LNVIROSMENTAL
RESEARCH
Increased
15, 57-64
(1978)
Susceptibility to Lead Toxicity Semipurified Diet+*
A. A. MYLROIE,
L. MOORE,
Received
B. OLYAI.
May
in Rats Fed
AND M. ANDERSON
25. 1976
Rats fed stock diets, like Purina Chow. are known to be relatively insensitive to the toxic effects of lead. The addition of lead acetate (1000 ppm of Pb) to the drinking water of S-week-old rats for 35 days produced toxicity (decreased growth rate and lead-induced anemia) in rats fed semipurified diet. but not in rats fed Purina Rat Chow (Chow). Blood and tissue lead levels also were higher in rats fed semipurified diet than in rats fed Chow. In order to determine whether rats fed semipurified diet could be used as animal models in the determination of permissible levels of lead in the environment. the susceptibility to lead toxicity of these rats to lead-based paints was tested. The addition of 1% paint chips containing lOpi lead octoate to the diets resulted in lead toxicity (reduction in growth rates and hematocrit values) only in rats fed the semipurified diet. Rats fed a semipurified diet that permits normal growth are more susceptible to the toxic effects of lead and can be used more effectively in lead toxicity studies than rats fed Chow.
INTRODUCTION
Rats are the most frequently used animals in studies of lead toxicity, yet, except for the infant rat, they are considered to be relatively insensitive to the toxic effects of lead (Pate1 et nl., 1974). According to Scharding and Oehme (1973), rats are able to tolerate as much as 60 mg of lead per day for periods of over 1 year, a dose equivalent to 8 g of lead in man. In order to produce lead toxicity in rats, high concentrations of lead salts ( l-5%) are usually added to the feed (Sobel et ul., 1940: Baernstein and Grand, 1942; Chiodi and Cardeza, 1949; Pardoe, 1952; Sobel and Burger, 1955: Goyer, 1968; Michaelson and Sauerhoff, 1974; Patelet nl., 1974). The resistance to lead toxicity has lessened the usefulness of rats as experimental animals. Nevertheless. permissible concentrations of lead in the environment have been based on lead toxicity studies using rats. Partially as a result of a research report entitled “Lead Paint Ingestion Studies” (Midwest Research Institute, 1974), the legal limit for lead in household paints has been set by Congress (PL 93-151) at 0.5’;; (National Paint and Coatings Association, 1975). In addition much information on lead toxicity, lead metabolism, and the interaction of lead with other environmental pollutants is based on experiments using rats (National Academy of Sciences, 1972). In most of these studies, the animals were fed stock diets like Purina Chow. ’ This investigation was supported by Grant RR-(08043-05) from General Research Support NIH. ’ Presented partly at the 170th American Chemical Society meeting, August. 1975 in Chicago, and partly at the Proceedings of the Fourth Annual Xavier MBS Biomedical Symposium, April. New Orleans. Louisiana.
Branch, Illinois, 1976, in
57 0013-9351/78/0151-0057$02.00/0 Copyright c-i 1978 by Academic Press. Inc. All rights of reproduction m any form reserve
58
MYLROIE
E7 AL
Semipurified diets have been used primarily to determine the effects of individual dietary components on lead metabolism and lead retention. Vitamin D. calcium, and phosphorus were reported to affect the concentration of lead in soft tissue and bone (Sobel of rrl., 1940: Sobel and Burger. 1955). Levels of calcium (Six and Goyer. 1970; Mahaffey c’r rrl.. 1973). iron (Six and Goyer, 1972). as well as copper(Klaudel and Petering, 1975) in the diet were found to affect lead retention and lead toxicity in the rat. The deliberate use of semipurified diets containing sufficient amounts of essential nutrients, in place of stock diets like Purina Rat Chow, has not yet been proposed in lead toxicity studies. In the present study, the susceptibility of rats to lead toxicity was examined in rats fed Chow and in rats fed Normal Protein Test diet.’ a commercially produced semipurified diet. Rats fed Normal Protein Test diet were found to be more sensitive to the toxic effects of lead (decreased growth rate and lead-induced anemia) than rats fed Chow. Rats fed semipurified diets can be useful as animal models for lead metabolism studies as well as for the study of permissible concentrations of lead in the environment, such as the determination of the legal limit for lead in paints. MATERIALS AND METHODS I. Dirt studies. Eighty 4-week-old male Holtzman rats weighing 60-80 g were fed
