- Email: [email protected]
Effects of 1,25-dihydroxicolecalciferol and dietary calcium–phosphate on distribution of lead to tissues during growth
Toxicology and Applied Pharmacology 210 (2006) 123 – 127 www.elsevier.com/locate/ytaap
Effects of 1,25-dihydroxicolecalciferol and dietary calcium–phosphate on distribution of lead to tissues during growth G.E. Cortina-Ramı´rez, J. Cerbo´n-Solorzano, J.V. Caldero´n-Salinas * Department of Biochemistry, Centro de Investigacio´n y de Estudios Avanzados del IPN, PO Box 14-740, Me´xico City 07000, Me´xico Received 3 May 2005; revised 10 August 2005; accepted 5 September 2005 Available online 11 October 2005
Abstract The susceptibility to the toxic effects of lead (Pb) is mainly mediated by age and nutritional and hormonal status, and children are among the most vulnerable to them. During growth, an increase in calcium, phosphate and vitamin D in diet is recommended to enhance calcium and phosphate intestinal absorption and bone deposit. Calcium and phosphate reduce lead intestinal absorption, and 1,25-dihydroxicolecalciferol (1,25(OH)2D3) (active metabolite of vitamin D) increases both lead and calcium intestinal absorption. However, the effects of 1,25(OH)2D3 on lead bone deposit and redistribution to soft tissues are not well known. In this study, we examined the effects of calcium – phosphate diet supplementation and the administration of 1,25(OH)2D3 on Pb distribution to soft tissue and bone in growing rats exposed to Pb. Rats (21 days old) were exposed for 28 days to 100 ppm of Pb solution in drinking water. Calcium and phosphate in diet were increased from 1 to 2.5% and from 0.65 to 1.8%, respectively, and 1,25(OH)2D3 was administrated by intraperitoneal injection of 7.2 ng/kg every 7 days. Between 21 and 49 days, the body weight increased about 5 times. The results showed that high calcium – phosphate diet led to lower Pb concentration in blood and in bone, but Pb liver and kidney concentrations increased, which indicates that absorption and bone deposit redistribution of Pb decreased. On the other hand, no effect of this diet rich in calcium – phosphate in Pb concentration was observed in brain. Blood and bone Pb concentrations increased even more when the high calcium – phosphate diet included 1,25(OH)2D3. In the rats treated only with 1,25(OH)2D3, blood and bone Pb concentrations were lower. Higher concentrations of lead in the soft organs were observed also in rats treated under a high calcium – phosphate diet plus 1,25(OH)2D3 administration. The above mentioned results suggested that 1,25(OH)2D3 induces an increased absorption and redistribution of Pb, and therefore, it may enhance systemic damage in Pb-exposed growing animals. D 2005 Elsevier Inc. All rights reserved. Keywords: Lead; Calcium; Phosphate; 1,25-Dihydroxicolecalciferol; Vitamin D
Introduction Lead (Pb) intoxication is still an important health problem for many populations (Needleman, 2004; Patocka and Cerny, 2003). The susceptibility to Pb effects is mainly mediated by age and nutritional and hormonal status (Mahaffey, 1983; DeMichelle, 1984). During growth, children are more susceptible to the toxic effects of Pb (Mykkanen et al., 1979; Bellinger, 2004); the intestinal absorption and body retention of Pb are higher than in adulthood (Mykkanen et al., 1979; Bellinger, 2004; Ziegler et al., 1978; Aungst et al., 1981). Minerals and vitamin supplements, particularly calcium, phosphate, and vitamin D, are recommended during growth (Flynn, 2003; Gartner and Greer, 2003; Abrams and Atkinson, * Corresponding author. Fax: +52 5 5061 3391. E-mail address: [email protected] (J.V. Caldero´n-Salinas). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.09.002
2003). The Pb absorption in the intestinal epithelium is carried out mainly through calbindin (Mykkanen et al., 1984; Barton et al., 1978), the calcium transport protein in the intestinal lumen (Fullmer, 1992); due to this property, higher calcium intake might be a way of reducing Pb absorption (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001). Increased dietary phosphate is another condition that limits Pb intestinal absorption (Quarterman et al., 1978; Barton and Conrad, 1981; Spickett and Bell, 1983). However, there are conflicting data about the effect of high calcium and phosphate intake on Pb tissue retention and distribution (Aungst and Fung, 1985). It is well known that 1,25(OH)2D3 (active metabolite of vitamin D) increases Pb absorption (Sobel and Burger, 1955; Moon, 1994; Barton et al., 1980; Hart and Smith, 1981; Edelstein et al., 1984) and also Pb bone uptake (Sobel and Burger, 1955; Hart and Smith, 1981). However, the effects of 1,25(OH)2D3 on Pb distribution and redistribution to other tissues besides bone are
124
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
Table 1 Pb concentration in the blood, brain, liver, kidney and bone, free erythrocyte protoporphyrins (FEP) concentration and body weight of rats [exposed to Pb (100 ppm during 28 days) and non-exposed]
Pb concentration Blood (Ag/dl) Brain (nmol/g) Liver (nmol/g) Kidney (nmol/g) Bone (nmol/g) FEP (Ag/dl) Body weight (g)
Group A
Group B
Non-exposed rats (n = 5)
Exposed rats (n = 5)
5.2 0.1 0.2 0.3 0.4 10.6 179.6
T T T T T T T
0.3 0.01 0.03 0.02 0.03 2.7 31.9
30.90 0.2 0.8 0.8 27.02 47.2 198.2
T T T T T T T
0.9* 0.01* 0.04* 0.03* 0.1* 2.2* 23.9
Tests were performed as indicated in Methods. Data represent mean T SD. * P < 0.05 (Student’s t test).
still unclear. The Pb quantification in human and experimental animal tissues has revealed that the highest concentration of Pb is found in the bone, followed by the liver and kidney (Gross et al., 1975; Miller and Massaro, 1983). This Pb tissue distribution could be modified when the Pb storage in bone is altered under conditions which increase the kinetics of calcium, as pregnancy, lactation (Maldonado-Vega et al., 1996, 2002) and growth (Rader et al., 1981). In the present study, we have analyzed the effects of high dietary calcium (2.5%), phosphate (1.8%) and 1,25(OH)2D3 administration (intraperitoneal injection of 7.2 ng/kg each 7 days) on the distribution of Pb to soft tissue and Pb bone deposit in growing rats exposed to Pb. Methods
Sampling and analysis. Blood samples were obtained on days 14 and 28 of lead exposure. Rats were then killed by cervical dislocation in order to remove the brain, liver, kidney and femur. Organ samples were stored at 70 -C, until assays were carried out. Pb concentrations were then determined by atomic absorption spectrophotometry, using a heated graphite atomizer (MaldonadoVega et al., 1996; D’Haese et al., 1991). Total calcium in plasma and bone calcium concentration were determined by flame atomic absorption. Pb and calcium determinations were run in triplicate. The standard curves were calculated with the method of addition in order to minimize the matrix effect on the absorption peak (recovery of 85 – 113%). Precision of analyses were 87 – 104% (using ESA Hi and Lo calibrators), and detection limit was 1 Ag/dl. In order to confirm Pb intoxication, increased free erythrocyte protoporphyrins (FEP) resulting from the interaction of Pb with ferrochelatase enzyme were analyzed on blood, and their concentration was measured with a microhematofluorometer (Maldonado-Vega et al., 1996; Redig et al., 1991). The differences with external standard references were less than 5%.
