FLORIAN Department Marquette Milwaukee,
L. CERKLEWSKI of Basic Science, Division of Biochemistry, University School of Dentistry, Wisconsin 53233
ABSTRACT Confirmed pregnant female albino rats received a purified diet containing either 12 or 120 ppm zinc with or without lead (0 or 500 ppm) from day 0 gestation through day 16 of lactation. An 11% reduction in dam weight gain during gestation and a 12% reduction in average pup weight in rats fed the low zinc plus lead diet was markedly different compared to controls not receiving lead and lead supplemented rats receiving supple mental zinc. Milk lead concentration was reduced in rats receiving addi tional dietary zinc and lead which was reflected in a 15% reduction in pup tibia lead concentration, a 20% reduction in inhibition of pup liver deltaaminolevulinic acid dehydratase activity and a 31% reduction in pup blood porphyrin concentration. Some of the beneficial effects of supple mental zinc on lead toxicity of pups may be explained by a reduction of both liver lead (40%) and blood lead (32%) concentration of dams. However an interaction between lead and zinc at the placental and mam mary barriers must also be considered. The results of this study suggest that nutritional status of zinc may be an important factor in modifying lead burden of infants borne by women occupationally and environmentally exposed to lead. J. Nutr. 109: 1703-1709, 1979. INDEXING KEY WORDS lead •zinc •reproduction The trace metal lead has two faces. On one hand it has many industrial applications (1) whereas on the other hand it is a toxic trace metal for man resulting in disruption of heme biosynthesis, renal funetion, nervous system development and reproductive function (2-8). Both environmental and occupational exposure contribute to lead sources of man (4, 9-17). Because it is probably not possible to completely eliminate lead exposure of man, greater emphasis has been placed on limiting lead exposure especially in terms of pediatrie lead poisoning (18-19). Experimental studies have indicated that nutrìtional status may be a modifying factor in this regard.
centrations min i
E,
shown
For example
of protein,
Selenium
and
to exaggerate
low
calcium,
dietary
Zinc have tt_ , •
COn-
iron, vitaall rr
been . r
the toxic effects
lead in rats (20-23). Lead absorption in rats has been shown to be increased for low dietary intakes of iron and zinc and for high intakes of vitamin D (24—26). In other cases susceptibility to biochemical effects of lead can be reduced especially in terms of decreased hematocrit, increased reticulocyte count and red cell fragility (27) as well as inhibition of lead sensitive enzymes such as delta-aminolevulinic acid dehydratase activity (6, 24, 28). At least one clinical trial has investigated the effects of zinc supplementation on blood lead of workers occupationally exReceivedfor publication.March12, 1979. 'Supported In part by the Marquette University
$gg$gS8? on Research and NIH No- I-SOS-BR* Presented °f American
In part at the OSrd Annual Societies for Experimental
of Dalias. Texas. 1703
Federation Biology,
Downloaded from https://academic.oup.com/jn/article-abstract/109/10/1703/4770604 by Tulane University Library, Serials Acquisitions Dept. user on 12 January 2019
Influence of Dietary Zinc on Lead Toxicity During Gestation and Lactation in the Female Rat1'2
1704
FLORIAN L. CERKLEWSKI
Component
g/kg
white*Cellulose Spray-dried egg powder2Vitamin mixture3Mineral mixture4Corn oil6Cornstarch2Anhydrous dextrose620040504060150460 1 Marshall Produce Co., Marshall, Minnesota. 2Teklad Test Diets, Madison, Wisconsin. 3 Milli gram per kilogram glucose except where noted: thiamin-HCl, 200; riboflavin, 200; pyridoxine-HCl, 200 ; calcium pantothenate, 400 ; d-biotin, 40 ; niacin, 500; folie acid, 40; vitamin Bi2 (0.1% in mannitol), 500 ; menadione, 10 ; choline chloride, 30 gm ; vitamin A palmitate (250,000 lU/gm), 1 gm; calciferol (500,000 lU/gm), 80 mg; d-alpha tocopheryl acid succinate (1,210 IU/gm), 800 mg. Made to 1 kg with glucose. 4Gram per kilogram mixture : CaHPOi, 594.23; NaCl, 126.92; K2S04, 45.35; K2CO3, 74.49; K3C,H5O,-H2O, 103.73; MnCl2-4H2O, 4.5030; FeC«H5O7-5HjO, 5.9991; MgCO3, 43.35; ZnCO,, 0.5274; C/iCl2-2H20,0.4024; KIO3,0.0088 ; NajSeO. -10H2O, 0.0117; CrK(Sq4)2-12H2O, 0.4802. By analysis this adjustment diet was found to contain 12 ppm zinc. 6 Mazóla, CPC Internati., Englewood Cliffs, New Jersey. 6 United States Bio chemical Corp., Cleveland, Ohio.
