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Concentrations of lead in the soft tissues of male rats during a long-term dietary exposure
ENVIRONMENTAL
RESEARCH
28, 147- 153 (1982)
Concentrations of Lead in the Soft Tissues of Male Rats during a Long-Term Dietary Exposure H. M. MYKKANEN,’ Division
of Nutrition
M. C. LANCASTER,~
AND J. W. T. DICKERSON
and Food Science, Department of Biochemistry, Surrey. Guildford. Surrey GU2 5XH. England
University
of
Received May 28, 1981 Newborn rats of albino Wistar strain were exposed to lead from birth, first indirectly through maternal milk and then directly through a diet containing 0.5% lead acetate. At 3,6, and 12 months of age some of the male rats were killed for the determination of the concentrations of lead in the blood, brain, kidneys, and liver. This long-term exposure resulted in a slight retardation of growth, yet no change in the food consumption was seen. Concentrations of lead in the blood and brain were similar at 3 and 6 months of age, but significantly higher at 12 months, while the concentrations of lead in the kidneys increased significantly with age. Of the brain parts examined the forebrain had the highest concentration of lead, which increased significantly with age. The present study, together with already published data, demonstrated clearly that lead accumulated in rat brain during the suckling period remains relatively unchanged throughout adult life, while the concentrations in the blood and other soft tissues fall rapidly after weaning. Thus the blood lead level cannot be used as a measure of the amount of lead in the brain, particularly in the case of long-term or intermittent exposure of the young.
INTRODUCTION
Knowledge of the levels of lead in various tissues during a period of lead intoxication is essential to the understanding of the diverse toxic effects of this heavy metal. Unfortunately, data on the basic metabolism of lead, particularly in children and after a long-term exposure, are rather limited. Barry and Mossman (1970) found 90% of the total body burden of lead in the bones. However, it is the soft tissue fraction which appears to be responsible for the known acute toxic effects of this metal (Chisolm, 1971). In humans, the highest concentrations of lead are found in the aorta, kidneys, and liver, whereas heart, muscle, and brain all show consistently low values (Schroeder and Balassa, 1961; Schroeder and Tipton, 1968; Barry and Mossman, 1970; Barry, 1975). Furthermore, the concentrations in these organs appear to be higher during the second decade of life, and remain relatively constant thereafter (Barry and Mossman, 1970). The above studies were, however, carried out on persons who did not have a history of abnormal lead exposure, and thus may not be relevant in cases of a long or intermittent increased exposure to this metal. In our earlier report we presented data on the soft tissue lead levels in young ’ To whom reprint requests should be sent. Present address: Department of Nutrition, University of Helsinki, 00710 Helsinki 71, Finland. 2 Present address: Boots Company Limited, Pennyfoot Street, Nottingham, England. 147 0013-9351/82/030147-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
148
MYKKANEN,
LANCASTER,
AND
DICKERSON
rats exposed to lead from birth, first indirectly through maternal milk, and then directly through a diet containing lead (Mykkanen et al., 1979). We found in paralyzed suckling rats concentrations of lead similar to those in children suffering from acute lead poisoning (Chisolm, 1968). The present study provides data on the concentrations of lead in the blood and soft tissues of older rats during a long-term dietary exposure to this metal. MATERIALS
AND
METHODS
The animals, diets, and conditions of housing were the same as those previously described (Mykkanen ef al., 1979). The experimental diets were fed to the mother rats starting from the day on which the litters were born. The food intake and weight gain of the lactating rats were monitored weekly. The litters were weaned at 21 days of age, the sexes were separated, and the young rats were caged in groups of three to five animals in community cages. After weaning the young rats were fed the same diets as those given to their mothers, and the food intakes (per cage) were determined periodically. The body weights were measured weekly. At 3,6, and 12 months of age some of the animals were killed for the determination of the concentrations of lead in the blood, brain, kidney, and liver. The brain was dissected into forebrain, cerebellum, and the rest (called “brain stem”). Blood samples were stored at +4”C and the tissues at -15°C until analyzed for lead. For the solubilization of the tissues a modification of the method of Mm-thy and co-workers (1973) was used, and the determination of the lead concentrations was carried out using the nickel-cup technique of Delves (1970). Data on the male rats only are included in this report. Means and standard deviations were calculated for each group. The pairs of group means were compared statistically using Student’s t test. RESULTS
The rats fed diets without added lead consistently weighed about 10% more than those that had received lead in their diet (Fig. 1). The difference in the body weights was not, however, due to the food intake, since this was similar for both
-150
150 . 0 0 2
4
AGE
6
6
10
12
(months)
FIG. 1. Growth of the male Wistar rats fed control diet (0) or diet containing 0.5% lead acetate (0) measured by mean body weights.