Results The animals exposed to Pb for 28 days (group B) showed a significant increase of Pb content in the blood, kidney, liver and bone, along with an increase in FEP concentration in comparison to the non-Pb-exposed rats, group A (Table 1), which indicates Pb toxicity. There were no differences in weight gain between Pb-exposed (group B) and non-exposed rats (group A). The consumption of drinking water and diet was similar among all Pb-exposed and non-exposed groups. In addition, the concentration of total plasma calcium (2.55 T 0.30 mmol/l) was not altered by the Pb exposure or the high calcium – phosphate diet or the 1,25(OH)2D3 administration. Pb concentration in blood
Animals. Male Wistar rats, 21 days old (an important growth period) and 35 T 6 g weight, were used at the beginning of all experiments, which took 28 days. Groups of five rats were housed in plastic cages with sterilized wood shavings as bedding, and 12 light/dark photoperiods (starting at 07:00 h). All animals were kept in the same room to maintain a constant environment. Diet. Rats were fed with pellet diet (Formulab Dieti 5008) containing 23% protein, 6.5% fat, 4% crude fiber, 1% vitamin mix (vitamin D 3.3 UI/g) and 2.5% mineral mix (1% calcium and 0.65% phosphorus). The calcium and phosphorus concentrations in this diet were increased by 2.5% (2.5 times) and 1.8% (2.8 times) wt./wt., respectively, with CaHPO4 to obtain the high calcium – phosphate diet. Drinking water was supplied either distilled water acidified (with HCl pH 5.5) or a 100 ppm solution of Pb acetate in acidified distilled water. Diet and water were provided ad libitum, and the consumption was closely recorded. Growth was determined by weight gain. Experimental groups. follows:
Group E Rats exposed to Pb, fed the high calcium (2.5%) – phosphate (1.8%) diet and 1,25(OH)2D3 IP administrated as mentioned above.
The Pb concentration in blood (PbB) increased significantly in the groups exposed to Pb, days 14 and 28 of exposure (Fig. 1). The lowest increase in PbB was found in Pb-exposed rats fed with the high calcium – phosphate diet (group C), in agreement with an inhibited intestinal absorption of Pb by high dietary
21-day-old rats were assigned into groups of 10 rats as
Group A Control rats not exposed to Pb and fed with normal diet (1% calcium and 0.65% phosphate). Rats exposed to Pb 100 ppm: Group B Rats exposed to Pb, fed with normal diet. Group C Rats exposed to Pb and fed the high calcium (2.5%)-phosphate (1.8%) diet. Group D Rats exposed to Pb under normal diet and 1,25(OH)2D3 (7.2 ng/kg) administrated by an intraperitoneal injection (IP) every 7 days (Sigma-Aldrich Co.).
Fig. 1. Pb concentration in blood for rats Pb exposed (group B, .); non-Pb exposed (group A, o); Pb exposed and fed with high calcium – phosphate diet (group C, g); Pb exposed and 1,25(OH)2D3 administration (group D, >); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E, q). Results are mean T SD (n = 5). Significant differences with respect to part A, *P < 0.05 (ANOVA).
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
calcium and phosphate (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001; Quarterman et al., 1978; Barton and Conrad, 1981). The administration of 1,25(OH)2D3 caused an important increment in PbB concentration in Pb-exposed animals fed with normal (group D) and high calcium – phosphate diets (group E); this was more evident for animals fed the normal diet, 6 times on day 14 and 7.7 times on day 28, with respect to control rats (group A). These increments in PbB concentration agree with the well known stimulated Pb absorption effect of 1,25(OH)2D3 (Sobel and Burger, 1955; Hart and Smith, 1981; Edelstein et al., 1984). Pb in the brain, liver, kidney and bone Pb tissue distribution was evaluated by determining Pb concentrations in the brain, liver, kidney and bone. The Pbexposed animals showed significant increments in Pb concen-
125
trations in soft tissues and bone. On day 14, the largest concentration of Pb was observed in the bone, followed by the liver, kidney and brain. After 28 days of Pb exposure, Pb concentration in kidney was similar to that found in the liver. The Pb concentrations in the bone, liver and kidney increased from day 14 to 28; in contrast, Pb concentration in brain was reduced (Fig. 2A). Enhanced dietary calcium –phosphate in Pb-exposed rats (group C) led to changes in Pb tissue distribution. The Pb concentration in bone was 25% lower than in Pb-exposed rats fed with a normal diet (Fig. 2A). At the same time, calcium concentration in bone increased for 11% and 24%, days 14 and 28 respectively (Fig. 2B). These results suggest a competition between calcium and Pb for the sites of mineral deposit available in the bone, which could limit the Pb bone deposit/accumulation; therefore, there is an increment of Pb available for soft tissue distribution, as can be observed by the increase in Pb
Fig. 2. (A) Pb concentration in the brain, liver, kidney and bone and (B) calcium concentration in the bone, for rats Pb exposed (group B, .); non-Pb exposed (group A, o); Pb exposed and fed with high calcium – phosphate diet (group C, g); Pb exposed and 1,25(OH)2D3 administration (group D, >); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E, q). Results are mean T SD (n = 5). Significant differences with respect to part (B), *P < 0.05 (ANOVA).