Chow for 1 week. They were then divided into two dietary groups: Half the animals were fed Chow, and the other half. Normal Protein Test Diet (NPT). Lead-treated rats in each dietary group had lead acetate added to the drinking water ( 1 mg of Pbiml) and the control animals received deionized water only. The NPT diet was commercially obtained in pelletized form and its composition is given in Table I. Animals were individually housed in suspended cages in a temperature-controlled room. Food and drinking water were given ad lihirum. Food intake and water consumption were recorded throughout the 5-week experimental period. Rats were placed in metabolic cages for urine collection during the fourth week of the experiment. Glacial acetic acid (0.1 ml) was added to the urine collection containers and the samples were frozen to prevent breakdown of urinary &aminolevulinic acid (ALA). ALA was analyzed by the method of Kornfield and Ullman (1972). At the end of the 5-week period, animals were anesthetized with ether and blood was collected by heart puncture into heparinized syringes. Liver, kidney. spleen,
Component of diet
(%) 27 56.8 IO 4
Casein Sucrose Oil
Salt mixture. USP XIV Vitamin fortification mixture ‘I Commercially z Obtained
obtained in pelletized
from form
ICN from
Pharmaceuticals, ICN
Pharmaceuticals,
2.2 Cleveland. Cleveland,
Ohio. Ohio.
LEAD
TOXICITY
IN
59
RATS
and femur were excised and weighed. These tissues were digested with concentrated HNO, and subsequently analyzed for lead using an Instrumentation Laboratory (IL 253) atomic absorption spectrophotometer in the A-B mode. employing a hydrogen continuum lamp for background correction. Matrix studies indicated negligible interference with Pb analysis due to incomplete digestion. Hemoglobin, hematocrit, and red blood cell determinations were made manually as well as with a Coulter counter. 2. PLI~‘I~Tirlgrstiorl studies. Paint chips containing 1057 lead in the form of lead octoate were added to either Chow or NPT diet. Twelve rats were divided into two groups; one group was fed Chow + 1% paint chips. and the other group was fed NPT diet + 1% paint chips. The animals were weighed twice a week and food consumption was measured. At the end of the 35day experimental period, rats were sacrificed as described above in the diet studies. Hematocrits were determined and blood and tissue lead concentrations were measured after appropriate nitric acid digestion. Standard statistical techniques were used in interpreting the data (Edwards, 1969). RESULTS I. Diet Stlidics Wright goin, jimd c,orl.rrrnzptiotl, cr~rtl Irrrd ingrstiorl.
Rats fed NPT diet exhibited normal weight gains in the absence of lead: however. in the presence of lead, the rats showed a 20% decrease in weight gain as well as a 10% decrease in food consumption (Table 2). In contrast, rats fed Chow grew equally well in the presence or absence of added lead: their food consumption was not affected by the addition of lead. Rats given lead acetate solution generally drank less than those given water. Rats on NPT + Pb drank slightly less than rats on Chow + Pb and thus ingested somewhat less lead (Table 2). No correlation was found in individual rats within a dietary group between fluctuations in lead consumption and lead retention in tissue. Blood trrltl tissrlcj l~rrti (.otl(.etltt.~ltio/l.~. Lead-treated rats fed NPT diet had significantly (P < 0.01) higher lead concentrations in bone as well as in soft tissues. liver. kidney. and spleen (Table 3). than did rats fed Chow. Blood lead concentrations of rats fed Chow fall within the “acceptable range” (0.4 pg of Pbiml) for human blood. despite the high intake of lead. Blood lead levels of rats fed NPT diet were significantly elevated (P < 0.01).
Weight gain Diet
Food consumed (g)
Solution consumed (ml)
+
214 -+ 6.1 216 + 6.0
930 i 945 i
1100 2 30 885 2 I@
3.8
+
221 2 6.1 177 _f 3.0
770 -t 20 600 t 14’
850 -+ 32 763 2 34h
3.5
Pb
Chow NPT
(kc)
” Data are expressed as the mean f SEM. ’ Significantly different from control animals
18 12
not receiving
lead.