SOFT TISSUE Pb DURING
LONG-TERM
149
EXPOSURE
TABLE 1 EFFECTS OF LONG-TERM LEAD” INGESTION ON THE BODY AND ORGAN WEIGHTS IN MALE WISTAR RATS
Diet
No. of animals
Body weight w
Brain
Kidney
Liver
3
Control Lead
10 10
399 * 48 359 2 34*
2.08 k 0.13 1.93 k 0.06**
2.49 + 0.21 2.45 ” 0.31
14.81 k 1.78 13.92 k 2.02
6
Control Lead
8 8
504 + 49 473 2 27
2.23 f 0.11 2.07 f 0.06**
2.67 2 0.23 2.97 k 0.31*
19.25 k 1.84 19.78 k 1.21
12
Control Lead
6 11
668 ? 36 587 k 51**
2.28 k 0.07 2.12 ” 0.06***
3.46 -t 0.40 4.11 f 0.98
19.59 2 1.77 18.64 f 2.50
Age (months)
Organ weight (g)
Note. Values are given as means I SD. Values significantly different from appropriate values are indicated by superscripts denoting the level of significance (Student’s t test). a 0.5% lead acetate in the diet. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.
control
groups. Likewise, the brain weights (calculated from the weights of the brain parts) were consistently lower in the lead-exposed animals (Table l), the difference being significant at all ages studied. The kidney weights were similar in the lead-exposed and control animals at 3 months, but slightly higher in the lead group at 6 and 12 months (P < 0.05 at 6 months, Table 1). The difference was not statistically significant at 12 months, when the kidney weights were rather variable in the lead-treated animals. The concentrations of lead in the blood and brain of the lead-exposed animals were similar at 3 and 6 months of age, but were significantly higher at 12 months (Table 2). The concentration of lead in the kidneys increased with age so that the TABLE 2 CONCENTRATIONS OF LEAD IN THE BLOOD AND SOFT TISSUES OF MALE WISTAR
RATS DURING
A LONG-TERM
DIETARY
EXPOSURE
TO LEADS
Lead concentration
Age
No. of animals
Blood (~p/l@J Mb
Brain h-%kY
Kidney bih3)’
Liver hdg)’
3 6 12
10 8 11
76t 11 78 k ll*** 103 2 17***
1.23 k 0.24” 1.11 f 0.24** 1.61 2 0.42***
13.6 k 1.2*** 18.9 + 2.7*** 35.9 + 6.6***
2.86 ~fr0.34*** 1.99 k 0.35*** 4.59 k 0.98***
(months)
Note. Values are given as means + SD. Significantly different values are indicated by superscripts, the levels of significance as in Table 1. Values not significantly different are connected by braces. n 0.5% lead acetate in the diet. b Whole blood. I’ Wet weight.