126
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
concentrations in the brain, liver and kidney. The brain showed a 46% increase on Pb concentration only after 28 days of trial compared to animals fed with a normal diet (group B); whereas the increase of Pb content in the liver and kidney was only 10%. Group D (with the administration of 1,25(OH)2D3) showed the largest Pb bone concentration, in comparison to all the other groups, in spite of an increased calcium bone uptake (Figs. 2A and B). This could be explained on the basis of an increment of calcium binding on bone, induced by 1,25(OH)2D3 (Hart and Smith, 1981). Despite the important deposit of Pb in bone, the Pb concentrations in soft tissues showed significant differences in Pb tissue distribution. Pb concentrations in the brain and kidney increased twofold on day 14, with slight increments from then to the end of the 28-day trial. The initial large increment could be a consequence of the enhanced intestinal absorption, as shown by the increased PbB induced by 1,25(OH)2D3, and the second one from the result of the equilibrium between absorption, excretion, distribution and bone deposit. Interestingly, liver Pb concentration decreased by 12% from day 14 on, when compared to animals with normal diet (group B). In Pb-exposed rats with 1,25(OH)2D3 administration and fed with high calcium – phosphate diet (group E), Pb concentration in bone was 16.2% and 20% lower, days 14 and 28 respectively, compared to that observed for rats with 1,25(OH)2D3 administration only (group D). However, the Pb bone concentration was higher than in the group Pb-exposed but fed a normal diet (group B). On the other hand, the calcium bone uptake was similar to that observed for group D. In the rats treated with 1,25(OH)2D3 and calcium and phosphate supplementation, after 14 days, the concentration of Pb was increased 2.3-fold in the brain and kidney and 1.2-fold in liver when compared to rats fed with a normal diet (group B). After 14 days, the concentration of Pb of rats treated with 1,25(OH)2D3 and calcium and phosphate supplementation (group E) increased 2.3-fold in the brain and kidney and 1.2fold in the liver, compared to rats fed with a normal diet (group B). Just a slight increment of Pb concentration was observed in the soft organs from day 14 to 28. Blood FEP concentration The increase of free erythrocyte protoporphyrins (FEP) is an index of Pb interaction with ferrochelatase enzyme, and it is a biochemical test to confirm Pb intoxication, and all Pb-exposed groups showed an important increment on FEP in blood (14 days). After 28 days of lead exposure, FEP concentration was increased even more in all Pb-exposed groups. Lower concentrations were observed in the groups fed high calcium and phosphate in the diet with or without 1,25(OH)2D3 administration, which suggests less Pb systemic toxicity. Although FEP concentration may indicate clinical Pb toxicity, it does not indicate changes in Pb tissue distribution or redistribution processes (Table 2). Discussion The role of 1,25(OH)2D3 and dietary calcium – phosphate on Pb intestinal absorption is better understood than the
Table 2 Free erythrocyte protoporphyrins (FEP) concentrations in the blood, for rats non-Pb exposed (group A); Pb exposed (group B); Pb exposed and fed with high calcium – phosphate diet (group C); Pb exposed and 1,25(OH)2D3 administration (group D); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E) (Groups)
FEP concentration (Ag/dl) 14 days of treatment
Non-lead exposed (A) +Pb (B) +Pb (C) +Pb + 1,25(OH)2D3 (D) +Pb + Ca-PO4+1,25(OH)2D3 (E)
10.0 34.8 32.8 35.4 31.6
T T T T T
2.7* 3.8 2.6 1.8 2.3
28 days of treatment 10.6 47.2 42.4 48.8 43.2
T T T T T
2.7* 2.1 1.5* 1.6 1.8*
Results are mean T SD (n = 5). Significant differences with respect to B. * P < 0.05 (ANOVA).
effects that these factors have on Pb tissue distribution during Pb exposure. The data reported herein demonstrate changes on tissue distribution of Pb by effect of high calcium (2.5%) – phosphate (1.8%) diet and 1,25(OH)2D3 administration. High calcium –phosphate diet led to a lower PbB, with respect to rats fed with a normal diet, which suggests a diminished intestinal absorption of Pb, in agreement with the inhibitory effect of a high calcium –phosphate diet on Pb intestinal absorption which has been well documented (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001). The low PbB observed may be due to the calcium –Pb competition for the calbindin (Edelstein et al., 1984; Fullmer, 1997; Simons, 1986) and probably to the phosphate –Pb complexes formation, which makes difficult the intestinal absorption of Pb (Mykkanen et al., 1984). Even though in the groups of rats fed with high calcium – phosphate diet this absorption diminished (low PbB), the Pb concentration in the brain, liver and kidney increased. This trend is consistent with a lower bone accumulation of Pb, 25% less compared with the Pb deposit observed in rats fed with a normal diet (group B), and it is probably a consequence of a competition between calcium and Pb to be deposited in bone. The reduced Pb bone deposit may increase the Pb availability to be distributed to soft tissues (Maldonado-Vega et al., 2002) as was suggested by the increase in Pb concentrations in the brain, liver and kidney. These results are also in agreement with the effect reported of high calcium diet which reduced blood Pb clearance and increased the Pb bioavailability (Aungst and Fung, 1985). It is well known that 1,25(OH)2D3 increases the intestinal calbindin synthesis and leads to a greater Pb uptake by the organism (Edelstein et al., 1984; Fullmer, 1997), which explains the increased PbB in rats with 1,25(OH)2D3 administration (group D) (Fig. 1). Therefore, an increased Pb distribution in the brain and kidney was observed on day 14 of the treatment. Interestingly, after 28 days of treatment, there was a trend to diminish the Pb concentration in brain. This might be explained as a consequence of a redistribution process, with an important bone deposit allowing a lower Pb organ accumulation. The effect of 1,25(OH)2D3 administration on calcium and lead intestinal absorption and bone deposit are well documented but are not known in the liver and kidney.
