Ca ) , i 16 However, additional study will be needed to address this hypothesis, 17 In summary, the present study demonstrates that high dietary calcium 18 does not protect against lead-induced increases in blood pressure. In 19 addition, high dietary calcium may itself increase blood pressure, produce 20 nephrocalcinosis, reduce tissue concentrations of some essential minerals, and 21 promote lead induced renal carcinogenesis in the rat. The relevance of these 22 findings to lead exposure of humans is uncertain. Nevertheless, these results 23 suggest caution in the use of calcium supplements, particularly by those 24 exposed to excessive quantities of lead, and suggest the need for additional 25 studies of tumor promotion by dietary calcium, ACKNOWLEDGEMENT 26 We thank .Wendell Niemann, D.V.M., for his advice and assistance in the 27 conduct of this study. DUPD40007094 16 REFERENCES 1. PERRY, H.M., ERLANGER, M.W., KOPP, S.J. & PERRY, E.F. {1986} Hypertension in man and animals as an asymptomatic manifestation of long-term low-level lead exposure. In: Trace Substances Envlronmental Health, (D, Hemphill, ed.), University of Missouri Press, Columbia, MO, pp. 80-92, 2. AVIV, A., JOHN, E,, BERNSTEIN, J., GOLDSMITH, D. & SPITZER, A, {1980) Lead intoxication during development: Its late effects on kidney function and " blood pressure. Kidney Inti. 17:430-437. 3. WEBB, R,, WINQUIST, R., VICTERY, W,, & VANDER, A.j. {1981) In vivo and in vitro effects pf lead on vascular reactivity in rats. Anu Physiol, 241:H2I1-H2I6. 4. 80SC0L0, P, & CARMIGNANI, M. {1988) Neurohumoral blood pressure regulation in lead exposure. Environ. Health Perspec. 78:101-106. 5. DIAI-RIVERA, R, & HORN, R. {1945) Postmortem studies on hypertensive rats chronically intoxicated with lead acetate. Proc. Soe. Exp. Biol. Med. 59; 161-16.3.. 6. FINE, B,, VETRANO, T., SKURNICK, J. & TY, A, {1988) Blood pressure elevation in young dogs during low-level lead poisoning, Tox, Appl> Pharm. 93:388-393. 7. VICTERY, W, {1988) Evidence for effects of chronic lead exposure on blood pressure in experimental animals. Environ. Health, Perspec, 78:71-76. 8. HARLAN, W.R., LANDIS, J.R., SCHMOUDER, R.L., GOLDSTEIN, N.G. & HARLAN, L.C. (1985) Blood lead and blood pressure. Amer. Med. Assoc, 253:530-534, 9. WEISS, $., MUNOZ, A., STEIN, A., SPARROW, D. & SPEIZER, F. {1986) The relationship of blood lead to blood pressure in a longitudinal study of workingmen. Am. J. Epidem. 123:800-808. 10. jDEKORT, W., VERSCHOOR, M., WIB0W0, A. & VAN HEMMEN J. (1987) Occupational exposure to lead and blood pressure: a study in 105 workers. Am. J. Indust. Med, II:145-156. DUP040007095 17 11. RABJNOWITZ, H., BELLINGER, D., LEVITON, A., NEEDLEMAN, H. & SCHOENBAUM, 5, (1987) Pregnancy hypertension, blood pressure during labor, and blood lead levels. Hypertension 10:447-451. 