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CNS lead toxicity in rabbit offspring
ENVIRONMENTAL
RESEARCH
17, 13l-
1% (1978)
CNS Lead Toxicity
in Rabbit Offspring
A. V. LORENZO,~ MYRNA GEWIRTZ, AND DAMON AVERILL with technical assistance of M. Mauer Department
of Neurology and Pharmacology, Harvard Medical and the Division of Neuroscience and Neuropathology, Children’s Hospital Medical Center. Boston, Massachusetts
School, 02115
Received September 8, 1977 An animal model for asymptomatic and overt lead toxicity is presented in which rabbit pups, 1 to 30 days of age, were fed a milk supplement containing Pb(NO& Reduced body growth, increased mortality, hematologic abnormalities, kidney and liver histopathology, as well as encephalopathy, were observed in rabbits 2 to 30 days of age ingesting more than 2.8 mg of Pb/day. At the highest dose, brain lesions were widespread, involving the frontal and occipital cortex, striatum, hippocampus, and cerebellum. At moderate doses, the region most affected was the cerebellum. At the lowest dose no alteration in body, brain growth, mortality, or tissue histology was evident: however, basophilic stippling was observed. AS a group, these asymptomatic rabbits generally exhibited higher activity than non-lead-fed littermates. The rabbit model presented here should provide many advantages in the study of lead toxicity on the developing nervous system, since (a) maximum brain development in the rabbit, like that in rhesus monkey and man, occurs perinatally, and(b) the effects of lead toxicity in the rabbit closely parallel those in man.
INTRODUCTION Accidental lead poisoning in children and lead toxicity in industrial workers have been, in the past, the primary concern of health officials dealing with the problem of lead intoxication. At present, as a result of increased contamination of the environment with heavy metals, it is the opinion of some (Cole, 1977; Lockeretz, 1975; Urban, 1976; Waldron and Stofen, 1974), but not others (Barry, 1975), that continuous exposure to low levels of lead in the environment may constitute the more serious problem. Indeed, numerous investigators have expressed grave concern regarding the possible impairment of mental development of children exposed to environmental concentrations of lead during infancy or early childhood (Baloh et al., 1975; Beattie et al., 1975; Chisolm, 1977; David et al., 1976; de la Burde and Choate, 1972, 1975). Childhood lead poisoning is recognized as one of the more serious health hazards in the United States. It is estimated that some 5 to 10% of all children in the U.S. have abnormally high blood lead levels (Gilsinn, 1972). Of the many children exposed to lead each year, only a small proportion (0.5%) exhibit overt clinical symptoms; the majority remain clinically asymptomatic (Browder et al., 1973). Many of the clinically asymptomatic children develop subtle neurological and behavioral defects (Browder et al., 1973; Byers and Lord, 1943; David et al., 1973; de la Burde and Choate, 1975). Unfortunately, other factors such as I Address reprint requests to: A. V. Lorenzo, Children’s Hospital Medical Center, Neuroscience Research Department, 300 Longwood Avenue, Boston, Massachusetts, 02115. 131 0013-9351/78/0171-0131$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
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nutritional and socioeconomic status confound the relationship of lead to mental development (Committee on Toxicology, 1976; Waldron and Stofen, 1974). It is difftcult, therefore, to ascribe the increased frequency of intellectual impairment and behavioral aberrations observed in these clinically asymptomatic children to low-level lead exposure only (Beattie et al., 1975; Committee on Toxicology, 1976). For this reason investigators have attempted to develop animal models that parallel the effects of lead toxicity on the developing human central nervous system (CNS) (Bornschein et al., 1977b; Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966; Silbergeld and Goldberg, 1973). In 1966, Pentschew and Garro introduced an animal model for lead encephalopathy which has become the prototype for subsequent experiments. In this model, lactating rats exposed to 4% lead carbonate in their diet transmit lead via the maternal milk to the suckling rat. The lead-exposed suckling animal has proven to be a valuable experimental model to study the effects of overt lead toxicity on the CNS (Goldstein et al., 1974; Krigman et al., 1974a,b; Rosenblum and Johnson, 1968; Thomas et al., 1971). Its use to investigate cognitive as well as behavioral abnormalities arising from asymptomatic lead poisoning is not devoid, however, of serious drawbacks (Sobotka et al., 1975). The ingestion of 1 to 5% lead salts by the dam leads to diminished food intake (Bornschein et al., 1977b), weight loss (Krigman et al; 