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
The decreased Pb concentration in the liver and kidney, compared to rats without 1,25(OH)2D3 administration (group B), is probably related to a lower Pb affinity as a result of several factors, such as vascularization, Pb intake mechanisms in hepatocytes and kidney cells, extracellular matrix composition or an earlier response to a redistribution process caused by 1,25(OH)2D3. The 1,25(OH)2D3 administration plus the high calcium – phosphate diet limited the Pb entrance to the organism, when compared to rats treated with 1,25(OH)2D3 administration only and fed with a normal calcium – phosphate diet. However, the Pb accumulation in the brain, liver and kidney increased, in coincidence with a lower bone deposit (20% less). These results showed that dietetic and hormonal factors affected the Pb tissue distribution, and consequently, they could increase or decrease the Pb toxic effects. Notably, these data showed that, at least under the conditions tested of increased dietary calcium and phosphate and 1,25(OH)2D3 administration, these elements must not be considered as a therapeutic treatment against Pb intoxication since the concentration of FEP was increased. Additional studies are necessary to evaluate not only the effects of the dietetic factors but also of other hormonal conditions on the redistribution of Pb during growing stage to provide the mechanistic framework to the alteration in the pharmacokinetic behavior of lead. Acknowledgments The authors thank M. Flores and M. Montes for technical assistance. This work was partially supported by CONACyT grant 28759N. References Abrams, S.A., Atkinson, S.A., 2003. Calcium, magnesium, phosphorus and vitamin D fortification of complementary foods. J. Nutr. 133 (9), 2994S – 29949S. Aungst, B.J., Fung, H.L., 1985. The effects of dietary calcium on lead absorption, distribution, and elimination kinetics in rats. J. Toxicol. Environ. Health 16, 147 – 159. Aungst, B.J., Dolce, J.A., Fung, H.L., 1981. The effect of dose on the disposition of lead in rats after intravenous and oral administration. Toxicol. Appl. Pharmacol. 61, 48 – 57. Barton, J.C., Conrad, M.E., 1981. Effect of phosphate on the absorption and retention of lead in the rat. Am. J. Clin. Nutr. 34 (10), 2192 – 2198. Barton, J.C., Conrad, M.E., Harrison, L., Nuby, S., 1978. Effects of calcium on the absorption and retention of lead. J. Lab. Clin. Med. 91 (3), 366 – 376. Barton, J.C., Conrad, M.E., Harrison, L., Nuby, S., 1980. Effects of vitamin D on the absorption and retention of lead. Am. J. Physiol. 238, G124 – G130. Bellinger, D.C., 2004. Lead. Pediatrics 113 (Suppl. 4), 1016 – 1022. Bogden, J.D., Gertner, S.B., Christakos, S., Kemp, F.W., Yang, Z., Katz, S.R., Chu, C., 1992. Dietary calcium modifies concentrations of lead and other metals and renal calbindin in rats. J. Nutr. 122, 1351 – 1360. DeMichelle, S.H., 1984. Nutrition of lead. Comp. Biochem. Physiol. 78A (3), 401 – 408. D’Haese, P.C., Lamberts, L.V., Liang, L., Van de Vyver, F.L., De Broe, M.E., 1991. Elimination of matrix spectral interferences in the measurement of
127
lead and cadmium in urine and blood by electrothermal atomic absorption spectrometry with deuterium background correction. Clin. Chem. 37, 1583 – 1588. Edelstein, S., Fullmer, C.S., Wasserman, R.H., 1984. Gastrointestinal absorption of lead in chicks: involvement of the cholecalciferol endocrine system. J. Nutr. 114, 692 – 700. Flynn, A., 2003. The role of dietary calcium in bone health. Proc. Nutr. Soc. 62 (4), 851 – 858. Fullmer, C.S., 1992. Intestinal calcium absorption: calcium entry. J. Nutr. 122, 644 – 650. Fullmer, C.S., 1997. Lead – calcium interactions: involvement of 1,25-dihydroxyvitamin D. Environ. Res. 72, 45 – 55. Gartner, L.M., Greer, F.R., 2003. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics. 111 (4), 908 – 910. Gross, S.B., Pfitzer, E.A., Yeager, D.W., Kehoe, R.A., 1975. Lead in human tissues. Toxicol. Appl. Pharmacol. 32, 638 – 651. Hart, M.H., Smith, J.L., 1981. Effect of vitamin D and low dietary calcium on lead uptake and retention in rats. J. Nutr. 111, 694 – 698. Mahaffey, K.R., 1983. Biotoxicity of lead: influence of various factors. Fed. Proc. 42 (6), 1730 – 1734. Maldonado-Vega, M., Cerbo´n-Solo´rzano, J., Albores-Medina, A., Herna´ndezLuna, C., Caldero´n-Salinas, J.V., 1996. Lead: intestinal absorption and bone mobilization during lactation. Hum. Exp. Toxicol. 15, 872 – 877. Maldonado-Vega, M., Cerbo´n-Solo´rzano, J., Caldero´n-Salinas, J.V., 2002. The effects of dietary calcium during lactation on lead in bone mobilization: implications for toxicology. Hum. Exp. Toxicol. 21, 409 – 414. Meredith, P.A., Moore, M.R., Goldberg, A., 1977. The effect of calcium on lead absorption in rats. Biochem. J. 166, 531 – 537. Miller, G.D., Massaro, T.F., 1983. Tissue distribution of lead in the neonatal rat exposed to multiple doses of lead acetate. J. Toxicol. Environ. Health. 11, 21 – 28. Moon, J., 1994. The role of vitamin D in toxic metal absorption: a review. J. Am. Coll. Nutr. 13 (6), 559 – 564. Mykkanen, H.M., Dickerson, W.T., Lancaster, M.C., 1979. Effect of age on the tissue distribution of lead in the rat. Toxicol. Appl. Pharmacol. 51, 447 – 454. Mykkanen, H.M., Fullmer, C.S., Wasserman, R.H., 1984. Effect of phosphate on the intestinal absorption of lead (203Pb) in chicks. J. Nutr. 114, 68 – 74. Needleman, H., 2004. Lead poisoning. Annu. Rev. Med. 55, 209 – 222. Patocka, J., Cerny, K., 2003. Inorganic lead toxicology. Acta Med. 46 (2), 65 – 72. Quarterman, J., Morrison, J.M., Humphries, W.R., 1978. The influence of high dietary calcium and phosphate on lead uptake and release. Environ. Res. 17, 60 – 67. Rader, J.I., Peeler, J.T., Mahaffey, K.R., 1981. Comparative toxicity and tissue distribution of lead acetate in weanling and adult rats. Environ. Health Perspect. 42, 187 – 195. Redig, P.T., Lawler, E.M., Schwartz, S., Dunnette, J.L., Stephenson, B., Duke, G.E., 1991. Effects of chronic exposure to sub lethal concentration of lead acetate on heme synthesis and immune function in red-tails hawks. Arch. Environ. Contam. Toxicol. 21, 72 – 77. Simons, T.J., 1986. Cellular interactions between lead and calcium. Br. Med. Bull. 42 (4), 431 – 434. Sobel, A.E., Burger, M., 1955. The influence of calcium, phosphorus, and vitamin D on the removal of lead from blood and bone. J. Biol. Chem. 212 (1), 105 – 110. Spickett, J.T., Bell, R.R., 1983. The influence of dietary phosphate on the toxicity of orally ingested lead in rats. Food Chem. Toxicol. 21 (2), 157 – 161. Varnai, V.M., Piasek, M., Blanusa, M., Saric, M.M., Simic, D., Kostial, K., 2001. Calcium supplementation efficiently reduces lead absorption in suckling rats. Pharmacol. Toxicol. 89 (6), 326 – 330. Ziegler, E.E., Edwards, B.B., Jensen, R.L., Mahaffey, K.R., Fomon, S.J., 1978. Absorption and retention of lead by infants. Pediatr. Res. 12, 29 – 34.