12. PlRKLE, 3.1., SCHWARTZ, J., LANDIS, J.R. & HARLAN, W.R. (1985) The relationship between blood lead levels and blood pressure and its cardiovascular risk implications, A" m..e...r... J. E...p..i-de"m. 121:246-258. s 13. HCCARRON, D.A., MORRIS, C.D. & COLE, C. (1982) Dietary calcium in human hypertension. Science 217:267-269, 14. HCCARRON, D.A., MORRIS, C.D,, HENRY, H.j, & STANTON, J.L. (1984) Blood pressure and nutrient intake in the United States, Science 224:1392-1398, 15. HCCARRON, D.A. (1983) Calcium and magnesium nutrition in human hypertension, Ann. Intern. Med. 98:800-805. 16. HENRY, H.J, &-MCCARRON, D.A. (1985) Increasing calcium intake lowers blood pressure: the literature reviewed. vL Anu Diet, Assoc. 85:182-185, 17. DRAPER, H. & ROBISON, G.A. (1986) Controversy in pharmacology: calcium and hypertension. Fed, Proc. 45:2732-2733, 18. SIX, K.M. & GOYER, R.A. (1970) Experimental enhancement of lead toxicity by low dietary calcium. Lab. Clin, Med, 76:933-942. 19. MAHAFFEY, K.R, (1974) Nutritional factors and susceptibility to lead toxicity. Environ, Health Perspeet. 7:107-112, 20. PICONINI, F., FAVALLI, L, & CHARI, M.C. (1977) Experimental investigations on the contraction induced by lead in arterial smooth muscle. Toxicology 8:43-51, 21. CHAI, S & WEBB, R.C. (1988) Effects of lead on vascular reactivity. Environ. Health Perspec. 78:85-89. 22. SCHWARTZ, J. (1988) The relationship between blood lead and blood pressure in the NHANES II survey. Environ, Health Perspec. 78:15-22. DUP040007096 18 23. CHDIE, P.P., & RICHTER, G.W. (1980) Effects of lead on the kidney. In: Lead Toxicity. R, L. SLnghal and 0.A. Thomas, Eds., Baltimore, Urban & Schwarzenberg, pp, 187-212, 24. VICTERY, W., VANDER, A.J., SHULAK, J.M., SCHOEPS, P., & JULIUS, S, (1982) Lead, hypertension, and the renin-angiotensin system in rats. JL Lab, Clin. Med. 99:354-362, 25. PERRY, H.M,, ERLANGER, M.W., & PERRY, E.F. (1988) Increase in the blood pressure of rats fed low levels of lead. Environ. Health Perspec. 78:107-111, 26. JONES, MX, & HUIB0NH0A, A. (1988) Effect of marginal calcium intake on blood pressure in the Wistar rat, Clin. Exper, Hyper. * Theory and Practice 410:801-810, 27. MCCARRQN, D.A. (1982) Calcium, magnesium, and phosphorus balance in human and experimental hypertension,. Hypertension 4 Supp. 111:111-27-III-33. 28. NATIONAL ACADEMY OF SCIENCES/NATIONAL RESEARCH COUNCIL. (1978) Nutrient requirements of the laboratory rat. In: Nutrient Requirements of Laboratory Animals. National Academy of Sciences, Washington, D.C., pp. 7-37, 29. NAVEH, Y., WEIS, P., CHUNG, H.R. & BOGDEN, J.D. (1987) Effect of cimetidine on tissue distribution of some trace elements in the rat. IK Nutr. 117:1576-1587. 30. WALLENSTEIN, S., ZUCKER, C.L. & FLEISS, J.L, (1980) Some statistical methods useful in circulation research. Circulation Res, 47:1-9. 31. HOEK, A.C., LEMMENS, A.G., MULLINK, J,W. & BEYNEN, A.C. (1988) Influence of dietary calcium: phosphorus ratio on mineral excretion and nephroealcinosis in female rats. Jt Nutr. 118:1210-1216, 32. GREGER, J.L., KRZYK0WSKI, C.E., KHAZEN, R.R. & KRASHOC, C.L. (1987) Mineral utilization by rats fed various commercially available calcium supplements or milk. J^ Nutr. 117:717-724. 33. HITCHMAN, A.J., HASANY, S.A., HITCHMAN, A., HARRISON, J.E. & TAM, C. (1979) Phosphate-induced renal calcification in the,rat. Can. J. Physiol. Pharmacol. 