1974a; Lampert et al., 1967; Michaelson and Sauerhoff, 1974), and alteration in the nutritional quality of the milk in the dam (Bornschein et al., 1977b). Since the dam’s milk constitutes the only source of nutriment available to the offspring before weaning, the pronounced growth retardation exhibited by the offspring (Bornschein et al., 1977a, b; Krigman et al., 1974a; Michaelson, 1973; Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966; Silbergeld and Goldberg, 1973) can, in part, be attributed to malnutrition (Baernstein and Grand, 1942), specific nutritional deficiencies, or both. Nutritional deficiencies are known to potentiate the toxic effects of lead (Jugo, 1976; Levander et al., 1975; Mahaffey et al., 1973; Six and Goyer, 1972). When they occur during the critical period of brain development, long-lasting neuropsychological abnormalities result (Davison and Dobbing, 1968). These secondary effects arising from lead-induced changes in the dam apparently can be circumvented if either the concentration of lead in the diet is drastically reduced (Brown, 1975), or if lead is fed directly to the neonatal animal (Sobotka et al., 1975). Direct administration of lead to young rats (Brown et al., 1971), dogs (Stowe et al., 1973), or rabbits (Hass et al., 1964), however, does not necessarily result in CNS dysfunction even when, in the case of the rabbit, lead exposure is extended for 9 months (Hass et al., 1964). Abnormal behavior in weanling rats that are otherwise asymptomatic can be produced, if lead administration is started at birth (Brown, 1975; Sobotka et al., 1975; Winneke et al., 1977). In the present study in which rabbit pups are fed lead directly, we present a model for overt and asymptomatic lead toxicity that (a) provides a more accurate assessment of lead dosage to the offspring and (b) avoids confounding variables arising from the effect of lead on the dam. METHODS
Sixty-five New Zealand female rabbits, weighing 3.5 to 5.0 kg, impregnated in our animal facility, were housed in individual cages provided with a nesting box
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containing straw. They were fed normal laboratory chow (Blue Seal Lab Pels, H.K. Webster Co., Lawrence, Mass.) and tap water ad libitum. Twelve to 24 hours after parturition, the progeny were weighed, and the litter size was reduced to six pups. Each day, from 1 to 30 days of age, offspring were examined, weighed, and fed 1 ml of milk formula consisting of 100 mg of “Unilac” (Upjohn Co., Kalamazoo, Mich.) powder dissolved in 1 ml of water warmed to 37°C to which was added 0 (controls), 1.0, 4.5, 9.0, or 18.0 mg/ml of Pb (NO,),. Spontaneous locomotor activity was measured daily for a total of 20 min in an activity cage (Lehigh Valley Electronics, Model 1487) which registered, during four consecutive 5 min periods, the number of times a photoelectric light beam path was interrupted. Blood, brain, and peripheral tissues were obtained from animals killed at 2, 10, 15, 20, and 30 days of age or at autopsy. Tissue samples were divided and were either fixed in 10% buffered formalin for histology, dried to constant weight in a vacuum oven at 100°C to determine water content, or stored for lead assay. Samples of blood were obtained by cardiac puncture using heparinized disposable syringes. Possible lead contamination of syringes and heparin was assessed by drawing aliquots from a lo-ml heparin vial into five individual syringes (Table 1). Other tissues were dissected free using acidwashed, stainless-steel surgical instruments. Blood samples were transferred to lead-free, loo-p1 capillary tubes. [Environmental Sciences Associates (ESA), Burlington, Mass.]. Blood and tissue samples were transferred to lead-free tubes provided by ESA, for digestion in a mixture of nitric, sulfuric, and perchloric acids. Concentrations of lead in tissues and in sources of lead other than the oral dose administered, which could potentially increase the total lead ingested or contaminate tissue samples, were assayed with an ESA anodic stripping voltammeter (Model 3010 for blood and Model 2014 for tissues) according to the method of Searle er al. (1972) (Table 1). A matrix of distilled HZ0 and perchloric acid (pH 1.8), containing various concentrations of lead, was used to calibrate the instruments daily. Analysis of 15 aliquots of blood drawn from a single rabbit 18 hours after iv injection of 5 mg of lead acetate/kg, by the above procedure, indicated a blood-lead concentration of 86.6 + 1.2 pg%. Blank level variability differed from day to day, ranging from 0.007 (n=4) to 0.018 (n=5) pg of lead. RESULTS
Lead Concentration in Tissues Concentration of lead in blood of dams and offspring fed 0, 1.O, and 4.5 mg of Pb TABLE POSSIBLE
SOURCES
Source
n*
Unilac Water Rabbit chow (pellets) Heparin
4 5 4 5
n n = number of samples assayed, b Values are means 2 SEM.