Effects of 1,25-dihydroxicolecalciferol and dietary calcium–phosphate on distribution of lead to tissues during growth G.E. Cortina-Ramı´rez, J. Cerbo´n-Solorzano, J.V. Caldero´n-Salinas * Department of Biochemistry, Centro de Investigacio´n y de Estudios Avanzados del IPN, PO Box 14-740, Me´xico City 07000, Me´xico Received 3 May 2005; revised 10 August 2005; accepted 5 September 2005 Available online 11 October 2005
Abstract The susceptibility to the toxic effects of lead (Pb) is mainly mediated by age and nutritional and hormonal status, and children are among the most vulnerable to them. During growth, an increase in calcium, phosphate and vitamin D in diet is recommended to enhance calcium and phosphate intestinal absorption and bone deposit. Calcium and phosphate reduce lead intestinal absorption, and 1,25-dihydroxicolecalciferol (1,25(OH)2D3) (active metabolite of vitamin D) increases both lead and calcium intestinal absorption. However, the effects of 1,25(OH)2D3 on lead bone deposit and redistribution to soft tissues are not well known. In this study, we examined the effects of calcium – phosphate diet supplementation and the administration of 1,25(OH)2D3 on Pb distribution to soft tissue and bone in growing rats exposed to Pb. Rats (21 days old) were exposed for 28 days to 100 ppm of Pb solution in drinking water. Calcium and phosphate in diet were increased from 1 to 2.5% and from 0.65 to 1.8%, respectively, and 1,25(OH)2D3 was administrated by intraperitoneal injection of 7.2 ng/kg every 7 days. Between 21 and 49 days, the body weight increased about 5 times. The results showed that high calcium – phosphate diet led to lower Pb concentration in blood and in bone, but Pb liver and kidney concentrations increased, which indicates that absorption and bone deposit redistribution of Pb decreased. On the other hand, no effect of this diet rich in calcium – phosphate in Pb concentration was observed in brain. Blood and bone Pb concentrations increased even more when the high calcium – phosphate diet included 1,25(OH)2D3. In the rats treated only with 1,25(OH)2D3, blood and bone Pb concentrations were lower. Higher concentrations of lead in the soft organs were observed also in rats treated under a high calcium – phosphate diet plus 1,25(OH)2D3 administration. The above mentioned results suggested that 1,25(OH)2D3 induces an increased absorption and redistribution of Pb, and therefore, it may enhance systemic damage in Pb-exposed growing animals. D 2005 Elsevier Inc. All rights reserved. Keywords: Lead; Calcium; Phosphate; 1,25-Dihydroxicolecalciferol; Vitamin D
Introduction Lead (Pb) intoxication is still an important health problem for many populations (Needleman, 2004; Patocka and Cerny, 2003). The susceptibility to Pb effects is mainly mediated by age and nutritional and hormonal status (Mahaffey, 1983; DeMichelle, 1984). During growth, children are more susceptible to the toxic effects of Pb (Mykkanen et al., 1979; Bellinger, 2004); the intestinal absorption and body retention of Pb are higher than in adulthood (Mykkanen et al., 1979; Bellinger, 2004; Ziegler et al., 1978; Aungst et al., 1981). Minerals and vitamin supplements, particularly calcium, phosphate, and vitamin D, are recommended during growth (Flynn, 2003; Gartner and Greer, 2003; Abrams and Atkinson, * Corresponding author. Fax: +52 5 5061 3391. E-mail address: [email protected] (J.V. Caldero´n-Salinas). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.09.002
2003). The Pb absorption in the intestinal epithelium is carried out mainly through calbindin (Mykkanen et al., 1984; Barton et al., 1978), the calcium transport protein in the intestinal lumen (Fullmer, 1992); due to this property, higher calcium intake might be a way of reducing Pb absorption (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001). Increased dietary phosphate is another condition that limits Pb intestinal absorption (Quarterman et al., 1978; Barton and Conrad, 1981; Spickett and Bell, 1983). However, there are conflicting data about the effect of high calcium and phosphate intake on Pb tissue retention and distribution (Aungst and Fung, 1985). It is well known that 1,25(OH)2D3 (active metabolite of vitamin D) increases Pb absorption (Sobel and Burger, 1955; Moon, 1994; Barton et al., 1980; Hart and Smith, 1981; Edelstein et al., 1984) and also Pb bone uptake (Sobel and Burger, 1955; Hart and Smith, 1981). However, the effects of 1,25(OH)2D3 on Pb distribution and redistribution to other tissues besides bone are
124
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
Table 1 Pb concentration in the blood, brain, liver, kidney and bone, free erythrocyte protoporphyrins (FEP) concentration and body weight of rats [exposed to Pb (100 ppm during 28 days) and non-exposed]
Pb concentration Blood (Ag/dl) Brain (nmol/g) Liver (nmol/g) Kidney (nmol/g) Bone (nmol/g) FEP (Ag/dl) Body weight (g)
Group A
Group B
Non-exposed rats (n = 5)
Exposed rats (n = 5)
5.2 0.1 0.2 0.3 0.4 10.6 179.6
T T T T T T T
0.3 0.01 0.03 0.02 0.03 2.7 31.9
30.90 0.2 0.8 0.8 27.02 47.2 198.2
T T T T T T T
0.9* 0.01* 0.04* 0.03* 0.1* 2.2* 23.9
Tests were performed as indicated in Methods. Data represent mean T SD. * P < 0.05 (Student’s t test).
still unclear. The Pb quantification in human and experimental animal tissues has revealed that the highest concentration of Pb is found in the bone, followed by the liver and kidney (Gross et al., 1975; Miller and Massaro, 1983). This Pb tissue distribution could be modified when the Pb storage in bone is altered under conditions which increase the kinetics of calcium, as pregnancy, lactation (Maldonado-Vega et al., 1996, 2002) and growth (Rader et al., 1981). In the present study, we have analyzed the effects of high dietary calcium (2.5%), phosphate (1.8%) and 1,25(OH)2D3 administration (intraperitoneal injection of 7.2 ng/kg each 7 days) on the distribution of Pb to soft tissue and Pb bone deposit in growing rats exposed to Pb. Methods
Sampling and analysis. Blood samples were obtained on days 14 and 28 of lead exposure. Rats were then killed by cervical dislocation in order to remove the brain, liver, kidney and femur. Organ samples were stored at 70 -C, until assays were carried out. Pb concentrations were then determined by atomic absorption spectrophotometry, using a heated graphite atomizer (MaldonadoVega et al., 1996; D’Haese et al., 1991). Total calcium in plasma and bone calcium concentration were determined by flame atomic absorption. Pb and calcium determinations were run in triplicate. The standard curves were calculated with the method of addition in order to minimize the matrix effect on the absorption peak (recovery of 85 – 113%). Precision of analyses were 87 – 104% (using ESA Hi and Lo calibrators), and detection limit was 1 Ag/dl. In order to confirm Pb intoxication, increased free erythrocyte protoporphyrins (FEP) resulting from the interaction of Pb with ferrochelatase enzyme were analyzed on blood, and their concentration was measured with a microhematofluorometer (Maldonado-Vega et al., 1996; Redig et al., 1991). The differences with external standard references were less than 5%.
