Influence of Dietary Selenium Lead Toxicity in the Rat1
on
FLORIAN L. CERKLEWSKI2 AND RICHARD M. FORBES3 Department of Animal Science, Nutritional Biochemistry Laboratory, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 ABSTRACT An investigation of the influence of dietary selenium (0.015, 0.05, 0.50, 1.0 ppm) on toxicity of dietary lead (0 and 200 ppm) in the young male rat indicated that selenium was mildly protective against the toxic effects of lead, but only up to 0.50 ppm selenium. At the excess selenium dietary level an exaggeration of lead toxicity was observed. Criteria employed to judge the effects of dietary selenium on lead toxicity included tissue lead concentration and urinary delta-aminolevulinic acid excretion. One exception to the exaggeration effect of excess selenium on lead toxicity was the protective effect of selenium on liver delta-amino levulinic acid dehydratase activity. Since lead depressed kidney selenium concentration, lead may act as an antagonist to selenium metabolism. J. Nutr. 206: 778-783, 1976. INDEXING KEY WORDS nium •lead toxicity
selenium-lead
Both selenium (1-6) and lead (7-12) have a high affinity for sulfur-containing proteins. Other metals such as cadmium, mercury and arsenic that likewise form strong bonds with sulfur (10, 13), have been reported to interact with selenium. Selenium was found to provide protection against both cadmium and mercury toxicities and arsenic provided protection against selenium toxicity. A review of the litera ture and discussion on possible mecha nisms of these interactions is contained in several articles ( 14-16 ). Lead has been found not to affect the excretion of volatile selenium in rats ( 17 ), but no data are available to describe the effect of dietary selenium on lead metab olism. It was therefore the purpose of the present study to investigate the possible interaction between selenium and lead in the rat.
•kidney sele
Torula yeast-containing diet shown in table 1 (diet 1) and distilled water for a period of 2 weeks. Experiment 1. After the 2-week period, five rats were randomly assigned to each of the four diets. Additions of lead as the acetate and selenium as sodium selenite were made to the semipurified diet shown in table 1 (diet 2) to give 0.05 and 0.50 ppm selenium and lead at 0 and 200 ppm. Experiment 2. The purpose of this sec ond study was to further investigate the interaction between selenium and lead by employing more extreme dietary selenium levels. After the 2-week period, six rats were randomly assigned to each of the four diets. Additions of lead as the acetate and selenium as sodium selenite were Received for publication December 9. 1075. 1 Work supported in part by USPHS Grant GM (mi;:>:t-14 and the Illinois Agricultural Experiment Station. Data originate from work submitted in par tial fulfillment of the requirements for the Doctor of Philosophy Nutritional Sciences at the University of Illinois. in I'rbana-Champaign. 2 USPHS Trainee. 3 To whom reprint requests should be sent. 4 Sprague-Dawley, Madison, Wisconsin.