1 OF LEAD
Lead concentration (mhl or g) 0.30 0.01 0.33 0.01
f 2 f f
0.04b 0.00 0.10 0.00
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(NO&/day is shown in Table 2. Even though dosage, based on kilogram body weight, decreased with age, blood lead concentrations kept increasing from 2 to 20 days. By 30 days, the lead concentration in blood was lower. Controls kept separate had lower lead values than those housed with lead-fed littermates. Presumably, this may be the result of eating contaminated straw, preening and licking behavior, or ingesting excreted lead transmitted via the dam’s milk or a combination thereof. The latter, however, does not appear likely, since the concentration of lead in the dam’s blood was relatively low (Table 2). The percentage of blood lead associated with plasma was highest at 2 days and decreased thereafter (Table 2). Nevertheless, the level at 20 and 30 days was somewhat higher than that reported for older animals and children (Rosen and Trinidad, 1974). The concentration and pattern of lead accumulation from 2 to 30 days in tissues of animals exposed to 0, 1.0, and 4.5 mg of Pb (NO&/day is shown in Fig. 1. At all doses and ages the concentration of lead in bone (ulna) exceeded the levels found in other tissues. Generally, lead accumulation in bone increased with continuing exposure to lead, even when concentrations in blood remained constant (Fig. 1A) or decreased (Figs. 1B and C). Lead concentration in liver was next highest. With TABLE CONCENTRATION
OF LEAD
(pglml)
2
IN BLOOD
OF DAMS
AND
NEWBORN
RABBITS~
Daily lead dose (mg of lead nitrate/day) Age (days) 2
0 0.56 k 0.13’ (7) 0.10 ‘- 0.01’
1 1.45 k 0.27
15
0.49 2. 0.01 (7) 0.08 +- 0.01’ (4) 0.56 f 0.05
(6) 20 30
0.75 -t 0.11 (10) 0.54 k 0.12
(8)
15.3d
(6)
(6)
10
4.5
1.54 2 0.25 (9)
6.1d
1.28 k 0.21 (5) 2.15 2 0.35 (10) 0.87 f 0.11
4.ld
(6)
3.ld l.8d
P/B x lOO*
2.00 2 0.62 (7)
76.2d
3.69 k 0.68 (5)
24.6d
3.99 f 1.24 (5) 6.60 + 0.73
18.6d
(6)
0.79 k 0.09 (7)
33.41
(20) 28.37
(21) 15.47
(16) 14.56 8.3d
8.01 (20) 9.09 (11)
0.14 2 0.05’ (3) Dams
0.06 f O.Olf
(8) n Values are means * SE; number of animals in parentheses. * P/B x 100, lead concentration in plasma as a percentage of blood lead concentration for all lead dosages. c Non-lead-fed animals housed with lead-fed littermates. d Average dose of lead nitrate (milligram per kilogram basis) fed orally to pups at designated age. p Non-lead-fed animals kept separate from lead-fed neonates. r Blood was obtained from the marginal ear vein of the dam on day last pup of litter died.
LEAD
TOXICITY
I A. w mg
B.
Pb mo&~w
In 0
5
18 lo
ls
11, 20
IN
26
m
0 0
a 6
1.0
RABBIT
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maPbtNO&dw
8 lo
1s
c.