150
MYKKANEN,
LANCASTER,
AND
DICKERSON
differences between all ages were highly significant (P < 0.001, Table 2). Liver lead concentrations were rather variable with age, and after a transient reduction from 3 to 6 months of age there was a considerable increase at 12 months (Table 2). Of the three parts of the brain studied, the forebrain had the highest concentration and this significantly increased its lead content with age (Table 3). Figure 2 shows the data on the blood and brain lead concentrations in l-, 3-, 5, and 7-week-old rats (data from Mykkanen et al., 1979) and in 3-, 6-, and 12month-old animals exposed to lead first indirectly through maternal milk and then directly through the diet. It is evident that the concentration of lead in the blood decreases rapidly after weaning until 7 weeks of age, and thereafter is maintained relatively unchanged. On the contrary, the brain appears to retain the lead accumulated during the suckling period throughout adult life. DISCUSSION
The long-term exposure to dietary lead in the present study produced a slight retardation of growth, yet no change in the food consumption was seen. A natural explanation for this result would be either increased excretion or decreased absorption of nutrients. Lead intoxication is known to enhance the urinary excretion of amino acids (Goyer, 1968), but there is some evidence that decreased weight gain is one of the most sensitive indices of the metabolic effects of lead, and becomes apparent at a smaller dose than that producing aminoaciduria (Goyer et al., 1970). In fact, there is some indication that lead may inhibit intestinal absorption of nutrients (Gruden et al., 1974; Wapnir et al., 1977), but the relevance of these findings, involving toxic quantities of lead, to the present environmental lead levels needs to be established. The concentrations of lead in the 3- and 6-month-old rats in the present study were comparable to those in wild rats from an urban environment. Mouw and co-workers (1975) reported that wild rats, weighing just under 400 g, had the TABLE CONCENTRATIONS WISTAR
Age (months) 3 6 12
OF LEAD RATS
IN THE DURING
No. of animals 10 8 10’
FOREBRAIN, A LONG-TERM
3 CEREBELLUM, DIETARY
Lead Forebrain 1.48 ‘- 0.30 1.45 + 0.38*” 2.02 ? 0.65**
AND EXPOSURE
concentration
BRAIN
STEM
OF MALE
TO LEADS
(pdg)b
Cerebellum
Brain
stem
0.80 5 0.24 0.70 ? 0.16 1.14 + 0.14
0.90 2 0.38 0.70 t 0.19 1.05 k 0.32
Note. Values are given as means + SD. Significantly different values are indicated by superscripts, the levels of significance as in Table 1. Values not significantly different are connected by braces. Differences in lead concentrations between forebrain and cerebellum and between forebrain and brain stem were highly significant (P < 0.001). Differences in lead concentrations between cerebellum and brain stem were not significant. a 0.5% lead acetate in the diet. * Wet weight. c Four cerebellum samples pooled from eight animals.
SOFT
TISSUE
Pb DURING
LONG-TERM
151
EXPOSURF
3,o
2.0 1.0
I
. * * 1357
13
26
AGE
52
(weeks)
FIG. 2. Concentrations of lead in the blood (0) and brain (m) of the male Wistar rats during a long-term dietary exposure, first indirectly via maternal milk and then directly through a diet containing 0.5% lead acetate (mean ? SD).
following tissue lead concentrations: blood, 55 pug/100 ml; brain, 1.11 pg/g; kidneys, 22.7 pglg; and liver, 3.34 &g. The respective values in the 3-month-old male rats fed 0.5% lead acetate in the diet were 76, 1.23, 13.6, and 2.86. Although Mouw and co-workers stressed the importance of the respiratory exposure to airborne lead as a cause for the accumulation of lead in urban rats, they did not deny the possible contribution of other factors such as street dirt and dust, and peeling paint. The occurrence of behavioral disorders as the result of exposure to present urban lead concentrations is a much debated subject. Evidence has been presented that children with only slightly increased blood lead levels show some behavioral abnormalities, such as hyperactivity, impaired tine-motor coordination, and learning problems (David et al., 1972; de la Burde and Choate, 1972; Pueschel et al., 1972; Beattie et al., 1975; Landrigan et al., 1975). In contrast are the reports claiming that these abnormalities were caused by social and environmental factors rather than by lead (Kotok, 1972; Lansdown et al., 1974). These controversies might be explained, at least partly, by the use of the blood lead concentration as an indicator of lead exposure. Nordberg (1976) has postulated that the