57:92-97, DUP040007097 19 34, EVANS, G.H, & WEAVER, C.M, (1989), Association of magnesium deficiency with the blood pressure lowering effects of calcium. Hypertension, in press, .35, BERNARD, B.P, & BECKER, C.E, (1988) Environmental lead exposure and the kidney, Clin, Toxicol. 26:1-34. 36. TSUCHIYA, K. (1979) Lead, In: Handbook on the Toxicology Of Metals, (Friberg, L., Nordberg, G.F., & Vouk, V.B., eds.), Elsevier, New York, pp.s' 451-484. 37. KASPRZAK, K.S., HOOVER, K.L. & POIRIER, L.A. (1985) Effects of dietary calcium acetate on lead subacetate carcinogenicity in kidneys of male Sprague-Dawley rats. Carcinogenesis 6:279-282. 38. KASPRZAK, K.S, & WAALKES, M,P, 1986. The role of calcium, magnesium, and zinc in carcinogenesis, Adv Exper Med Biol 206:497-515. 39. VAN E.SCH, G.J., VAN GENDEREN, H., VINK, H.H, (1962) The induction of renal tumors by feeding basic lead acetate to rats. Brit, J. Cancer 16:289-297. 40. MAO, P, MOLNAR, J.J. (1967) The fine structure and histochemistry of lead-induced renal tumors in rats. Aij u Pathol, 50:571-603, 41. GOLDSWORTHY, T.L., LYGHT, 0., BURNETT, V.L. & POPP, J.A, (1988) Potential role of alpha-2u-g1obulin, protein droplet accumulation, and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene, and pentachloroethane. Toxicol. Appl. Pharmacol, 90:367-379. 42. SWENBERG, J.A. SHORT, B., BORGHOFF, S,, STRASSER, J,, & CHARBONNEAU, M, (1989) The comparative pathobiology of 2u-globulin nephropathy. Toxicol, Appl. Pharm. 97:35-46, 43. SHORT, B.G., BURWETT, V.L., COX, M.G., RUS, J.S. & SWENBERG, O.A. (1987) Site-specific renal cytotoxicity and cell proliferation in male rats exposed to petroleum hydrocarbons. Lab, Invest, 57:564-577. 44. DONNELLY, j.O. & K00NTZ, W.W. (1975) Carcinoma of the renal pelvis. South. Med, vh 68:943-946, DUP040007098 20 45. REDDY, D.B., RAO, N., YENKATESWARARAO, K. & VENKATESWARARAO, N. (1969) Squamous cell carcinoma of the renal pelvis. Indian jL Cancer 6:27-33, 46. MH1RI, M.N., TORKI. R.L. & SMIDA, M.L. (1987) Association between squamous cell carcinoma of the renal pelvis and calculi, Brit, J, Urol, 64:201- 202. 47. REED, H.M. & ROBINSON, N,D, (1984) Horseshoe kidney with simultaneous occurrence of calculi, transitional cell and squamous cell carcinoma, ^ Urology 23:62-64. 48. MAHAFFEY, K,R. (1981) Nutritional factors in lead poisioning. Nutr. Reviews . 39;353-362, 49. SIX, K, & GOYER, R,A, (1970) Experimental enhancement of lead toxicity by low dietary calcium, <1 Lab. Clin. Med, 76:933-942, 50. MYLROIE, A.A., MOORE, 1., OLYAI, B. & ANDERSON, M, (1978) Increased susceptibility to lead toxicity in rats fed semipurified diets. Environ, Res. 15:57-64. 51. REEVES, P.G. (1989) AIN 76 diet: Should we change the formulation, J. Nutr, 119:1081-1082. 52. SHARP, D.S, et al (1988) Blood pressure and blood lead concentration in bus drivers. Environ. Health Perspect. 78:131-137. 53. GUSTAFSON, A., HEDNER, P., SCHUTZ, A. & SKERFV1NG, S. (1989) Occupational lead exposure and pituitary function. Int. Arch, Occup, Environ. Health 61:277-281 54. TRUMP, B.F., & BEREZESKY, I.K. (1987) Ion regulation, cell injury, and carcinogenesis. Carcinogenesis 8:1027-1031. 