MATERIALSAND METHODS Male albino rats * initially weighing 45 to 55 g were allowed free access to a 778
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
interaction
SELENIUM
779
AND LEAD INTERACTION
made to the semipurified diet shown in table 1 (diet 3) to give 0.015 and 1.0 ppm selenium and lead at 0 and 200 ppm. In each study, rats were housed in indi vidual stainless steel cages in an isolated temperature-controlled animal room with free access to food and distilled water. Control diets did not have lead addition and were found to contain less than 2 ppm lead. Diets were mixed in stainless steel bowls that had been rinsed with dis tilled water and all glassware used in the two experiments was made of pyrex. Glassware was soaked overnight in 10% nitric acid and rinsed with deionized water. All rats were killed by drawing blood into acid-washed syringes from the ab dominal aorta under light ether anesthesia after 4 weeks of consuming individual diets. Blood, kidneys, liver and tibias were removed from each rat. Soft tissues and diet samples were wet ashed in concentrated nitric acid followed by 30% hydrogen peroxide on a tempera ture-controlled hot plate in a fume hood. Tibias were dry ashed in an electric muffle furnace in 30-ml pyrex beakers at about 590°.The white ash of all samples was dis solved and made to appropriate volumes in 3 N hydrochloric acid. Analyses for lead were made by atomic absorption spectrophotometry with simultaneous correction for background interference.5 Selenium analyses of kidney and diets were made by the fluorometric procedure of Watkinson ( 18) with the modification that 100-ml Kjeldahl flasks were used for digestion of samples (19). Air condensers were not employed and spectrograde cyclohexane 6 was the organic solvent. Samp ling of kidney was simplified by homog enizing tissue in 3 volumes of 20 HIM phos phate buffer for 10 seconds.7 Proper sample size of pipetted homogenate was deter mined by referring to the data of Burk et al. (20). The instrument for selenium analysis was a fluorometer 8 equipped with a high sensitivity sample chamber. To supplement the mineral analyses, weekly determinations of urinary deltaaminolevulinic acid (ALA) and terminal determinations of tissue delta-aminolevulinic acid dehydratase activity (ALAD) were made.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
TABLE 1 Composition of diets Component
Diet 1
Diet 2
Diet 3
casein1Tonila Vitamin-free yeast1DL-Methionine*Cellulose3Vitamin
mixture4Mineral mixture6Corn oil6Anhydrous dextrose19/kg0.0300.03.030.050.017.050.0550.0Q/kg150.00.03.030.050.030 1General Biochemicals, Chagrin Falls, Ohio. 2 Grand Island Biological, Grand Island, New York. 3»Solka-floc,Brown Co., Boston. ' nig/kg glucose except where noted: thiamin-HCl, 200; ribonavin, 120; pyridoxine • HC1, 80; calcium pantothenate, 320; biotin, 4; niacin, 500; folie acid, 10; vitamin B12, 0.1% in mannitol, 400; menaquinone, 10; calciferol (850,000 IU/g), 50; d-alpha tocopheryl acid succinate, 908; eholine chloride, 30 g; retinyl palmitate (250,000 IU/g), 1 g. 'g/kg (diet 2): CaHPO,, 556.48; CaCO3, 90.08; NaCl, 111.53; K2CO3, 62.80; K2SO,, 106.50; MnCl2-4H,O, 6.605; FeC,H6O7-3H2O. 6.242; MgCO3, 57.80; ZnCO3, 1.342; CuCl2-2HA 0.58; KIO3, 0.027; Na2SeO3, 0.003. Alterations were made in this mineral mixture for diets 1 and 3 in order to account for minerals present in tonila yeast, so that all diets would pro vide the following: 0.6% Ca, 0.5% P, 0.3% K, 35 ppm Fe, 30 ppm Zn, 6.5 ppm Cu. Selenium content in ppm of the three diets was as follows: diet 1, liver. Dietary selenium (191 level had little effect upon ALAD activity II in blood and kidney; however, selenium 100 did decrease inhibition of ALAD by lead in the liver. Tissue lead. Results are summarized in 15 50 5OO IODO table 2. Excess dietary selenium increased tissue lead concentration of lead-supple ppb Dietary Selenium (log scole) mented rats, but at lower selenium levels Fig. I The effect of dietary selenium and 200 there was a trend for liver and tibia lead Epm lead on the excretion of urinary delta-aminoconcentration to decrease as dietary sele ivulinic acid. Control (0 lead) values ranged nium increased. Tissue lead was found to from 10 to 25 /ig ALA/mg creatinine. Each point be correlated with ALA excretion as evirepresents the number of observations cited in parentheses, mean ±SEM. a versus c: P < 0.01, b versus c: P < 0.001, b versus d: P < 0.05, c versus d: P < 0.001.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
10Bausch and Lomb spectrophotometer, Rochester, New York.