levels of lead in critical organs, such as brain, kidneys, and bone marrow, relate more closely to the harmful effects in these organs than blood lead levels. In extension of the previous results (Goldstein er al., 1974; Mykkanen et al., 1979), the present data clearly demonstrate that the amount of lead in rat brain accumulated during the suckling period is maintained relatively unchanged throughout adult life, while the concentration of lead in the blood falls with increasing age. Thus the blood lead level cannot be used as a measure of the amount of lead in the brain, particularly in the case of a long-term exposure of the young. Furthermore, retention of lead by the brain after the levels in the blood and in
152
MYKKANEN,
LANCASTER,
AND DICKERSON
other soft tissues fall may be responsible for the chronic neurologic disorders after an intermittent exposure to this metal. The distribution of lead within the brain may be of importance concerning the harmful effects of this metal on the nervous system. The present findings that the highest concentration of lead was in the forebrain appear to conflict with the results of Niklowitz and Yeager (1973) and Goldstein and co-workers (1974), who reported no differences in lead concentration between various parts of the brains of acutely intoxicated rabbits and suckling rats. On the other hand, Michaelson (1973) reported that the concentration of lead in the cerebellum was twice that in the cerebrum of 3-week-old rats exposed to lead via maternal milk. Extensive hemorrhage in the cerebellar cortex may have contributed to the latter finding. In fact, in the study by Goldstein and co-workers (1974), the concentration of lead in the cerebrum of the adult rats slightly exceeded the concentrations in the cerebellum and pons. The kidneys were slightly enlarged and, although tests of renal function were not done, it is possible that impaired renal function may partly explain the increased concentrations of lead in the blood and tissues of the lZmonth-old animals in the present study. Goyer (1968) found a positive correlation between the increase in kidney weight of lead-intoxicated rats and aminoaciduria. Cramer and co-workers (1974) observed a lower excretion of lead in the urine, and lack of intranuclear inclusion bodies in the kidneys, of the workers with longer periods of exposure to lead, and they suggested that severe prolonged lead exposure may result in frank renal malfunction. The present findings of rats with body burdens of lead similar to those of wild urban rats clearly demonstrate the persistence into adult life of lead accumulated in the brain early in life. These observations are particularly significant concerning the controversial behavioral disorders resulting from long-term lead exposure of the young. It seems that unless one can define the relation between blood and brain lead concentrations at various ages and after various exposures, the use of blood lead concentration as an indicator of soft tissue lead in a long-term or intermittent increased exposure may be misleading. Naturally, the levels of lead in the soft tissues other than blood cannot be measured readily in humans, and therefore an animal model such as described in the present study is needed and may be of considerable value in helping to understand the diverse toxic effects of lead.
REFERENCES Barry, P. S. I. (1975). A comparison of concentrations of lead in human tissues. Bn’f. J. Ind. Med. 32, 119-139. Barry, P. S. I., and Mossman, D. B. (1970). Lead concentrations in human tissues. Bit. J. Ind. Med. 27, 339-351. Beattie, A. D., Moore, M. R., Goldberg, A., Finlayson, M. J. W., Graham, J. F., Mackie, E. M., Main, J. C., McLaren, D. A., Murdoch, R. M., and Stewart, G. T. (1975). The role of chronic low-level lead exposure in the etiology of mental retardation. Lancet 1, 589-592. de la Burde, B., and Choate, M. L. (1972). Does asymptomatic lead exposure in children have latent sequelae? J. Pediatr. (St. Louis) 81, 1088- 1091.
SOFT
TISSUE
Pb DURING
LONG-TERM
EXPOSURE
153