posed to lead (29). However workers also received a vitamin C supplement and there were no controls for workers occupationally exposed to lead but not treated with vitamin C and zinc. The effect of nutritional status on lead metabolism during pregnancy however has not been investigated despite the fact that lead crosses both the placental and mam mary barriers (30-32). Three recent clini cal reports for example have indicated the dangers of environmental and occupational lead exposure of women during pregnancy in terms of blood lead concentration and erythrocyte protoporphyrin of offspring (33-35). Although currently available De partment of Labor statistics do not detail the number of women employed in leadrelated industries (36-37) women do ac count for almost half of the total work force (36). It may therefore be important to know the possible influence of nutrition on lead transfer from mother to infant especially in women environmentally and occupationally exposed to low levels of lead.
The purpose of the present study is to report the effects of modifying dietary status of zinc on transfer of lead from mother to offspring utilizing the albino rat as the animal model. MATERIALS
AND METHODS
Forty 70 day old virgin female albino rats 3 initially weighing 188 ±2 g, mean ± SEM, were individually housed in sus pended stainless steel cages in a tempera ture controlled animal room with a 12 nour light cycle. Each rat had free access to the purified diet described in table 1 and deionized water for approximately 5 weeks. Precautions were taken to minimize trace metal contamination including use of acidwashed glass food jars and water bottles, silicone rubber stoppers (38) fitted with stainless steel sippers, use of a low metal containing dietary protein egg white as well as crystalline vitamins and reagent grade mineral salts. Furthermore, diets were mixed with a commercial food mixer 4 equipped with stainless steel bowl, splash guard and mixing paddle all of which had been rinsed with deionized water prior to use. After reaching an average weight of 235 ±2 g, three females were mated over night with a stock-fed male.5 The breed ing cages (20 X 48 X 27 cm) which were also used throughout gestation and lacta tion were made of polypropylene fitted with stainless steel wire tops and raised wire floors.6 Wood chips were used to absorb waste. Presence of spermatozoa in the vagina ( 39 ) the following morning was called day 0 gestation. Individual diets containing either 12 or 120 ppm zinc as the carbonate with or without lead as the acetate (0 or 500 ppm) as determined by atomic ab sorption spectrophotometry 7 ( 24 ) were randomly assigned at this time. These diets were prepared by adding appropriate amounts of the specified reagent salts to batches of diet using glucose as a carrier. At day 20 gestation each rat was weighed and placed in a clean cage without a raised 3 Sprague-Duwley, Madison, Wisconsin. • Hobart Corp., Troy. Ohio. 5 Wayne Lab Blox, Chicago, Illinois. « Lab Products, Rochelle Park, New Jersey. 7 Perkln-Elmer model #460, Norwalk, Connecticut.
Downloaded from https://academic.oup.com/jn/article-abstract/109/10/1703/4770604 by Tulane University Library, Serials Acquisitions Dept. user on 12 January 2019
TABLE l Composition of diet
ZINC AND LEAD INTERACTION
DURING
1705
REPRODUCTION
ppm Pb ZnDam ppm weight gain after (6)36.5*±0.6 (4)35.9±1.4 gAverage 20 days of gestation, (6)(6) 129.7 ±8.2 pup weight at (5)120139.2±6.2 (4)12116.0b±5.529.9b±l.l500120(6) 33.8"±0.4 (6) day 16 of lactation, g012137.8"±5.7 1MeandhsEM (n). There were eight pups per group.