0 20
’ 22
II 20
0
4.Sm(1 PbO40&14
t 5
8 lo
t Is
“1 20
2s
20
ms
FIG. 1. Lead concentration in tissues of lead- and non-lead-fed rabbit neonates from 2 to 30 days of age receiving (A) 0 (B) 1.0, and (C) 4.5 mg of lead nitrate/day. Ordinate scale is logarithmic. Values represent means, and bars indicate SEM.
the exception of levels in 20- and 30-day-old control animals (Fig. IA), liver concentrations consistently exceeded concentrations in blood. In contrast to lead accumulation in bone, that in liver tended to decrease after 15 or 20 days more or less paralleling the fall observed in blood lead levels. The lead concentration in kidneys of controls was lower than that of other tissues assayed; however, with lead exposure [l.O and 4.5 mg of Pb (NO&/day] lead concentration in kidneys became markedly elevated, sometimes exceeding levels observed in blood and CNS of these animals. Interestingly, accumulation of lead in kidneys appeared to abruptly increase after 10 and plateau after 20 days, a pattern which may be related to initiation of lead excretion by the kidney. The concentration of lead in cerebellum and whole brain was usually lower or equal to that of blood. At all doses, and nearly at all ages, the concentration of lead in cerebellum exceeded that of whole brain. Even though accumulation in cerebellum and whole brain fluctuated with continuing lead exposure, in general, accumulation in cerebellum was highest during the first 15 days of lead exposure, while that in whole brain was highest from 15 to 30 days of age. Lead concentrations in selected tissues of pups receiving 4.5 mg of lead nitrate/day and which succumbed to the toxic effects
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of lead are shown in Table 3. Lead concentrations were considerably higher in tissues of autopsied noenates than in tissues of littermates fed the same daily doses but which were killed at 2, 10, or 15 days. Appearance
of Offspring
At all doses, lead-fed offspring could be distinguished from control littermates by the ruffled appearance of their pelts (Fig. 2). At the highest doses some of the lead-fed animals exhibited a grayish pallor and a splayed stance. The spontaneous motor activity (motility) of 11 lead-fed (1 mg of lead nitrate/day) and six non-lead-fed animals was generally highest during the first and second Sminute intervals (Fig. 3) (except in the 20- to 25day-old control group). During the second or third period, the activity declined sharply and thereafter tended to level off or increase slightly. In general, the average motility of the leadtreated animals was always greater than their non-lead-fed littermates, except at Day 10 when the activity was equal to or lower than that of controls. No significant differences were found between individual mean activities (Colquhoun, 1971) of the two groups; however, using the nonparametric test of Smimov (Conover, 1971), the activity during the first two Sminute periods of lead-fed pups was found to be significantly different (P
3
OF LEAD IN TISSUES OF NEONATES NITRATE PER DAY THAT DIED OF LEAD
Concentration Age
(days)
0 - 56
6 - 10 11 -15 Average for all ages (autopsied) Values for neonates killed’
FED 4.5 MC OF LEAD TOXICITY~
(&g)
Kidney
Liver
Brain
Cerebellum
42.3 (1) 67.5 k 17.1 (3) 35.1 r 11.7 (3) 50.6 k 11.2 (7)
306.2 (1) 419.5 2 146.0 (3) 96.5 2 28 (3) 264.9 f 84.1 (7)
0.5 (1) 4.2; 2.0
-
6.1 ? 1.0
19.9 2 2.1
(18) P <
0.001”
(18) P <
0.001
9.2; 9.9
4.5 k 2.2 (3) 3.4 k 1.2
9.5 2 -
(6)
(2)
1.1 + 0.1
5.7 T 1.1 (13)
(16) P < 0.05
-
a Values are means 2 SE; number of neonates in each group in parentheses. b Age span during which neonates died as a result of lead toxicity. c Values for littermates killed from 2 to 15 days of age which also were fed 4.5 mg of lead nitrate/day. d P values determined by Student’s I test.
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FIG. 2. Typical appearance of rabbit neonates fed 0 and 4.5 mg of lead nitrate/day for 15 days. The ruffled appearance of the pelt of rabbits receiving concentrations of lead higher than 1 mg of lead nitrate/day was usually accompanied by growth retardation and splayed stance. Similar observations have been reported for rats (Michaelson, 1973) and mice (Rosenblum and Johnson, 1968) exposed to lead via the dam’s milk. Young rabbits fed 1 mg of lead nitrate/day, however, exhibited a ruffled pelt but no other overt signs of lead exposure. Since it is known that in humans lead concentrates in hair, it is possible that the altered appearance of the pelt of neonatal rabbits may reflect an accumulation of lead.