Chisolm, J. J., Jr. (1968). The use of chelating agents in the treatment of acute and chronic lead intoxication in childhood. J. Pediatr. (St. Louis) 73, l-38. Chisolm, J. J., Jr. (1971). Lead poisoning. Sci. Amer. 224, 15-23. Cramer, K., Goyer, R. A., Jagenburg, R., and Wilson, M. H. (1974). Renal ultrastructure, renal function and parameters of lead toxicity in workers with different periods of lead exposure. Brit. J. Ind. Med. 31, 113-127. David, O., Clark, J., and Voeller, K. (1972). Lead and hyperactivity. Lancer 2, 900-903. Delves, H. T. (1970). A micro-sampling method for the rapid determination of lead in blood by atomic absorption spectrophotometry. Analyst 95, 431-438. Goldstein, G. W., Asbury, A. K., and Diamond, I. (1974). Pathogenesis of lead encephalopathy. Uptake of lead and reaction of brain capillaries. Arch. Neurol. 31, 382-389. Goyer, R. A. (1968). The renal tubule in lead poisoning. I. Mitochondrial swelling and aminoaciduria. Lab. Invest. 19, 71-77. Goyer, R. A., Leonard, D. L., Moore, J. F., Rhyne, B., and Krigman, M. R. (1970). Lead dosage and the role of intranuclear inclusion body. Arch. Environ. Health 20, 705-711. Gruden, N., Stantic, M., and Buben, M. (1974). Influence of lead on calcium and strontium transfer through the duodenal wall in rats. Environ. Res. 8, 203-206. Kotok, D. (1972). Development of children with elevated blood lead levels. A controlled study. J. Pediatr. (St. Louis) 80, 57-61. Landrigan, P. J., Whitworth, R. H., Baloh, R. W., Stachling, N. W., Barthel, W. F., and Rosenblum, B. F. (1975). Neuropsychological dysfunction in children with chronic low-level lead absorption. Lancet 1, 708-712. Lansdown, R. G., Shepherd, J., Clayton, B. E., Delves, H. T., Graham, P. J., and Turner, W. L. (1974). Blood-lead levels, behaviour, and intelligence-A population study. Lancet 1, 538-541. Michaelson, I. A. (1973). Effects of inorganic lead on RNA, DNA and protein content in the developing neonatal rat brain. Toxicol. Appl. Pharmacol. 26, 539-548. Mouw, D., Kalitis, K., Anver, M., Schwartz, J., Constan, A., Hartung, R., Cohen, B., and Ringler, D. (1975). Lead. Possible toxicity in urban vs. rural rats. Arch. Environ. Health 30, 276-280. Murthy, L., Menden, E. E., Eller, P. M., and Petering, H. G. (1973). Atomic absorption determination of zinc, copper, cadmium and lead in tissues solubilized by aqueous tetramethyl ammonium hydroxide. Anal. Biochem. 53, 365-372. Mykkanen, H. M., Dickerson, J. W. T., and Lancaster, M. C. (1979). Effect of age on tissue distribution of lead in the rat. Toxicol. Appl. Pharmacol. 51, 447-454. Niklowitz, W. J., and Yeager, D. W. (1973). Interference of Pb with essential brain tissue Cu, Fe, and Zn as main determinant in experimental tetraethyl lead encephalopathy. Life Sci. 13, 897-905. Nordberg, G. F. (1976). Effects and dose-response relationships of toxic metals. In “Proceedings, International Meeting on Occupational Health, Tokyo, November 18-23, 1974.” Elsevier, Amsterdam. Pueschel, S. M., Kopito, L., and Schwachman, H. (1972). Children with increased lead burden. A screening and follow-up study. J. Amer. Med. Assoc. 222, 462-466. Schroeder, H. A., and Balassa, J. J. (1961). Abnormal trace metals in man: Lead. J. Chronic Dis. 14, 408-425. Schroeder, H. A., and Tipton, I. H. (1968). The human body burden of lead. Arch. Environ. Health 17, 965-977. Wapnir, R. A., Exeni, R. A., McVicar, M., and Lifshitz, F. (1977). Experimental lead poisoning and intestinal transport of glucose, amino acids, and sodium. Pediatr. Res. 11, 153- 157.
RESEARCH
28, 147- 153 (1982)
Concentrations of Lead in the Soft Tissues of Male Rats during a Long-Term Dietary Exposure H. M. MYKKANEN,’ Division
of Nutrition
M. C. LANCASTER,~
AND J. W. T. DICKERSON
and Food Science, Department of Biochemistry, Surrey. Guildford. Surrey GU2 5XH. England
University
of
Received May 28, 1981 Newborn rats of albino Wistar strain were exposed to lead from birth, first indirectly through maternal milk and then directly through a diet containing 0.5% lead acetate. At 3,6, and 12 months of age some of the male rats were killed for the determination of the concentrations of lead in the blood, brain, kidneys, and liver. This long-term exposure resulted in a slight retardation of growth, yet no change in the food consumption was seen. Concentrations of lead in the blood and brain were similar at 3 and 6 months of age, but significantly higher at 12 months, while the concentrations of lead in the kidneys increased significantly with age. Of the brain parts examined the forebrain had the highest concentration of lead, which increased significantly with age. The present study, together with already published data, demonstrated clearly that lead accumulated in rat brain during the suckling period remains relatively unchanged throughout adult life, while the concentrations in the blood and other soft tissues fall rapidly after weaning. Thus the blood lead level cannot be used as a measure of the amount of lead in the brain, particularly in the case of long-term or intermittent exposure of the young.