Results The animals exposed to Pb for 28 days (group B) showed a significant increase of Pb content in the blood, kidney, liver and bone, along with an increase in FEP concentration in comparison to the non-Pb-exposed rats, group A (Table 1), which indicates Pb toxicity. There were no differences in weight gain between Pb-exposed (group B) and non-exposed rats (group A). The consumption of drinking water and diet was similar among all Pb-exposed and non-exposed groups. In addition, the concentration of total plasma calcium (2.55 T 0.30 mmol/l) was not altered by the Pb exposure or the high calcium – phosphate diet or the 1,25(OH)2D3 administration. Pb concentration in blood
Animals. Male Wistar rats, 21 days old (an important growth period) and 35 T 6 g weight, were used at the beginning of all experiments, which took 28 days. Groups of five rats were housed in plastic cages with sterilized wood shavings as bedding, and 12 light/dark photoperiods (starting at 07:00 h). All animals were kept in the same room to maintain a constant environment. Diet. Rats were fed with pellet diet (Formulab Dieti 5008) containing 23% protein, 6.5% fat, 4% crude fiber, 1% vitamin mix (vitamin D 3.3 UI/g) and 2.5% mineral mix (1% calcium and 0.65% phosphorus). The calcium and phosphorus concentrations in this diet were increased by 2.5% (2.5 times) and 1.8% (2.8 times) wt./wt., respectively, with CaHPO4 to obtain the high calcium – phosphate diet. Drinking water was supplied either distilled water acidified (with HCl pH 5.5) or a 100 ppm solution of Pb acetate in acidified distilled water. Diet and water were provided ad libitum, and the consumption was closely recorded. Growth was determined by weight gain. Experimental groups. follows:
Group E Rats exposed to Pb, fed the high calcium (2.5%) – phosphate (1.8%) diet and 1,25(OH)2D3 IP administrated as mentioned above.
The Pb concentration in blood (PbB) increased significantly in the groups exposed to Pb, days 14 and 28 of exposure (Fig. 1). The lowest increase in PbB was found in Pb-exposed rats fed with the high calcium – phosphate diet (group C), in agreement with an inhibited intestinal absorption of Pb by high dietary
21-day-old rats were assigned into groups of 10 rats as
Group A Control rats not exposed to Pb and fed with normal diet (1% calcium and 0.65% phosphate). Rats exposed to Pb 100 ppm: Group B Rats exposed to Pb, fed with normal diet. Group C Rats exposed to Pb and fed the high calcium (2.5%)-phosphate (1.8%) diet. Group D Rats exposed to Pb under normal diet and 1,25(OH)2D3 (7.2 ng/kg) administrated by an intraperitoneal injection (IP) every 7 days (Sigma-Aldrich Co.).
Fig. 1. Pb concentration in blood for rats Pb exposed (group B, .); non-Pb exposed (group A, o); Pb exposed and fed with high calcium – phosphate diet (group C, g); Pb exposed and 1,25(OH)2D3 administration (group D, >); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E, q). Results are mean T SD (n = 5). Significant differences with respect to part A, *P < 0.05 (ANOVA).
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
calcium and phosphate (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001; Quarterman et al., 1978; Barton and Conrad, 1981). The administration of 1,25(OH)2D3 caused an important increment in PbB concentration in Pb-exposed animals fed with normal (group D) and high calcium – phosphate diets (group E); this was more evident for animals fed the normal diet, 6 times on day 14 and 7.7 times on day 28, with respect to control rats (group A). These increments in PbB concentration agree with the well known stimulated Pb absorption effect of 1,25(OH)2D3 (Sobel and Burger, 1955; Hart and Smith, 1981; Edelstein et al., 1984). Pb in the brain, liver, kidney and bone Pb tissue distribution was evaluated by determining Pb concentrations in the brain, liver, kidney and bone. The Pbexposed animals showed significant increments in Pb concen-
125
trations in soft tissues and bone. On day 14, the largest concentration of Pb was observed in the bone, followed by the liver, kidney and brain. After 28 days of Pb exposure, Pb concentration in kidney was similar to that found in the liver. The Pb concentrations in the bone, liver and kidney increased from day 14 to 28; in contrast, Pb concentration in brain was reduced (Fig. 2A). Enhanced dietary calcium –phosphate in Pb-exposed rats (group C) led to changes in Pb tissue distribution. The Pb concentration in bone was 25% lower than in Pb-exposed rats fed with a normal diet (Fig. 2A). At the same time, calcium concentration in bone increased for 11% and 24%, days 14 and 28 respectively (Fig. 2B). These results suggest a competition between calcium and Pb for the sites of mineral deposit available in the bone, which could limit the Pb bone deposit/accumulation; therefore, there is an increment of Pb available for soft tissue distribution, as can be observed by the increase in Pb
Fig. 2. (A) Pb concentration in the brain, liver, kidney and bone and (B) calcium concentration in the bone, for rats Pb exposed (group B, .); non-Pb exposed (group A, o); Pb exposed and fed with high calcium – phosphate diet (group C, g); Pb exposed and 1,25(OH)2D3 administration (group D, >); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E, q). Results are mean T SD (n = 5). Significant differences with respect to part (B), *P < 0.05 (ANOVA).