The mortality determined at 30 days of age for pups fed 0, 1.0,4.5,9.0, and 18.0 mg of Pb (NO,)$day was approximately 16, 17,50,85, and lOO%, respectively. At 4.5,9.0, and 18.0 mg of Pb (NO&day, 50% of the offspring had died by 11,7, and 5 days, respectively. While the mortality for pups fed 1 mg of Pb (NO&/day was
400 I
OL58T6%3 is-
4?36%eQ Tiicc-
-
5MY
10
IMvts-
s’w GG5
FIG. 3. Locomotor activity in counts per minute of rabbit pups was always measured prior to feeding 1 mg of lead nitrate/day.
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o--o
GEWIRTZ,
AND
AVERILL
CONTROL
125
18 mg Pb/day
FIG. 4. Average weight gain as a function of age and lead exposure. Scale for animals treated with 18 mg of lead nitrate/day is different from others.
approximately 17%, a seemingly high rate, it should be noted that untreated, hutch-raised rabbit pups generally have a mortality which exceeds 11.2% (Whitney et al., 1976). The high mortality obtained in the asymptomatic [l mg of Pb (NO&/day] groups can probably be attributed to the daily handling of pups by the investigators, as reflected by the mortality of 16% observed for non-lead-fed offspring. Hematologic Studies Basophilic stippling of erythrocytes was observed in all lead-fed siblings, even those receiving 1 mg of lead nitrate/day. Stippling occurred as early as Day 2, but thereafter, the total number of stippled red blood cells declined with age so that by Day 30 stippling was absent. Anisocytosis and poikilocytosis were marked in lead-fed rabbits, though present too, to some extent, in controls. “Thornapple” forms of erythrocytes were noted only in lead-fed animals; Roscoe and associates (1975) reported, however, that these are common in normal rabbit blood smears as well. The mean hematocrit of rabbit pups fed 4.5 mg of Pb (NO&day fell conspicuously only at Day 20 (Fig. 5) when blood lead levels were maximum. With 1 mg of Pb (NO&/day, there was no dramatic change in the hematocrit
LEAD
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1
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4.5
Fro 5. Mean hematocrit of neonatal rabbits fed 0, 1, and 4.5 mg of lead nitrate/day. Numbers appearing within bars represent age of groups while those appearing above the bars represent averages and SEM of blood lead concentrations for the respective group. A dagger (t) denotes statistically significant differences (P
values determined at 10, 15, 20, or 30 days. Furthermore, at 1 mg of Pb (NO&/day, the hematocrit values, determined at 10, 1.5, 20, and 30 days, did not differ significantly from those of control littermates. By Day 30, when blood lead levels of pups exposed to 1 and 4.5 mg of Pb (NO&day was approximately equal to that of the control littermates. Tissue Histology
The principle renal changes observed in rabbit neonates fed 4.5, 9.0, and 18.0 mg of Pb (NO&day were acute tubular necrosis, proteinuria, and intranuclear inclusion bodies. Mild to moderate hepatocellular changes, which included focal fatty degeneration and occasional intranuclear inclusion bodies, were present in animals fed 4.5, 9.0 and 18.0 mg of Pb (NO&/day. No histopathological changes in kidney or liver were present in controls or pups fed 1.0 mg of Pb (NO&day. Hematoxylinand eosin-stained sections of brains from rabbits exposed to 4.5,
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FIG. 6. Hematoxylin- and eosin-stained sections of (A) striatum, (B) hippocampus, (C) occipital cortex, and (D) cerebellum from a 6-day-old rabbit neonate exposed to 18 mg of lead nitrate/day. (A) Widespread petechiation and hemorrhagic necrosis as shown in the section of caudate nucleus was pronounced throughout the striatal area. (B) Disseminated petechiation and ecchymosis is prominent
LEAD TOXICITY
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throughout the pyramidal cell layer and less so m the molecular and granular cell layer of the hippocampus. Clear fluid spaces and cavitation are observed throughout all layers. In (C) disseminated petechiae are observed in the deep cortical cell layers (polymorphous). In the cerebellum (D) cellular infiltration and hemorrhagic necrosis of molecular, Purkinje, and granular layers is extensive. Magnification, x 360.