Model 400,
781
SELENIUM AND LEAD INTERACTION
TABLE 2
100 i Blood
The effect of dietary selenium and lead on tissue lead concentration1 Lead content of tissue
-^Kidney
Blood (fresh)
Treatment*
20
0.015 0.05
0.50 ppm Dietary Selenium
Fig. 2 The effect of dietary selenium and 200 ppm lead on tissue delta-aminolevulinic acid dehyclratase activity. Control (0 lead) values for blood, kidney and liver were 0.2, 0.9 and 2.2 /¿moles porphobilinogen/g tissue/hour, respec tively. The points show the mean ±SEM for five observations each at 0.05 and 0.50 ppm Se and for six observations each at 0.015 and 1.0 ppm Se. a versus c: P < 0.01.
denced by the following significant corre lation coefficients: liver, r = 0.81; and tibia, r = 0.98. Similar correlations for blood, r = 0.68 and kidney, r = 0.66 were not significant. Kidney selenium. The results for the fluorometric determination of kidney se lenium at the end of the experiments are shown in figure 3. Kidney selenium in creased with increasing dietary selenium level which supports the observation that kidney selenium concentration is a good indicator of selenium status in the rat (20). The effect of 200 ppm dietary lead was to decrease kidney selenium concen tration by approximately 0.2 ppm irrespec tive of whether rats received 0.05, 0.50 or 1.0 ppm dietary selenium. Depression of kidney selenium by lead was not observed in rats fed the selenium-deficient diet. The mere presence of lead in the kidney homogenate did not interfere with the assay, since deliberate additions of lead up to 20 fig did not affect the standard fluores cence. The actual kidney digest contained less than l /ig lead. Other analyses. Neither 1 ppm selenium nor 200 ppm dietary lead affected liver zinc or copper; however, rats fed the combination of these dietary concentra tions had a definite depression of both liver zinc (15%, P < 0.001) and liver cop-
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
Controls (22)200 (6)0.015 ppm Se200 ppm (5)0.05ppm Se200 ppm (5)0.50ppm Se200 ppm (6)1.0 ppm ppm
Liver (fresh)
Kidney (fresh)
Tibia (ash)
Pb Pb Pb
Pb Seppm
on
FLORIAN L. CERKLEWSKI2 AND RICHARD M. FORBES3 Department of Animal Science, Nutritional Biochemistry Laboratory, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 ABSTRACT An investigation of the influence of dietary selenium (0.015, 0.05, 0.50, 1.0 ppm) on toxicity of dietary lead (0 and 200 ppm) in the young male rat indicated that selenium was mildly protective against the toxic effects of lead, but only up to 0.50 ppm selenium. At the excess selenium dietary level an exaggeration of lead toxicity was observed. Criteria employed to judge the effects of dietary selenium on lead toxicity included tissue lead concentration and urinary delta-aminolevulinic acid excretion. One exception to the exaggeration effect of excess selenium on lead toxicity was the protective effect of selenium on liver delta-amino levulinic acid dehydratase activity. Since lead depressed kidney selenium concentration, lead may act as an antagonist to selenium metabolism. J. Nutr. 206: 778-783, 1976. INDEXING KEY WORDS nium •lead toxicity
selenium-lead
Both selenium (1-6) and lead (7-12) have a high affinity for sulfur-containing proteins. Other metals such as cadmium, mercury and arsenic that likewise form strong bonds with sulfur (10, 13), have been reported to interact with selenium. Selenium was found to provide protection against both cadmium and mercury toxicities and arsenic provided protection against selenium toxicity. A review of the litera ture and discussion on possible mecha nisms of these interactions is contained in several articles ( 14-16 ). Lead has been found not to affect the excretion of volatile selenium in rats ( 17 ), but no data are available to describe the effect of dietary selenium on lead metab olism. It was therefore the purpose of the present study to investigate the possible interaction between selenium and lead in the rat.