Pb consumed/ average body weight (mdg)
60
ET AL
ILIYLROIE
Lead Diet Chow NFT
Pb
Pb in blood
(&ml)
in tissues
Live1
Bone
@gig
wet wt)
Kidney
Spleen
+
RESEARCH
Increased
15, 57-64
(1978)
Susceptibility to Lead Toxicity Semipurified Diet+*
A. A. MYLROIE,
L. MOORE,
Received
B. OLYAI.
May
in Rats Fed
AND M. ANDERSON
25. 1976
Rats fed stock diets, like Purina Chow. are known to be relatively insensitive to the toxic effects of lead. The addition of lead acetate (1000 ppm of Pb) to the drinking water of S-week-old rats for 35 days produced toxicity (decreased growth rate and lead-induced anemia) in rats fed semipurified diet. but not in rats fed Purina Rat Chow (Chow). Blood and tissue lead levels also were higher in rats fed semipurified diet than in rats fed Chow. In order to determine whether rats fed semipurified diet could be used as animal models in the determination of permissible levels of lead in the environment. the susceptibility to lead toxicity of these rats to lead-based paints was tested. The addition of 1% paint chips containing lOpi lead octoate to the diets resulted in lead toxicity (reduction in growth rates and hematocrit values) only in rats fed the semipurified diet. Rats fed a semipurified diet that permits normal growth are more susceptible to the toxic effects of lead and can be used more effectively in lead toxicity studies than rats fed Chow.
INTRODUCTION
Rats are the most frequently used animals in studies of lead toxicity, yet, except for the infant rat, they are considered to be relatively insensitive to the toxic effects of lead (Pate1 et nl., 1974). According to Scharding and Oehme (1973), rats are able to tolerate as much as 60 mg of lead per day for periods of over 1 year, a dose equivalent to 8 g of lead in man. In order to produce lead toxicity in rats, high concentrations of lead salts ( l-5%) are usually added to the feed (Sobel et ul., 1940: Baernstein and Grand, 1942; Chiodi and Cardeza, 1949; Pardoe, 1952; Sobel and Burger, 1955: Goyer, 1968; Michaelson and Sauerhoff, 1974; Patelet nl., 1974). The resistance to lead toxicity has lessened the usefulness of rats as experimental animals. Nevertheless. permissible concentrations of lead in the environment have been based on lead toxicity studies using rats. Partially as a result of a research report entitled “Lead Paint Ingestion Studies” (Midwest Research Institute, 1974), the legal limit for lead in household paints has been set by Congress (PL 93-151) at 0.5’;; (National Paint and Coatings Association, 1975). In addition much information on lead toxicity, lead metabolism, and the interaction of lead with other environmental pollutants is based on experiments using rats (National Academy of Sciences, 1972). In most of these studies, the animals were fed stock diets like Purina Chow. ’ This investigation was supported by Grant RR-(08043-05) from General Research Support NIH. ’ Presented partly at the 170th American Chemical Society meeting, August. 1975 in Chicago, and partly at the Proceedings of the Fourth Annual Xavier MBS Biomedical Symposium, April. New Orleans. Louisiana.
Branch, Illinois, 1976, in
57 0013-9351/78/0151-0057$02.00/0 Copyright c-i 1978 by Academic Press. Inc. All rights of reproduction m any form reserve
58
MYLROIE
E7 AL
Semipurified diets have been used primarily to determine the effects of individual dietary components on lead metabolism and lead retention. Vitamin D. calcium, and phosphorus were reported to affect the concentration of lead in soft tissue and bone (Sobel of rrl., 1940: Sobel and Burger. 1955). Levels of calcium (Six and Goyer. 1970; Mahaffey c’r rrl.. 1973). iron (Six and Goyer, 1972). as well as copper(Klaudel and Petering, 1975) in the diet were found to affect lead retention and lead toxicity in the rat. The deliberate use of semipurified diets containing sufficient amounts of essential nutrients, in place of stock diets like Purina Rat Chow, has not yet been proposed in lead toxicity studies. In the present study, the susceptibility of rats to lead toxicity was examined in rats fed Chow and in rats fed Normal Protein Test diet.’ a commercially produced semipurified diet. Rats fed Normal Protein Test diet were found to be more sensitive to the toxic effects of lead (decreased growth rate and lead-induced anemia) than rats fed Chow. Rats fed semipurified diets can be useful as animal models for lead metabolism studies as well as for the study of permissible concentrations of lead in the environment, such as the determination of the legal limit for lead in paints. MATERIALS AND METHODS I. Dirt studies. Eighty 4-week-old male Holtzman rats weighing 60-80 g were fed