126
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
concentrations in the brain, liver and kidney. The brain showed a 46% increase on Pb concentration only after 28 days of trial compared to animals fed with a normal diet (group B); whereas the increase of Pb content in the liver and kidney was only 10%. Group D (with the administration of 1,25(OH)2D3) showed the largest Pb bone concentration, in comparison to all the other groups, in spite of an increased calcium bone uptake (Figs. 2A and B). This could be explained on the basis of an increment of calcium binding on bone, induced by 1,25(OH)2D3 (Hart and Smith, 1981). Despite the important deposit of Pb in bone, the Pb concentrations in soft tissues showed significant differences in Pb tissue distribution. Pb concentrations in the brain and kidney increased twofold on day 14, with slight increments from then to the end of the 28-day trial. The initial large increment could be a consequence of the enhanced intestinal absorption, as shown by the increased PbB induced by 1,25(OH)2D3, and the second one from the result of the equilibrium between absorption, excretion, distribution and bone deposit. Interestingly, liver Pb concentration decreased by 12% from day 14 on, when compared to animals with normal diet (group B). In Pb-exposed rats with 1,25(OH)2D3 administration and fed with high calcium – phosphate diet (group E), Pb concentration in bone was 16.2% and 20% lower, days 14 and 28 respectively, compared to that observed for rats with 1,25(OH)2D3 administration only (group D). However, the Pb bone concentration was higher than in the group Pb-exposed but fed a normal diet (group B). On the other hand, the calcium bone uptake was similar to that observed for group D. In the rats treated with 1,25(OH)2D3 and calcium and phosphate supplementation, after 14 days, the concentration of Pb was increased 2.3-fold in the brain and kidney and 1.2-fold in liver when compared to rats fed with a normal diet (group B). After 14 days, the concentration of Pb of rats treated with 1,25(OH)2D3 and calcium and phosphate supplementation (group E) increased 2.3-fold in the brain and kidney and 1.2fold in the liver, compared to rats fed with a normal diet (group B). Just a slight increment of Pb concentration was observed in the soft organs from day 14 to 28. Blood FEP concentration The increase of free erythrocyte protoporphyrins (FEP) is an index of Pb interaction with ferrochelatase enzyme, and it is a biochemical test to confirm Pb intoxication, and all Pb-exposed groups showed an important increment on FEP in blood (14 days). After 28 days of lead exposure, FEP concentration was increased even more in all Pb-exposed groups. Lower concentrations were observed in the groups fed high calcium and phosphate in the diet with or without 1,25(OH)2D3 administration, which suggests less Pb systemic toxicity. Although FEP concentration may indicate clinical Pb toxicity, it does not indicate changes in Pb tissue distribution or redistribution processes (Table 2). Discussion The role of 1,25(OH)2D3 and dietary calcium – phosphate on Pb intestinal absorption is better understood than the
Table 2 Free erythrocyte protoporphyrins (FEP) concentrations in the blood, for rats non-Pb exposed (group A); Pb exposed (group B); Pb exposed and fed with high calcium – phosphate diet (group C); Pb exposed and 1,25(OH)2D3 administration (group D); Pb exposed, fed with high calcium – phosphate diet and 1,25(OH)2D3 administration (group E) (Groups)
FEP concentration (Ag/dl) 14 days of treatment
Non-lead exposed (A) +Pb (B) +Pb (C) +Pb + 1,25(OH)2D3 (D) +Pb + Ca-PO4+1,25(OH)2D3 (E)
10.0 34.8 32.8 35.4 31.6
T T T T T
2.7* 3.8 2.6 1.8 2.3
28 days of treatment 10.6 47.2 42.4 48.8 43.2
T T T T T
2.7* 2.1 1.5* 1.6 1.8*
Results are mean T SD (n = 5). Significant differences with respect to B. * P < 0.05 (ANOVA).
effects that these factors have on Pb tissue distribution during Pb exposure. The data reported herein demonstrate changes on tissue distribution of Pb by effect of high calcium (2.5%) – phosphate (1.8%) diet and 1,25(OH)2D3 administration. High calcium –phosphate diet led to a lower PbB, with respect to rats fed with a normal diet, which suggests a diminished intestinal absorption of Pb, in agreement with the inhibitory effect of a high calcium –phosphate diet on Pb intestinal absorption which has been well documented (Meredith et al., 1977; Bogden et al., 1992; Varnai et al., 2001). The low PbB observed may be due to the calcium –Pb competition for the calbindin (Edelstein et al., 1984; Fullmer, 1997; Simons, 1986) and probably to the phosphate –Pb complexes formation, which makes difficult the intestinal absorption of Pb (Mykkanen et al., 1984). Even though in the groups of rats fed with high calcium – phosphate diet this absorption diminished (low PbB), the Pb concentration in the brain, liver and kidney increased. This trend is consistent with a lower bone accumulation of Pb, 25% less compared with the Pb deposit observed in rats fed with a normal diet (group B), and it is probably a consequence of a competition between calcium and Pb to be deposited in bone. The reduced Pb bone deposit may increase the Pb availability to be distributed to soft tissues (Maldonado-Vega et al., 2002) as was suggested by the increase in Pb concentrations in the brain, liver and kidney. These results are also in agreement with the effect reported of high calcium diet which reduced blood Pb clearance and increased the Pb bioavailability (Aungst and Fung, 1985). It is well known that 1,25(OH)2D3 increases the intestinal calbindin synthesis and leads to a greater Pb uptake by the organism (Edelstein et al., 1984; Fullmer, 1997), which explains the increased PbB in rats with 1,25(OH)2D3 administration (group D) (Fig. 1). Therefore, an increased Pb distribution in the brain and kidney was observed on day 14 of the treatment. Interestingly, after 28 days of treatment, there was a trend to diminish the Pb concentration in brain. This might be explained as a consequence of a redistribution process, with an important bone deposit allowing a lower Pb organ accumulation. The effect of 1,25(OH)2D3 administration on calcium and lead intestinal absorption and bone deposit are well documented but are not known in the liver and kidney.