wire floor. Rats were allowed to normally deliver their pups on the wood chip bed ding material, ingest the placenta and clean the young. Litters were standardized to eight within 2 days of birth. On day 16 of lactation each dam was weighed and a milk sample was collected as described by Navia (40). Rats were killed by drawing blood from the abdomi nal aorta under light sodium pentobarbital anesthesia. Livers were also removed from each dam and stored at —20°for later analysis. Litter weights were also recorded at this time. Two pups were randomly selected from each group on day 16 of lactation and a blood sample was drawn as previously described. The remaining pups were de capitated. Livers and tibias were removed from each pup and stored at —20° for later analysis. Blood porphyrins were determined in pup blood as well as pup liver delta-aminolevulinic acid dehydratase activity (24). All blood, liver and milk samples were wet-ashed in concentrated reagent grade nitric acid followed by 30% hydrogen peroxide and analyzed for lead by atomic absorption spectrophotometry. All samples
2 a versus b: P < 0.05.
were corrected for interference by simul taneous deuterium-arc background correc tion. Tibias were dry-ashed in an electric muffle furnace at 590°in preparation for lead analysis as described above. The experimental design involved two factors, zinc and lead, with two levels of each factor. In each case an F-test was cal culated and interactions between treat ments were evaluated by least significant difference (41 ). Differences between treat ments were considered to be statistically significant for P < 0.05. A dietary zinc concentration of 12 ppm was selected because it has been reported to be the lowest level that will support normal reproduction in the rat.8 The other zinc level was arbitrarily set at 10 times this concentration. The lead level of 500 ppm was primarily selected to produce elevated milk lead concentrations (32). RESULTS AND DISCUSSION
Because no attempt was made to syn chronize estrus cycles, it was necessary to 8Williams, K. B., Demertzis, P. & Mills. C. F. (1973) The effects of dietary zinc concentration on reproduction in the rat. Proc. Nutr. Soc. ii, 3A-4A.
TABLE 3 Influence of dietary zinc and lead on dam milk lead concentration and pup tibia lead concentration1 500
ppm Pb ppm Zn
12
120
Microgram Pb/ml milk Pup tibia Pb Mg/gra ash
L. CERKLEWSKI of Basic Science, Division of Biochemistry, University School of Dentistry, Wisconsin 53233
ABSTRACT Confirmed pregnant female albino rats received a purified diet containing either 12 or 120 ppm zinc with or without lead (0 or 500 ppm) from day 0 gestation through day 16 of lactation. An 11% reduction in dam weight gain during gestation and a 12% reduction in average pup weight in rats fed the low zinc plus lead diet was markedly different compared to controls not receiving lead and lead supplemented rats receiving supple mental zinc. Milk lead concentration was reduced in rats receiving addi tional dietary zinc and lead which was reflected in a 15% reduction in pup tibia lead concentration, a 20% reduction in inhibition of pup liver deltaaminolevulinic acid dehydratase activity and a 31% reduction in pup blood porphyrin concentration. Some of the beneficial effects of supple mental zinc on lead toxicity of pups may be explained by a reduction of both liver lead (40%) and blood lead (32%) concentration of dams. However an interaction between lead and zinc at the placental and mam mary barriers must also be considered. The results of this study suggest that nutritional status of zinc may be an important factor in modifying lead burden of infants borne by women occupationally and environmentally exposed to lead. J. Nutr. 109: 1703-1709, 1979. INDEXING KEY WORDS lead •zinc •reproduction The trace metal lead has two faces. On one hand it has many industrial applications (1) whereas on the other hand it is a toxic trace metal for man resulting in disruption of heme biosynthesis, renal funetion, nervous system development and reproductive function (2-8). Both environmental and occupational exposure contribute to lead sources of man (4, 9-17). Because it is probably not possible to completely eliminate lead exposure of man, greater emphasis has been placed on limiting lead exposure especially in terms of pediatrie lead poisoning (18-19). Experimental studies have indicated that nutrìtional status may be a modifying factor in this regard.