•kidney sele
Torula yeast-containing diet shown in table 1 (diet 1) and distilled water for a period of 2 weeks. Experiment 1. After the 2-week period, five rats were randomly assigned to each of the four diets. Additions of lead as the acetate and selenium as sodium selenite were made to the semipurified diet shown in table 1 (diet 2) to give 0.05 and 0.50 ppm selenium and lead at 0 and 200 ppm. Experiment 2. The purpose of this sec ond study was to further investigate the interaction between selenium and lead by employing more extreme dietary selenium levels. After the 2-week period, six rats were randomly assigned to each of the four diets. Additions of lead as the acetate and selenium as sodium selenite were Received for publication December 9. 1075. 1 Work supported in part by USPHS Grant GM (mi;:>:t-14 and the Illinois Agricultural Experiment Station. Data originate from work submitted in par tial fulfillment of the requirements for the Doctor of Philosophy Nutritional Sciences at the University of Illinois. in I'rbana-Champaign. 2 USPHS Trainee. 3 To whom reprint requests should be sent. 4 Sprague-Dawley, Madison, Wisconsin.
MATERIALSAND METHODS Male albino rats * initially weighing 45 to 55 g were allowed free access to a 778
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
interaction
SELENIUM
779
AND LEAD INTERACTION
made to the semipurified diet shown in table 1 (diet 3) to give 0.015 and 1.0 ppm selenium and lead at 0 and 200 ppm. In each study, rats were housed in indi vidual stainless steel cages in an isolated temperature-controlled animal room with free access to food and distilled water. Control diets did not have lead addition and were found to contain less than 2 ppm lead. Diets were mixed in stainless steel bowls that had been rinsed with dis tilled water and all glassware used in the two experiments was made of pyrex. Glassware was soaked overnight in 10% nitric acid and rinsed with deionized water. All rats were killed by drawing blood into acid-washed syringes from the ab dominal aorta under light ether anesthesia after 4 weeks of consuming individual diets. Blood, kidneys, liver and tibias were removed from each rat. Soft tissues and diet samples were wet ashed in concentrated nitric acid followed by 30% hydrogen peroxide on a tempera ture-controlled hot plate in a fume hood. Tibias were dry ashed in an electric muffle furnace in 30-ml pyrex beakers at about 590°.The white ash of all samples was dis solved and made to appropriate volumes in 3 N hydrochloric acid. Analyses for lead were made by atomic absorption spectrophotometry with simultaneous correction for background interference.5 Selenium analyses of kidney and diets were made by the fluorometric procedure of Watkinson ( 18) with the modification that 100-ml Kjeldahl flasks were used for digestion of samples (19). Air condensers were not employed and spectrograde cyclohexane 6 was the organic solvent. Samp ling of kidney was simplified by homog enizing tissue in 3 volumes of 20 HIM phos phate buffer for 10 seconds.7 Proper sample size of pipetted homogenate was deter mined by referring to the data of Burk et al. (20). The instrument for selenium analysis was a fluorometer 8 equipped with a high sensitivity sample chamber. To supplement the mineral analyses, weekly determinations of urinary deltaaminolevulinic acid (ALA) and terminal determinations of tissue delta-aminolevulinic acid dehydratase activity (ALAD) were made.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
TABLE 1 Composition of diets Component
Diet 1
Diet 2
Diet 3
casein1Tonila Vitamin-free yeast1DL-Methionine*Cellulose3Vitamin