INTRODUCTION
Knowledge of the levels of lead in various tissues during a period of lead intoxication is essential to the understanding of the diverse toxic effects of this heavy metal. Unfortunately, data on the basic metabolism of lead, particularly in children and after a long-term exposure, are rather limited. Barry and Mossman (1970) found 90% of the total body burden of lead in the bones. However, it is the soft tissue fraction which appears to be responsible for the known acute toxic effects of this metal (Chisolm, 1971). In humans, the highest concentrations of lead are found in the aorta, kidneys, and liver, whereas heart, muscle, and brain all show consistently low values (Schroeder and Balassa, 1961; Schroeder and Tipton, 1968; Barry and Mossman, 1970; Barry, 1975). Furthermore, the concentrations in these organs appear to be higher during the second decade of life, and remain relatively constant thereafter (Barry and Mossman, 1970). The above studies were, however, carried out on persons who did not have a history of abnormal lead exposure, and thus may not be relevant in cases of a long or intermittent increased exposure to this metal. In our earlier report we presented data on the soft tissue lead levels in young ’ To whom reprint requests should be sent. Present address: Department of Nutrition, University of Helsinki, 00710 Helsinki 71, Finland. 2 Present address: Boots Company Limited, Pennyfoot Street, Nottingham, England. 147 0013-9351/82/030147-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
148
MYKKANEN,
LANCASTER,
AND
DICKERSON
rats exposed to lead from birth, first indirectly through maternal milk, and then directly through a diet containing lead (Mykkanen et al., 1979). We found in paralyzed suckling rats concentrations of lead similar to those in children suffering from acute lead poisoning (Chisolm, 1968). The present study provides data on the concentrations of lead in the blood and soft tissues of older rats during a long-term dietary exposure to this metal. MATERIALS
AND
METHODS
The animals, diets, and conditions of housing were the same as those previously described (Mykkanen ef al., 1979). The experimental diets were fed to the mother rats starting from the day on which the litters were born. The food intake and weight gain of the lactating rats were monitored weekly. The litters were weaned at 21 days of age, the sexes were separated, and the young rats were caged in groups of three to five animals in community cages. After weaning the young rats were fed the same diets as those given to their mothers, and the food intakes (per cage) were determined periodically. The body weights were measured weekly. At 3,6, and 12 months of age some of the animals were killed for the determination of the concentrations of lead in the blood, brain, kidney, and liver. The brain was dissected into forebrain, cerebellum, and the rest (called “brain stem”). Blood samples were stored at +4”C and the tissues at -15°C until analyzed for lead. For the solubilization of the tissues a modification of the method of Mm-thy and co-workers (1973) was used, and the determination of the lead concentrations was carried out using the nickel-cup technique of Delves (1970). Data on the male rats only are included in this report. Means and standard deviations were calculated for each group. The pairs of group means were compared statistically using Student’s t test. RESULTS
The rats fed diets without added lead consistently weighed about 10% more than those that had received lead in their diet (Fig. 1). The difference in the body weights was not, however, due to the food intake, since this was similar for both
-150
150 . 0 0 2
4
AGE
6
6
10
12
(months)
FIG. 1. Growth of the male Wistar rats fed control diet (0) or diet containing 0.5% lead acetate (0) measured by mean body weights.
SOFT TISSUE Pb DURING
LONG-TERM
149
EXPOSURE
TABLE 1 EFFECTS OF LONG-TERM LEAD” INGESTION ON THE BODY AND ORGAN WEIGHTS IN MALE WISTAR RATS
Diet
No. of animals
Body weight w
Brain
Kidney
Liver
3
Control Lead
10 10
399 * 48 359 2 34*
2.08 k 0.13 1.93 k 0.06**
2.49 + 0.21 2.45 ” 0.31
14.81 k 1.78 13.92 k 2.02
6
Control Lead
8 8
504 + 49 473 2 27
2.23 f 0.11 2.07 f 0.06**
2.67 2 0.23 2.97 k 0.31*
19.25 k 1.84 19.78 k 1.21
12
Control Lead
6 11
668 ? 36 587 k 51**
2.28 k 0.07 2.12 ” 0.06***
3.46 -t 0.40 4.11 f 0.98
19.59 2 1.77 18.64 f 2.50
Age (months)
Organ weight (g)
Note. Values are given as means I SD. Values significantly different from appropriate values are indicated by superscripts denoting the level of significance (Student’s t test). a 0.5% lead acetate in the diet. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.