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9.0, and 18.0 mg of Pb (NO&day contained disseminated lesions which were absent in brain sections from rabbits that received 0 or 1 mg of Pb (NO&day. At 4.5 and 9.0 mg of Pb (NO&/day, the cerebral cortex contained microhemorrhages and acute neuronal necrosis, while at 18.0 mg of Pb (NO&/day there were hemorrhagic necrosis, gliosis, and cellular depletion in striatal areas, olfactory lobe, hippocampus, and occipital cortex (Fig. 6). The telencephalic white matter manifested erythrophagocytosis and diffuse petechiation, while the medulla, pons, midbrain, thalamus, and hippocampus exhibited mild to moderate disseminated petechiae and ecchymosis. Extensive lesions were also noted in the cerebella of animals fed 4.5, 9.0, and 18.0 mg of Pb (NO&/day (Fig. 7). At 4.5 mg Pb (NO&/day, multifocal petechiae were observed in the molecular, Purkinje, and internal granular cell layers. Following 9.0 mg of Pb (NO&/day, Purkinje cell loss, cellular depletion in the internal granular layer, focal gliosis within the molecular cell layer, as well as cystic degeneration of the white matter and focal Purkinje cell calcification were observed in one animal surviving to 24 days of age. At 18.0 mg of Pb (NO&day, there was hemorrhagic necrosis of the white matter with neuronal loss and gliosis in animals surviving more than 1 week. No significant lesions were present in brains of animals fed 1 mg of Pb (NO&day. Brain Water Content The mean water content of six brain areas in all lead (n=36)- and non-lead (n =36)-treated pups was highest at 2 days of age and generally declined thereafter (Fig. 8). In most brain areas the decrease was greatest between 10 and 20 days and minimal or absent between 20 and 30 days. At all ages, the mean water content of the medulla and the cervical spinal cord was significantly lower [P
In order to assess the relative vulnerability of organ systems in developing animals to lead toxicity, the dose, the distribution, and tissue concentrations of lead should be known. In the present study, we have attempted to provide simultaneous measurements of blood and tissue lead concentrations of rabbit
LEAD
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143
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80-
75I I
2
I
10
20
30 AGE
1
I,
2
10
20
30
(days)
FIG. 8. The mean water content of six brain areas in as a percentage of the wet tissue weight. Because of the dose groups and the independence of the percentage [means + SEM (bars)] for all lead-treated rabbits were 18 mg of Pb (NO&/day. P
lead (0)- and non-lead (.)-fed pups expressed relatively small number of rabbits in the higher water content relative to lead dosage, values combined; (i.e., those receiving 1, 4.5, 9, and
pups at various ages of development. Lead concentration in blood and tissues of rabbits exposed to lead increased up to 20 days. Thereafter, with exception of bone and kidney, tissue levels declined, possibly signaling the development of a more efficient process of excretion, deposition into bone, or both. The fact that lead levels in bone can be markedly increased at 2 and 10 days of age by feeding higher lead concentrations (Fig. 1) suggests that, in the rabbit as in the rat (Momcilovic and Kostial, 1974), an efficient process for lead deposition is present very early in development. Consequently, such a process would not be expected to cause an abrupt decrease in tissue lead levels after 20 days of age. Severe lead toxicity, resulting in early mortality, progressively eliminating from consideration at later ages animals with the highest tissue lead levels (Table 3), may be another factor. This process as hypothesized by Bornschein ef al. (1977b) would be important in the high lead dose groups experiencing significant mortality rates. In the low dose lead group, therefore, the development onset of a more efficient excretory process appears to be the more plausible explanation for the decrease
LEAD
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in tissue lead levels observed after 20 days. The declining tissue lead concentrations could also be attributed to decreasing dose per kilogram with age (Table 2). It should be noted, however, that the greatest decrease in dosage (64%) occurs from 2 to 10 days of age when tissue lead levels are increasing, and not from 20 to 30 days (42%) when levels are falling. The high plasma to blood lead ratios observed in the younger rabbits, which subsequently declined with age (Table 2), cannot be attributed to the relatively higher lead dosage fed to the younger offspring, since a similar plasma to blood lead distribution was observed in control littermates. If we assume that lead in plasma, as opposed to that in erythrocytes, is