1987. DUP040007099 21 table I Composition of Diets Ingredient Casein Di-methionine Salt mix Vitamin mix Choline bitartrate Cel1 ulose Cornstarch - Sucrose Corn oil Calciurn carbonate Total Weight Low Ca diet 200 3 30 10 2 50 550 100 50 5 lour grams High Ca diet 200 3 30 10 2 50 550 100 50 no HOT 1* Salt mix contained (in gm/857 gm): monobasic potassium phosphate - 500, magnesium sulfate heptahydrate - 72.5, magnesium oxide - 11.8, sodium chloride - 74, chromium potassium sulfate (12 H 0) - 0.55, cupric 2. carbonate - 0.3, potassium iodate - 0.01, ferric citrate monohydrate -6.0, manganous carbonate - 3.5, sodium selenite - 0.01, zinc carbonate -1.6 and sucrose - 186.8. 2. The vitamin mix consisted of the following components in gm/kg: vitamin A palmitate - 0.8, vitamin D3 - 0.25, vitamin E acetate - 10.0; menadione sodium bisulfate - 0.08, biotin (1.0%) - 2.0, cyanocobalamin (0.1%) - 1.0, folic acid - 0,2, niacin - 3.0, calcium pantothenate - 1.6, pyridoxine hydrochloride - 0.7, riboflavin - 0.6, thiamin hydrochloride - 0.6, and sucrose - 979.2. DUP040007100 22 Table 2 BLOOD CONCENTRATIONS Drinking Water Pb Cone. 0 ppm Lead 1.87 + 0.723 1 ppm 3.50 + 0,60 1D0 ppm 39,1 + 3.87 Low Calcium Diet Plasma Calcium a 9.73 + 0.24 9.61+0.19 9.55 + 0.29 Plasma Magnesium a 1.66 + 0.10 1.65 + 0.10 1.58 + 0.04 y N (8) (8) (8) 0 jppm 1 ppm 100 ppm Lead 2.00.+ 1.00 3.20 + 0.583 c 53.3 + 19.4 High Calcium Diet Plasma Calcium 'a 9.65 + 0.55 10,79 + 0.40 b 13.18 + 1.26 Plasma Maqnesium b 0.69 + 0.07 0.89 + 0.10^ a 1,36 + 0.39 (8) (3) (3) Data are mean + SE, n 3-8 Pb - ug/dl, Ca"and Mg - mg/di Values with different superscripts are significantly (p < 0.05) different. DUP040007101 23 Table 3 Brain Metal Concentrations Drinking Water ' Lead Concentration Low Calcium Plot ...............................;.......... ............ ................ Lead Copper Iron Magnesium 0 ppm 1 ppm 100 ppm a 0.03+0.02 a 0.03+0.01 ^b 0.44+0.09 2.67+0.09 2.90+0.14 2.68+0.07 22.1+0.8 21.9+0.8 23.3+0.8 160+3 161+3 155+3 Zinc 11.4+0.4 11.5+0.4 11,2+0.2 High Calcium Diet 0 ppm 1 ppm 100 ppm Lead Copper Iron Magnesium Zinc a 0.02+0.01 a 0.03+0.01 b 0.79+0.14 2.86+0.13 2.56+0.06 2.57+0.09 21.0+1.7 23.0+1.7 18.6+1.3 152+3 147+3 153+3 12.3+0.4 11.2+0.2 11.5+0.2 Concentration units are>g/g wet tissue weight. Data are mean SE n * 6-8 for alT values. . . .r.___,.v,, Values with different superscripts are significantly (p < 0.05) different. DUP040007102 Kidney M etal C oncentrations co in in o Q...O c +1 +1 +| N .01 "i* o n cn in n c m CM H3 0to) m CO in +1 P 4-1 VO e+-1 ft O(0' o o> os CM p-l H s 10 to 10 eo TO TO in M* 3 4*1 +1 + 1 O ft CTJ r> i. H CO 00 cn TO MTO* +fHfjl P' JO m so tO SO "3* 3 TO TO ft * 9 I -ft O OOo ft ft +1 fr- +ft 1 i+n1 p: f--i O D r> os in * u eo r* r> 3 J0 JO (0 e y3ft TO. r- os CM fsot O ft m+1 +i "M* +1 M* JO . . O CM u in ft ft TO J0 X) 0 r> so cn o 0 so 9 >0 o 0 CM (0 ft +:l m o C+M 1 TO* +1 O vp poH 3 f0t ft 10 Oc' ft