control
groups. Likewise, the brain weights (calculated from the weights of the brain parts) were consistently lower in the lead-exposed animals (Table l), the difference being significant at all ages studied. The kidney weights were similar in the lead-exposed and control animals at 3 months, but slightly higher in the lead group at 6 and 12 months (P < 0.05 at 6 months, Table 1). The difference was not statistically significant at 12 months, when the kidney weights were rather variable in the lead-treated animals. The concentrations of lead in the blood and brain of the lead-exposed animals were similar at 3 and 6 months of age, but were significantly higher at 12 months (Table 2). The concentration of lead in the kidneys increased with age so that the TABLE 2 CONCENTRATIONS OF LEAD IN THE BLOOD AND SOFT TISSUES OF MALE WISTAR
RATS DURING
A LONG-TERM
DIETARY
EXPOSURE
TO LEADS
Lead concentration
Age
No. of animals
Blood (~p/l@J Mb
Brain h-%kY
Kidney bih3)’
Liver hdg)’
3 6 12
10 8 11
76t 11 78 k ll*** 103 2 17***
1.23 k 0.24” 1.11 f 0.24** 1.61 2 0.42***
13.6 k 1.2*** 18.9 + 2.7*** 35.9 + 6.6***
2.86 ~fr0.34*** 1.99 k 0.35*** 4.59 k 0.98***
(months)
Note. Values are given as means + SD. Significantly different values are indicated by superscripts, the levels of significance as in Table 1. Values not significantly different are connected by braces. n 0.5% lead acetate in the diet. b Whole blood. I’ Wet weight.
150
MYKKANEN,
LANCASTER,
AND
DICKERSON
differences between all ages were highly significant (P < 0.001, Table 2). Liver lead concentrations were rather variable with age, and after a transient reduction from 3 to 6 months of age there was a considerable increase at 12 months (Table 2). Of the three parts of the brain studied, the forebrain had the highest concentration and this significantly increased its lead content with age (Table 3). Figure 2 shows the data on the blood and brain lead concentrations in l-, 3-, 5, and 7-week-old rats (data from Mykkanen et al., 1979) and in 3-, 6-, and 12month-old animals exposed to lead first indirectly through maternal milk and then directly through the diet. It is evident that the concentration of lead in the blood decreases rapidly after weaning until 7 weeks of age, and thereafter is maintained relatively unchanged. On the contrary, the brain appears to retain the lead accumulated during the suckling period throughout adult life. DISCUSSION
The long-term exposure to dietary lead in the present study produced a slight retardation of growth, yet no change in the food consumption was seen. A natural explanation for this result would be either increased excretion or decreased absorption of nutrients. Lead intoxication is known to enhance the urinary excretion of amino acids (Goyer, 1968), but there is some evidence that decreased weight gain is one of the most sensitive indices of the metabolic effects of lead, and becomes apparent at a smaller dose than that producing aminoaciduria (Goyer et al., 1970). In fact, there is some indication that lead may inhibit intestinal absorption of nutrients (Gruden et al., 1974; Wapnir et al., 1977), but the relevance of these findings, involving toxic quantities of lead, to the present environmental lead levels needs to be established. The concentrations of lead in the 3- and 6-month-old rats in the present study were comparable to those in wild rats from an urban environment. Mouw and co-workers (1975) reported that wild rats, weighing just under 400 g, had the TABLE CONCENTRATIONS WISTAR
Age (months) 3 6 12
OF LEAD RATS
IN THE DURING
No. of animals 10 8 10’
FOREBRAIN, A LONG-TERM
3 CEREBELLUM, DIETARY
Lead Forebrain 1.48 ‘- 0.30 1.45 + 0.38*” 2.02 ? 0.65**
AND EXPOSURE
concentration
BRAIN
STEM
OF MALE
TO LEADS
(pdg)b
Cerebellum
Brain
stem
0.80 5 0.24 0.70 ? 0.16 1.14 + 0.14
0.90 2 0.38 0.70 t 0.19 1.05 k 0.32
Note. Values are given as means + SD. Significantly different values are indicated by superscripts, the levels of significance as in Table 1. Values not significantly different are connected by braces. Differences in lead concentrations between forebrain and cerebellum and between forebrain and brain stem were highly significant (P < 0.001). Differences in lead concentrations between cerebellum and brain stem were not significant. a 0.5% lead acetate in the diet. * Wet weight. c Four cerebellum samples pooled from eight animals.