G.E. Cortina-Ramı´rez et al. / Toxicology and Applied Pharmacology 210 (2006) 123 – 127
The decreased Pb concentration in the liver and kidney, compared to rats without 1,25(OH)2D3 administration (group B), is probably related to a lower Pb affinity as a result of several factors, such as vascularization, Pb intake mechanisms in hepatocytes and kidney cells, extracellular matrix composition or an earlier response to a redistribution process caused by 1,25(OH)2D3. The 1,25(OH)2D3 administration plus the high calcium – phosphate diet limited the Pb entrance to the organism, when compared to rats treated with 1,25(OH)2D3 administration only and fed with a normal calcium – phosphate diet. However, the Pb accumulation in the brain, liver and kidney increased, in coincidence with a lower bone deposit (20% less). These results showed that dietetic and hormonal factors affected the Pb tissue distribution, and consequently, they could increase or decrease the Pb toxic effects. Notably, these data showed that, at least under the conditions tested of increased dietary calcium and phosphate and 1,25(OH)2D3 administration, these elements must not be considered as a therapeutic treatment against Pb intoxication since the concentration of FEP was increased. Additional studies are necessary to evaluate not only the effects of the dietetic factors but also of other hormonal conditions on the redistribution of Pb during growing stage to provide the mechanistic framework to the alteration in the pharmacokinetic behavior of lead. Acknowledgments The authors thank M. Flores and M. Montes for technical assistance. This work was partially supported by CONACyT grant 28759N. References Abrams, S.A., Atkinson, S.A., 2003. Calcium, magnesium, phosphorus and vitamin D fortification of complementary foods. J. Nutr. 133 (9), 2994S – 29949S. Aungst, B.J., Fung, H.L., 1985. The effects of dietary calcium on lead absorption, distribution, and elimination kinetics in rats. J. Toxicol. Environ. Health 16, 147 – 159. Aungst, B.J., Dolce, J.A., Fung, H.L., 1981. The effect of dose on the disposition of lead in rats after intravenous and oral administration. Toxicol. Appl. Pharmacol. 61, 48 – 57. Barton, J.C., Conrad, M.E., 1981. Effect of phosphate on the absorption and retention of lead in the rat. Am. J. Clin. Nutr. 34 (10), 2192 – 2198. Barton, J.C., Conrad, M.E., Harrison, L., Nuby, S., 1978. Effects of calcium on the absorption and retention of lead. J. Lab. Clin. Med. 91 (3), 366 – 376. Barton, J.C., Conrad, M.E., Harrison, L., Nuby, S., 1980. Effects of vitamin D on the absorption and retention of lead. Am. J. Physiol. 238, G124 – G130. Bellinger, D.C., 2004. Lead. Pediatrics 113 (Suppl. 4), 1016 – 1022. Bogden, J.D., Gertner, S.B., Christakos, S., Kemp, F.W., Yang, Z., Katz, S.R., Chu, C., 1992. Dietary calcium modifies concentrations of lead and other metals and renal calbindin in rats. J. Nutr. 122, 1351 – 1360. DeMichelle, S.H., 1984. Nutrition of lead. Comp. Biochem. Physiol. 78A (3), 401 – 408. D’Haese, P.C., Lamberts, L.V., Liang, L., Van de Vyver, F.L., De Broe, M.E., 1991. Elimination of matrix spectral interferences in the measurement of
127
lead and cadmium in urine and blood by electrothermal atomic absorption spectrometry with deuterium background correction. Clin. Chem. 37, 1583 – 1588. Edelstein, S., Fullmer, C.S., Wasserman, R.H., 1984. Gastrointestinal absorption of lead in chicks: involvement of the cholecalciferol endocrine system. J. Nutr. 114, 692 – 700. Flynn, A., 2003. The role of dietary calcium in bone health. Proc. Nutr. Soc. 62 (4), 851 – 858. Fullmer, C.S., 1992. Intestinal calcium absorption: calcium entry. J. Nutr. 122, 644 – 650. Fullmer, C.S., 1997. Lead – calcium interactions: involvement of 1,25-dihydroxyvitamin D. Environ. Res. 72, 45 – 55. Gartner, L.M., Greer, F.R., 2003. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics. 111 (4), 908 – 910. Gross, S.B., Pfitzer, E.A., Yeager, D.W., Kehoe, R.A., 1975. Lead in human tissues. Toxicol. Appl. Pharmacol. 32, 638 – 651. Hart, M.H., Smith, J.L., 1981. Effect of vitamin D and low dietary calcium on lead uptake and retention in rats. J. Nutr. 111, 694 – 698. Mahaffey, K.R., 1983. Biotoxicity of lead: influence of various factors. Fed. Proc. 42 (6), 1730 – 1734. Maldonado-Vega, M., Cerbo´n-Solo´rzano, J., Albores-Medina, A., Herna´ndezLuna, C., Caldero´n-Salinas, J.V., 1996. Lead: intestinal absorption and bone mobilization during lactation. Hum. Exp. Toxicol. 15, 872 – 877. Maldonado-Vega, M., Cerbo´n-Solo´rzano, J., Caldero´n-Salinas, J.V., 2002. The effects of dietary calcium during lactation on lead in bone mobilization: implications for toxicology. Hum. Exp. Toxicol. 21, 409 – 414. Meredith, P.A., Moore, M.R., Goldberg, A., 1977. The effect of calcium on lead absorption in rats. Biochem. J. 166, 531 – 537. Miller, G.D., Massaro, T.F., 1983. Tissue distribution of lead in the neonatal rat exposed to multiple doses of lead acetate. J. Toxicol. Environ. Health. 11, 21 – 28. Moon, J., 1994. The role of vitamin D in toxic metal absorption: a review. J. Am. Coll. Nutr. 13 (6), 559 – 564. Mykkanen, H.M., Dickerson, W.T., Lancaster, M.C., 1979. Effect of age on the tissue distribution of lead in the rat. Toxicol. Appl. Pharmacol. 51, 447 – 454. Mykkanen, H.M., Fullmer, C.S., Wasserman, R.H., 1984. Effect of phosphate on the intestinal absorption of lead (203Pb) in chicks. J. Nutr. 114, 68 – 74. Needleman, H., 2004. Lead poisoning. Annu. Rev. Med. 55, 209 – 222. Patocka, J., Cerny, K., 2003. Inorganic lead toxicology. Acta Med. 46 (2), 65 – 72. Quarterman, J., Morrison, J.M., Humphries, W.R., 1978. The influence of high dietary calcium and phosphate on lead uptake and release. Environ. Res. 17, 60 – 67. Rader, J.I., Peeler, J.T., Mahaffey, K.R., 1981. Comparative toxicity and tissue distribution of lead acetate in weanling and adult rats. Environ. Health Perspect. 42, 187 – 195. Redig, P.T., Lawler, E.M., Schwartz, S., Dunnette, J.L., Stephenson, B., Duke, G.E., 1991. Effects of chronic exposure to sub lethal concentration of lead acetate on heme synthesis and immune function in red-tails hawks. Arch. Environ. Contam. Toxicol. 21, 72 – 77. Simons, T.J., 1986. Cellular interactions between lead and calcium. Br. Med. Bull. 42 (4), 431 – 434. Sobel, A.E., Burger, M., 1955. The influence of calcium, phosphorus, and vitamin D on the removal of lead from blood and bone. J. Biol. Chem. 212 (1), 105 – 110. Spickett, J.T., Bell, R.R., 1983. The influence of dietary phosphate on the toxicity of orally ingested lead in rats. Food Chem. Toxicol. 21 (2), 157 – 161. Varnai, V.M., Piasek, M., Blanusa, M., Saric, M.M., Simic, D., Kostial, K., 2001. Calcium supplementation efficiently reduces lead absorption in suckling rats. Pharmacol. Toxicol. 89 (6), 326 – 330. Ziegler, E.E., Edwards, B.B., Jensen, R.L., Mahaffey, K.R., Fomon, S.J., 1978. Absorption and retention of lead by infants. Pediatr. Res. 12, 29 – 34.