Chow for 1 week. They were then divided into two dietary groups: Half the animals were fed Chow, and the other half. Normal Protein Test Diet (NPT). Lead-treated rats in each dietary group had lead acetate added to the drinking water ( 1 mg of Pbiml) and the control animals received deionized water only. The NPT diet was commercially obtained in pelletized form and its composition is given in Table I. Animals were individually housed in suspended cages in a temperature-controlled room. Food and drinking water were given ad lihirum. Food intake and water consumption were recorded throughout the 5-week experimental period. Rats were placed in metabolic cages for urine collection during the fourth week of the experiment. Glacial acetic acid (0.1 ml) was added to the urine collection containers and the samples were frozen to prevent breakdown of urinary &aminolevulinic acid (ALA). ALA was analyzed by the method of Kornfield and Ullman (1972). At the end of the 5-week period, animals were anesthetized with ether and blood was collected by heart puncture into heparinized syringes. Liver, kidney. spleen,
Component of diet
(%) 27 56.8 IO 4
Casein Sucrose Oil
Salt mixture. USP XIV Vitamin fortification mixture ‘I Commercially z Obtained
obtained in pelletized
from form
ICN from
Pharmaceuticals, ICN
Pharmaceuticals,
2.2 Cleveland. Cleveland,
Ohio. Ohio.
LEAD
TOXICITY
IN
59
RATS
and femur were excised and weighed. These tissues were digested with concentrated HNO, and subsequently analyzed for lead using an Instrumentation Laboratory (IL 253) atomic absorption spectrophotometer in the A-B mode. employing a hydrogen continuum lamp for background correction. Matrix studies indicated negligible interference with Pb analysis due to incomplete digestion. Hemoglobin, hematocrit, and red blood cell determinations were made manually as well as with a Coulter counter. 2. PLI~‘I~Tirlgrstiorl studies. Paint chips containing 1057 lead in the form of lead octoate were added to either Chow or NPT diet. Twelve rats were divided into two groups; one group was fed Chow + 1% paint chips. and the other group was fed NPT diet + 1% paint chips. The animals were weighed twice a week and food consumption was measured. At the end of the 35day experimental period, rats were sacrificed as described above in the diet studies. Hematocrits were determined and blood and tissue lead concentrations were measured after appropriate nitric acid digestion. Standard statistical techniques were used in interpreting the data (Edwards, 1969). RESULTS I. Diet Stlidics Wright goin, jimd c,orl.rrrnzptiotl, cr~rtl Irrrd ingrstiorl.
Rats fed NPT diet exhibited normal weight gains in the absence of lead: however. in the presence of lead, the rats showed a 20% decrease in weight gain as well as a 10% decrease in food consumption (Table 2). In contrast, rats fed Chow grew equally well in the presence or absence of added lead: their food consumption was not affected by the addition of lead. Rats given lead acetate solution generally drank less than those given water. Rats on NPT + Pb drank slightly less than rats on Chow + Pb and thus ingested somewhat less lead (Table 2). No correlation was found in individual rats within a dietary group between fluctuations in lead consumption and lead retention in tissue. Blood trrltl tissrlcj l~rrti (.otl(.etltt.~ltio/l.~. Lead-treated rats fed NPT diet had significantly (P < 0.01) higher lead concentrations in bone as well as in soft tissues. liver. kidney. and spleen (Table 3). than did rats fed Chow. Blood lead concentrations of rats fed Chow fall within the “acceptable range” (0.4 pg of Pbiml) for human blood. despite the high intake of lead. Blood lead levels of rats fed NPT diet were significantly elevated (P < 0.01).
Weight gain Diet
Food consumed (g)
Solution consumed (ml)
+
214 -+ 6.1 216 + 6.0
930 i 945 i
1100 2 30 885 2 I@
3.8
+
221 2 6.1 177 _f 3.0
770 -t 20 600 t 14’
850 -+ 32 763 2 34h
3.5
Pb
Chow NPT
(kc)
” Data are expressed as the mean f SEM. ’ Significantly different from control animals
18 12
not receiving
lead.
Pb consumed/ average body weight (mdg)
60
ET AL
ILIYLROIE
Lead Diet Chow NFT
Pb
Pb in blood
(&ml)
in tissues
Live1
Bone
@gig
wet wt)
Kidney
Spleen
+