SOFT
TISSUE
Pb DURING
LONG-TERM
151
EXPOSURF
3,o
2.0 1.0
I
. * * 1357
13
26
AGE
52
(weeks)
FIG. 2. Concentrations of lead in the blood (0) and brain (m) of the male Wistar rats during a long-term dietary exposure, first indirectly via maternal milk and then directly through a diet containing 0.5% lead acetate (mean ? SD).
following tissue lead concentrations: blood, 55 pug/100 ml; brain, 1.11 pg/g; kidneys, 22.7 pglg; and liver, 3.34 &g. The respective values in the 3-month-old male rats fed 0.5% lead acetate in the diet were 76, 1.23, 13.6, and 2.86. Although Mouw and co-workers stressed the importance of the respiratory exposure to airborne lead as a cause for the accumulation of lead in urban rats, they did not deny the possible contribution of other factors such as street dirt and dust, and peeling paint. The occurrence of behavioral disorders as the result of exposure to present urban lead concentrations is a much debated subject. Evidence has been presented that children with only slightly increased blood lead levels show some behavioral abnormalities, such as hyperactivity, impaired tine-motor coordination, and learning problems (David et al., 1972; de la Burde and Choate, 1972; Pueschel et al., 1972; Beattie et al., 1975; Landrigan et al., 1975). In contrast are the reports claiming that these abnormalities were caused by social and environmental factors rather than by lead (Kotok, 1972; Lansdown et al., 1974). These controversies might be explained, at least partly, by the use of the blood lead concentration as an indicator of lead exposure. Nordberg (1976) has postulated that the levels of lead in critical organs, such as brain, kidneys, and bone marrow, relate more closely to the harmful effects in these organs than blood lead levels. In extension of the previous results (Goldstein er al., 1974; Mykkanen et al., 1979), the present data clearly demonstrate that the amount of lead in rat brain accumulated during the suckling period is maintained relatively unchanged throughout adult life, while the concentration of lead in the blood falls with increasing age. Thus the blood lead level cannot be used as a measure of the amount of lead in the brain, particularly in the case of a long-term exposure of the young. Furthermore, retention of lead by the brain after the levels in the blood and in
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AND DICKERSON
other soft tissues fall may be responsible for the chronic neurologic disorders after an intermittent exposure to this metal. The distribution of lead within the brain may be of importance concerning the harmful effects of this metal on the nervous system. The present findings that the highest concentration of lead was in the forebrain appear to conflict with the results of Niklowitz and Yeager (1973) and Goldstein and co-workers (1974), who reported no differences in lead concentration between various parts of the brains of acutely intoxicated rabbits and suckling rats. On the other hand, Michaelson (1973) reported that the concentration of lead in the cerebellum was twice that in the cerebrum of 3-week-old rats exposed to lead via maternal milk. Extensive hemorrhage in the cerebellar cortex may have contributed to the latter finding. In fact, in the study by Goldstein and co-workers (1974), the concentration of lead in the cerebrum of the adult rats slightly exceeded the concentrations in the cerebellum and pons. The kidneys were slightly enlarged and, although tests of renal function were not done, it is possible that impaired renal function may partly explain the increased concentrations of lead in the blood and tissues of the lZmonth-old animals in the present study. Goyer (1968) found a positive correlation between the increase in kidney weight of lead-intoxicated rats and aminoaciduria. Cramer and co-workers (1974) observed a lower excretion of lead in the urine, and lack of intranuclear inclusion bodies in the kidneys, of the workers with longer periods of exposure to lead, and they suggested that severe prolonged lead exposure may result in frank renal malfunction. The present findings of rats with body burdens of lead similar to those of wild urban rats clearly demonstrate the persistence into adult life of lead accumulated in the brain early in life. These observations are particularly significant concerning the controversial behavioral disorders resulting from long-term lead exposure of the young. It seems that unless one can define the relation between blood and brain lead concentrations at various ages and after various exposures, the use of blood lead concentration as an indicator of soft tissue lead in a long-term or intermittent increased exposure may be misleading. Naturally, the levels of lead in the soft tissues other than blood cannot be measured readily in humans, and therefore an animal model such as described in the present study is needed and may be of considerable value in helping to understand the diverse toxic effects of lead.
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