more readily available to soft tissues, then high plasma lead concentrations may constitute an important factor in the greater susceptibility of the young neonatal animal to the toxic effects of lead. Indeed, plasma lead concentrations have been reported to be higher in younger than in older rats injected iv with lead citrate (Jugo, 1976). No age-related alterations in plasma lead concentrations, however, have been observed in infants or children (Rosen and Trinidad, 1974). The relative distribution of lead in immature rabbit tissues parallels, in some respects, the lead tissue distribution observed in other animals, as well as in humans (Barry, 1975; Krigman et al., 1974a; Stowe et al., 1973). Thus, with lead exposure, the highest concentrations of lead were found in bone, followed by liver, kidney, blood, and brain. Lead concentration in the cerebellum was, for the most part, always greater than in whole brain and could account for the marked susceptibility of the cerebellum to the toxic effects of lead. A similar relative lead distribution in brain has been reported for rats (Michaelson, 1973) and dogs (Stowe et al., 1973) by some investigators, but not by others (Goldstein et al., 1974). It is of interest, in this respect, that in our study, the difference in lead concentration between cerebellum and brain was greatest at 2 and 10 days but, thereafter, decreased with age. Possibly, the disparity in results reported in the literature may, in part, be due to sacrificing animals and assaying their tissue levels at different ages. Most manifestations of blood dyscrasia associated with lead toxicity in man are closely modeled in the rabbit (Hass et al., 1964; Roscoe et al., 1975). Rabbit and man are among the few species in which lead interferes with porphyrin metabolism (Hass et al., 1964) and produces punctate basophilic stippling (Hass et al., 1964; Roscoe et al., 1975) as well as K+ loss from erythrocytes (Joyce et al., 1954). The recognized relationship between the hematocrit and lead concentrations in blood is demonstrated by the decrease in hematocrit seen at 20 days in rabbits fed 4.5 mg of Pb (NO&/day. The conspicuous renal changes in human lead poisoning, such as the presence of intranuclear inclusion bodies in the tubular lining cells, tubular necrosis (Henderson, 1955), and aminoaciduria paralleled findings in young rabbits (Hass et al., 1964; von Studnitz and Haeger-Aronsen, 1962). In humans, pathologic changes, with regard to their location and sequence of development, are closely modeled by rabbits (Hass et al., 1964). The absence of statistically significant increases in water content in most brain areas of rabbits fed 1.0 or 4.5 mg of lead nitrate/day at 2, 10, 15, 20, or 30 days of
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age (Fig. 8) confirms the results of some (Krigman et al., 1974b; Michaelson, 1973), but not others (Clasen et al., 1974). In white matter, medulla, and cerebellum of 20-day-old, lead-fed rabbits, the water content was greater than that of littermate controls, confirming the results of Goldstein et al. (1974) and Michaelson (1973). The decline of water content with age in the non-lead-fed rabbit is reminiscent of developmental changes in brain water content of other species (Agrawal et al., 1968 a,b; Vernadakis and Woodbury, 1965; Barlow et al., 1961). The slower decrease in water content observed in lead-fed littermates, also noted by Michaelson in rats (1973), suggests that lead retards the maturation of this process. Since the fall in brain water content in animals during the period of rapid brain development is thought to reflect the most active phase of myelination (Agrawal et al. 1968a; Barlow et al., 1961), and lead exposure is known to retard myelination (Krigman et al., 1974a), it is likely that these results (Fig. 8) represent the effect of lead on myelination. The inability to detect a difference in water content between lead and control 30-day-old rabbit offspring may be attributable (as described above) to the early mortality of those offspring most susceptible to lead. It is interesting that marked increases in water volume, indicating possible brain edema, were noted only in a small percentage of lead-exposed animals. The lack of frank edema in most of our lead-intoxicated rabbits parallels the data of Pentschew (1965), who noted an ostensible increase in brain volume in only 6 out of 20 cases of acute lead encephalopathy in infants and small children. Many of the histopathologic CNS lesions observed by us in the lead-exposed rabbit were similar to those found in brains of other species, including man. These results stand in contradistinction to those of others who failed to produce encephalopathy ‘in rabbits, even