centrations min i
E,
shown
For example
of protein,
Selenium
and
to exaggerate
low
calcium,
dietary
Zinc have tt_ , •
COn-
iron, vitaall rr
been . r
the toxic effects
lead in rats (20-23). Lead absorption in rats has been shown to be increased for low dietary intakes of iron and zinc and for high intakes of vitamin D (24—26). In other cases susceptibility to biochemical effects of lead can be reduced especially in terms of decreased hematocrit, increased reticulocyte count and red cell fragility (27) as well as inhibition of lead sensitive enzymes such as delta-aminolevulinic acid dehydratase activity (6, 24, 28). At least one clinical trial has investigated the effects of zinc supplementation on blood lead of workers occupationally exReceivedfor publication.March12, 1979. 'Supported In part by the Marquette University
$gg$gS8? on Research and NIH No- I-SOS-BR* Presented °f American
In part at the OSrd Annual Societies for Experimental
of Dalias. Texas. 1703
Federation Biology,
Downloaded from https://academic.oup.com/jn/article-abstract/109/10/1703/4770604 by Tulane University Library, Serials Acquisitions Dept. user on 12 January 2019
Influence of Dietary Zinc on Lead Toxicity During Gestation and Lactation in the Female Rat1'2
1704
FLORIAN L. CERKLEWSKI
Component
g/kg
white*Cellulose Spray-dried egg powder2Vitamin mixture3Mineral mixture4Corn oil6Cornstarch2Anhydrous dextrose620040504060150460 1 Marshall Produce Co., Marshall, Minnesota. 2Teklad Test Diets, Madison, Wisconsin. 3 Milli gram per kilogram glucose except where noted: thiamin-HCl, 200; riboflavin, 200; pyridoxine-HCl, 200 ; calcium pantothenate, 400 ; d-biotin, 40 ; niacin, 500; folie acid, 40; vitamin Bi2 (0.1% in mannitol), 500 ; menadione, 10 ; choline chloride, 30 gm ; vitamin A palmitate (250,000 lU/gm), 1 gm; calciferol (500,000 lU/gm), 80 mg; d-alpha tocopheryl acid succinate (1,210 IU/gm), 800 mg. Made to 1 kg with glucose. 4Gram per kilogram mixture : CaHPOi, 594.23; NaCl, 126.92; K2S04, 45.35; K2CO3, 74.49; K3C,H5O,-H2O, 103.73; MnCl2-4H2O, 4.5030; FeC«H5O7-5HjO, 5.9991; MgCO3, 43.35; ZnCO,, 0.5274; C/iCl2-2H20,0.4024; KIO3,0.0088 ; NajSeO. -10H2O, 0.0117; CrK(Sq4)2-12H2O, 0.4802. By analysis this adjustment diet was found to contain 12 ppm zinc. 6 Mazóla, CPC Internati., Englewood Cliffs, New Jersey. 6 United States Bio chemical Corp., Cleveland, Ohio.
posed to lead (29). However workers also received a vitamin C supplement and there were no controls for workers occupationally exposed to lead but not treated with vitamin C and zinc. The effect of nutritional status on lead metabolism during pregnancy however has not been investigated despite the fact that lead crosses both the placental and mam mary barriers (30-32). Three recent clini cal reports for example have indicated the dangers of environmental and occupational lead exposure of women during pregnancy in terms of blood lead concentration and erythrocyte protoporphyrin of offspring (33-35). Although currently available De partment of Labor statistics do not detail the number of women employed in leadrelated industries (36-37) women do ac count for almost half of the total work force (36). It may therefore be important to know the possible influence of nutrition on lead transfer from mother to infant especially in women environmentally and occupationally exposed to low levels of lead.