ft -Afttf ft <c0 3 0*0 O Qfftt fSnts fJOt U3o fftt fftt ft ft O ft 0 f0t 24 CM * p r-i p g -NH. CM H CN f+t 1 c+pi * +i cn n o ft c n OS CM 0ft Pft H I TOH +1 4*1 r* TO CO GO r-l ft H 0S Si ** 0> c M* in ip +0HJ) uo +1 to .9 H Ao+O*1 M* o+s1 CcoO Q IH fyomt u ft Si Si in eo r> 0) in so ft ofot 0 O ,0. +1 +1 +1 os rm** os cm * * . ro <n co ft ft ft so ion ic>m- CM CM ft o o+sJ f+t1 +i u in er iwn cno JO rrf . ft TfOt o rt 1 3 o r> 4-1 H fl cn ft 4-1 -r* n o0H ft OH' Q> 0) -a3tHo ft ft O' O' u<0 <0 w *fHt 3 3 f3Ot f<td ft f3t O OGO ft ft ft ft ft ft P ft O Pft (nVDpaalt=ua<e6 -sa0 .r80ewf5i)tomhreda iadnfiflfl+fe SE. evraelunets re n t. *s u p e r s c r i p t s are s ig n ific a n tly DUP040007103 are pdeifrfsecrriep tnst . u (p < 0.05) . s co n ce n tra tio n u n its are g /g wet tis s u e w eight / L iv e r M etal C oncentrations D oo ocH *3 a t> o+1 9 H.** o +1 O:' ' r+o! (0 cm CHM* pj H 0A r* A cn ro c i+n1 <+1' c+n 1 in PI in CM CM c m s A so Mk CM CM HrH1 in <H + 1 + 1 +1 CO rH cn .rH CO i--i +> QH CM H* E PH o raiHd k H r-* o a. pfot oOo +n] N4! O+1' so. o H* in > 3 I rH ,* O' * rH H rOH 41 +1 in +1 in VO P N c m CN o cn 0 cn b .. rH oo a 3 +o1 +OJ' .in Q .0 <n oo 25 P> AO cn O' H 0 H n O HN n+*! r4.f1 p+-i Hn cPrv* pna 0O 5 P A' O O Hta m H rH O+'1 +h *i t+>1 6 pi H 'f sO' M PI N 0 O1 AO O PI H 0$ rH. Hk +1 +1 i+n1 A -<H o E P H rOH P *AOH k ,ft fot p <n cn in cn .rH *r IN o i+n1 O+ 1 V+O1 rsi t>* .* in in M. E PH H* rH ' in* CO CM o rH O+.'1 p+*i +CM1 P cn n csnf A CM c) - CM. oO tJ 3 +i co+n1 +1 eoH -ucH* 4knJs +f>i A4 k c Tf a -QkH 4S<>0 APCO E f&t E ft ft Eft ft rH o rH E EE fftt ft .ft ft ft H rH s SE. evraelunet ilfl+f iaagltuane6i f-sai8rcewfai tonmhrtelayadn D n sV DUP040007104 26 Femur M etal C o n ce n tra tio n s o VO tn in NcH +1 vo H+1 +! in in in *H h rH a3 e3 r- .00 Cf OOP *H . * e pOO + 1 + 1 +f 3 o VO O' (0 CO in p> s c h P3 CM id id `m a H p 0 c *3* O fj 4-1 + 1 p ** G\ r H . 3e. <3 CO O' 4* ft in oH r--{ t" VO n p p oO 9 aa* ooO + 1 4-1 +1 O CO m t--1 p c** r> CO CM CM CM -s 3H H VO O H +.1 co O+1' its in *4 C4 P H H rH -i ja n p rH O' P 0 .0 00 H +1 n, *0 itS r> r> oo sS . * o o nn c Drl P O' p c rl pc X C P *0 o rl p C aP~ sffcl: (i4 o P E aa aE G< Eft ft o r--f. oo rH in C+O1 VO *44-1 r> m+i Hin Hin rH aoo VO r H rH o . {Q P o 4 H-l + 1 44 3 VO t" nj O' m *0 * <M H rH rri J3;* J3W a id Si P as jn . m 0* , O' CO H C +1 +1 + 1 9rH O P H p* H *9 CJ *1* # Sf co rn roH 10 p r- CS CM P nj H I a ,,* ' * OoO a o r+H 1 +1 4-1 C\ p CVJ P 10 n ca IN g3 o H H H rH roH + 1 +j CO in in r> ca P rH H H 0 XJ 0 in VO p vr P o o P, +1 + 1 3 in VO +1 p in w p CNJ r >1 H P 0P 44 0 c P0 p.CP H 4-1 0V3 rH ' O' c IS O' rl ffl 3 3 P rl 4-3P-H P pPffl H3ffl ft rH P >> o \.O0'>rH O'S^H O' p cu P '30*HPo a3 Pr *PriCtsj vCioOmtnpCfflPpC .c 44 3tJ ii +j4H<P c c 4 44 G D-H o , *0 rH A P EPr fe O' in P ffl o P~ P C 3tS P PC O 3V G * P H O JQ ffl .ft PftPO>v- fEt s ft E ft ft ft ft o H o orH DUP040007105 H eart H etal C oncentrations w ith d iffe re n t superscripts ate s ig n ific a n tly 05) d iffe re n t* m n r> O o+j +o1 +! r> ei o