mixture4Mineral mixture6Corn oil6Anhydrous dextrose19/kg0.0300.03.030.050.017.050.0550.0Q/kg150.00.03.030.050.030 1General Biochemicals, Chagrin Falls, Ohio. 2 Grand Island Biological, Grand Island, New York. 3»Solka-floc,Brown Co., Boston. ' nig/kg glucose except where noted: thiamin-HCl, 200; ribonavin, 120; pyridoxine • HC1, 80; calcium pantothenate, 320; biotin, 4; niacin, 500; folie acid, 10; vitamin B12, 0.1% in mannitol, 400; menaquinone, 10; calciferol (850,000 IU/g), 50; d-alpha tocopheryl acid succinate, 908; eholine chloride, 30 g; retinyl palmitate (250,000 IU/g), 1 g. 'g/kg (diet 2): CaHPO,, 556.48; CaCO3, 90.08; NaCl, 111.53; K2CO3, 62.80; K2SO,, 106.50; MnCl2-4H,O, 6.605; FeC,H6O7-3H2O. 6.242; MgCO3, 57.80; ZnCO3, 1.342; CuCl2-2HA 0.58; KIO3, 0.027; Na2SeO3, 0.003. Alterations were made in this mineral mixture for diets 1 and 3 in order to account for minerals present in tonila yeast, so that all diets would pro vide the following: 0.6% Ca, 0.5% P, 0.3% K, 35 ppm Fe, 30 ppm Zn, 6.5 ppm Cu. Selenium content in ppm of the three diets was as follows: diet 1, liver. Dietary selenium (191 level had little effect upon ALAD activity II in blood and kidney; however, selenium 100 did decrease inhibition of ALAD by lead in the liver. Tissue lead. Results are summarized in 15 50 5OO IODO table 2. Excess dietary selenium increased tissue lead concentration of lead-supple ppb Dietary Selenium (log scole) mented rats, but at lower selenium levels Fig. I The effect of dietary selenium and 200 there was a trend for liver and tibia lead Epm lead on the excretion of urinary delta-aminoconcentration to decrease as dietary sele ivulinic acid. Control (0 lead) values ranged nium increased. Tissue lead was found to from 10 to 25 /ig ALA/mg creatinine. Each point be correlated with ALA excretion as evirepresents the number of observations cited in parentheses, mean ±SEM. a versus c: P < 0.01, b versus c: P < 0.001, b versus d: P < 0.05, c versus d: P < 0.001.
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
10Bausch and Lomb spectrophotometer, Rochester, New York.
Model 400,
781
SELENIUM AND LEAD INTERACTION
TABLE 2
100 i Blood
The effect of dietary selenium and lead on tissue lead concentration1 Lead content of tissue
-^Kidney
Blood (fresh)
Treatment*
20
0.015 0.05
0.50 ppm Dietary Selenium
Fig. 2 The effect of dietary selenium and 200 ppm lead on tissue delta-aminolevulinic acid dehyclratase activity. Control (0 lead) values for blood, kidney and liver were 0.2, 0.9 and 2.2 /¿moles porphobilinogen/g tissue/hour, respec tively. The points show the mean ±SEM for five observations each at 0.05 and 0.50 ppm Se and for six observations each at 0.015 and 1.0 ppm Se. a versus c: P < 0.01.
denced by the following significant corre lation coefficients: liver, r = 0.81; and tibia, r = 0.98. Similar correlations for blood, r = 0.68 and kidney, r = 0.66 were not significant. Kidney selenium. The results for the fluorometric determination of kidney se lenium at the end of the experiments are shown in figure 3. Kidney selenium in creased with increasing dietary selenium level which supports the observation that kidney selenium concentration is a good indicator of selenium status in the rat (20). The effect of 200 ppm dietary lead was to decrease kidney selenium concen tration by approximately 0.2 ppm irrespec tive of whether rats received 0.05, 0.50 or 1.0 ppm dietary selenium. Depression of kidney selenium by lead was not observed in rats fed the selenium-deficient diet. The mere presence of lead in the kidney homogenate did not interfere with the assay, since deliberate additions of lead up to 20 fig did not affect the standard fluores cence. The actual kidney digest contained less than l /ig lead. Other analyses. Neither 1 ppm selenium nor 200 ppm dietary lead affected liver zinc or copper; however, rats fed the combination of these dietary concentra tions had a definite depression of both liver zinc (15%, P < 0.001) and liver cop-
Downloaded from https://academic.oup.com/jn/article-abstract/106/6/778/4768841 by Washington University School of Medicine Library user on 22 March 2018
Controls (22)200 (6)0.015 ppm Se200 ppm (5)0.05ppm Se200 ppm (5)0.50ppm Se200 ppm (6)1.0 ppm ppm
Liver (fresh)
Kidney (fresh)
Tibia (ash)
Pb Pb Pb
Pb Seppm