The purpose of the present study is to report the effects of modifying dietary status of zinc on transfer of lead from mother to offspring utilizing the albino rat as the animal model. MATERIALS
AND METHODS
Forty 70 day old virgin female albino rats 3 initially weighing 188 ±2 g, mean ± SEM, were individually housed in sus pended stainless steel cages in a tempera ture controlled animal room with a 12 nour light cycle. Each rat had free access to the purified diet described in table 1 and deionized water for approximately 5 weeks. Precautions were taken to minimize trace metal contamination including use of acidwashed glass food jars and water bottles, silicone rubber stoppers (38) fitted with stainless steel sippers, use of a low metal containing dietary protein egg white as well as crystalline vitamins and reagent grade mineral salts. Furthermore, diets were mixed with a commercial food mixer 4 equipped with stainless steel bowl, splash guard and mixing paddle all of which had been rinsed with deionized water prior to use. After reaching an average weight of 235 ±2 g, three females were mated over night with a stock-fed male.5 The breed ing cages (20 X 48 X 27 cm) which were also used throughout gestation and lacta tion were made of polypropylene fitted with stainless steel wire tops and raised wire floors.6 Wood chips were used to absorb waste. Presence of spermatozoa in the vagina ( 39 ) the following morning was called day 0 gestation. Individual diets containing either 12 or 120 ppm zinc as the carbonate with or without lead as the acetate (0 or 500 ppm) as determined by atomic ab sorption spectrophotometry 7 ( 24 ) were randomly assigned at this time. These diets were prepared by adding appropriate amounts of the specified reagent salts to batches of diet using glucose as a carrier. At day 20 gestation each rat was weighed and placed in a clean cage without a raised 3 Sprague-Duwley, Madison, Wisconsin. • Hobart Corp., Troy. Ohio. 5 Wayne Lab Blox, Chicago, Illinois. « Lab Products, Rochelle Park, New Jersey. 7 Perkln-Elmer model #460, Norwalk, Connecticut.
Downloaded from https://academic.oup.com/jn/article-abstract/109/10/1703/4770604 by Tulane University Library, Serials Acquisitions Dept. user on 12 January 2019
TABLE l Composition of diet
ZINC AND LEAD INTERACTION
DURING
1705
REPRODUCTION
ppm Pb ZnDam ppm weight gain after (6)36.5*±0.6 (4)35.9±1.4 gAverage 20 days of gestation, (6)(6) 129.7 ±8.2 pup weight at (5)120139.2±6.2 (4)12116.0b±5.529.9b±l.l500120(6) 33.8"±0.4 (6) day 16 of lactation, g012137.8"±5.7 1MeandhsEM (n). There were eight pups per group.
wire floor. Rats were allowed to normally deliver their pups on the wood chip bed ding material, ingest the placenta and clean the young. Litters were standardized to eight within 2 days of birth. On day 16 of lactation each dam was weighed and a milk sample was collected as described by Navia (40). Rats were killed by drawing blood from the abdomi nal aorta under light sodium pentobarbital anesthesia. Livers were also removed from each dam and stored at —20°for later analysis. Litter weights were also recorded at this time. Two pups were randomly selected from each group on day 16 of lactation and a blood sample was drawn as previously described. The remaining pups were de capitated. Livers and tibias were removed from each pup and stored at —20° for later analysis. Blood porphyrins were determined in pup blood as well as pup liver delta-aminolevulinic acid dehydratase activity (24). All blood, liver and milk samples were wet-ashed in concentrated reagent grade nitric acid followed by 30% hydrogen peroxide and analyzed for lead by atomic absorption spectrophotometry. All samples
2 a versus b: P < 0.05.
were corrected for interference by simul taneous deuterium-arc background correc tion. Tibias were dry-ashed in an electric muffle furnace at 590°in preparation for lead analysis as described above. The experimental design involved two factors, zinc and lead, with two levels of each factor. In each case an F-test was cal culated and interactions between treat ments were evaluated by least significant difference (41 ). Differences between treat ments were considered to be statistically significant for P < 0.05. A dietary zinc concentration of 12 ppm was selected because it has been reported to be the lowest level that will support normal reproduction in the rat.8 The other zinc level was arbitrarily set at 10 times this concentration. The lead level of 500 ppm was primarily selected to produce elevated milk lead concentrations (32). RESULTS AND DISCUSSION
Because no attempt was made to syn chronize estrus cycles, it was necessary to 8Williams, K. B., Demertzis, P. & Mills. C. F. (1973) The effects of dietary zinc concentration on reproduction in the rat. Proc. Nutr. Soc. ii, 3A-4A.
TABLE 3 Influence of dietary zinc and lead on dam milk lead concentration and pup tibia lead concentration1 500
ppm Pb ppm Zn
12
120
Microgram Pb/ml milk Pup tibia Pb Mg/gra ash