n *jf <? *r rl ri H I fHfl r> n cd <U 3 Oid ' 5+s"1f +1 cm o+1 SST H rl r-l S to H O CM co sr f0t) oP3 H "H cm C+OJ r+li \D CSl in o CO co ri Q I \D H Hsi* ro-l -H P iU-f a p o. o O offtt o 4O-J1 r+>1 + 1 to 9 to in -'a* o >4 -3bH 00 o.ri r-i o r-l +\ a-Ht +1 u r-l n CM Oi IN <d <0 ft CO m in o TJ o <0 J+O1 I+N.1 c+o1 H Ol 9 ffl ir o ft ft CP iPd 3H f3t *i P 5) 3 0*0 O -Hft id 3 QP SCqfjtOo s ee ft ft ft a ft ft o rft r-i 27 rr osH :N o +1 +1 +1 SO ,e *r m yf CO *4 rl H ffl CO CO 0) +J +1 +1 sCP in CO o* m sId rl r-l H P H 9 CM m 3 +i -H +1 o Cl CM S> pft 9 . QQ)ri H 03 r-l t> so p> S 3 r- *rt r-l <rt f4 >0-l 3 M 9 <D o o O ft +1 +1 +1 P 0ft m ! rl in CO M .03H1, P * 9 9 j>r a I -cf H o .H nj H CM +1 +i +.1 SO OId ' in in in CM CM CM 3 n 3 .r--l irn-.9l T33 a p+91 +in 1 o+1 CO.9 P 43 ft CP -rt 0) & & 3 to ffl ri +> ft 0 > On N O' .0 * ft ffl (d 0) 3 ffl H ft W id ft m > 3 3+H H *0cH 3fcfl id P f(t0 B <0M P jj U0) co Cfl i o (D to 0) o3o fiiddt II >34d v ft CJ Q 3 > s-' eeB ft ft ft ft ft ft r-l rl DUF040007106 T e s tis M etal C oncentrations G 28 m D o. o o G -fl +1 f 1 N sr CM 03 .fl 4 fO fl CO oa oa CM H CO CO cn CO fl) + 1 +1 +1 e in \x> rH 0> 4 in 3 rH H i-i S' 333 P vp p \p a0H Pi c H oa p 41 4i 41 =*? in 04 1 H o jH r> ra p cn pi p r-i u3 SI SI u 0 A 03 Q o CM roa ; . ooo + 1 f 1 +1 A in CM rH OO P CMfl O H CM e CO CO av 3 . +-rH rH 04 o p 1 4| fl ar-H 3 flCO VO CM fl CO VO in 04 CM CM -Q H CM 10 H OoO * o Ooo s3 +1 + 1 fl on CO r O oo EE E aaa aaa o rH o o H OG' :QSCl 03 CO fl .fl co o H o tNH r tfl fi rH f1 in fl n in co 04 04 CM E -3H IS o ri ro rH 0 +i 41 f 1 G CM in CD pi in in M1 (0 rH S 0p |X) t> A vo A vp in. h h a G +1 +1 +1 8 vp vp CM I H H VO 03 o03 o pi p rH 10 y JG o dp P cm oin ro> OOP A +1 fl +1 A O rH P o a CO OM CO 04 O* *"* rE3H o fO P> PI cO n h + 1 A1 A* ry<H0 P CM n*r p03 O O <0 O JQ OO OCO ' o o o +1 +1 +1 (0 O rH PI O OH cp 3 o P -H H -rH G 0 O' s -rH m 0 3<a 0n 33 a 4J P a I' -rl Po O' 8 O' o a 0 33 um (0 30 -GP-P 3 rH 0 C -P W 3 U 0 H CO > 0 P G <P 0 0 fH *w *w rH-H<H G G 3 *0*H *0h 30 p,c: O P u3 B a <0w 4-shJoiPn p P oo CD I 3 0 0 to 0 OC -3P il H3 V OO Q3 .G3>a E aaa aaa o rH o o rH DUP040007107 29 FIGURES 1. Growth curves for rats in the six treatment groups* LoCa = 0.2% diet calcium content. HiCa = 4.0% diet Ca content. 2. Mean blood pressures (MBP) + SE of rats in the six treatment groups after 31 weeks. 3. Photomicrograph of renal proximal convoluted tubules with scattered tubular epithelial cells showing nuclear hypertrophy, hyperchromasia, coarse chromatic clumping and enlarged nucleoli, (hematoxylin - eosin, x400) 4. Histopathologic Section of transitional cell carcinoma of the renal pelvis. The tumor is papillary in configuration in the right upper portion of photograph and confluent tumor masses are compressing underlying stroma, (hematoxylin - eosin, xlOO) DUP040007108 30 WEIGHT (grams) LO Ca 0 ppm LO Ca 1 ppm LO Ca lOPppm HI Ca 0. ppm HI Ca 1 ppm HI Ca 1OOpprh Figure 1 DUP040007109 Mean Blood Pressure mm. 31 PPM LEAD Figure 2 DUP040007110 32 Figure 3 DUP040007111 33 Figure 4 DUP040007112