when lead exposure was from 3 to 12 months of age (Hass et al., 1964). The inability to produce lead encephalopathy in young rabbits possibly is related to the fact that rabbit brain, like that of man (Davison and Dobbing, 1968) and rhesus monkey (Portman et al., 1972), undergoes its maximal development around parturition (Hare1 et al., 1972). Since the brain is most vulnerable to perinatal stress during the period of maximum brain development (Davison and Dobbing, 1968), the threshold to lead toxicity also could be lower during this period. Other factors predisposing to greater CNS lead toxicity in the neonatal animal are the higher gastrointestinal absorption of lead (Forbes and Reina, 1972; Jugo, 1976), the lower overall excretion (Forbes and Reina, 1972), and the greater CNS uptake and retention of lead (Jugo, 1976; Momcilovic and Kostial, 1974). The critical dependency on age in the ability to produce lead encephalopathy is apparent also in the rat. Suckling rats exposed to lead via dams whose diet contains 4% lead carbonate, develop fatal encephalopathy by 4 weeks of age (Goldstein et al., 1974; Krigman et al., 1974a; Pentschew and Garro, 1966; Thomas et al., 1971). The encephalopathy can be avoided if at the time of weaning offspring are prevented from eating the chow containing 4% lead salt (Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966), or if the concentration of lead in the chow is reduced lOOO-fold, or to that found in the dam’s milk (Michaelson and Sauerhoff, 1974). Paradoxically, encephalopathy also is avoided if weanlings are started on a diet of 4% Pb (NO&/day beginning at 3 weeks and continuing to
LEAD
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11 weeks of age (Goldstein et al., 1974). The above results suggest that exposure to lead, even at extremely low concentrations, must occur shortly after birth, if the neonatal CNS is to become vulnerable to concentrations of lead which produce encephalopathy. This critical dependency on age, during which time the neonatal animal must first be exposed to lead, also appears to be an important factor in developing cognitive and behavioral deficits following exposure to low concentrations of lead (Brown, 1975; de la Burde and Choate, 1972, 1975; Sobotka er al., 1975). Increased locomotor activity over age-matched controls also has been reported in mice following exposure to lead from birth to 40 days of age (Silbergeld and Goldberg, 1973). In these studies, however, growth retardation was evident in the lead-treated offspring. When weight-matched controls were used, the sustained hyperactivity was found to be attributable to early undernutrition (Bomschein et al., 1977b). Nutritional deficiency arising from the effect of lead on the dam, however, is not a necessary factor in the induction of behavioral deficits in the offspring. This is indicated by the occurrence of behavioral alterations in adult and weanling rats which were initially exposed, from birth to 3 weeks of age, to lead directly (Brown, 1975; Sobotka et al., 1975). Increases in activity, ranging from 40 to 93% greater than pair-fed controls, have been noted in neonatal rats exposed to 25 ppm of dietary lead (Michaelson and Sauerhoff, 1974). In our studies, in which low concentrations of lead were fed directly to rabbit pups, activity generally was higher in lead than in non-lead-fed littermates (except at IO days of age). In agreement with Winneke et al. (1976), we have interpreted the generally higher activity exhibited by the lead-fed offspring to be a hyperresponsiveness to a novel situation. Indeed, the response of lead-fed asymptomatic pups to handling by investigators always appeared to be more exaggerated and of longer duration than that of littermate controls. This was most apparent when animals retreated from the approaching hand of the investigators. Hyperactivity, also exhibited by mice exposed to lead has been attributed to retardation of brain development (Maker et al., 1975) and not to specific neurological effects of lead. Even though serotonin levels in the CNS of lead-exposed rats have been found to be unaltered (Sobotka et al., 1975), such behavior would be consistent with alteration in serotonergic function. Increased locomotor activity following administration of d-amphetamine is potentiated by reducing the availability of tryptophan and/or interfering with serotonin synthesis (Hollister et al., 1976). It is known that serotonin turnover, dependent on tryptophan availability from blood, as well as other factors (Femstrom and Wurtman, 1971), may change without concurrent alterations in serotonin steady-state levels. Since low concentrations of lead [l mg of Pb (NO&day] are known to interfere with the passage of tryptophan into the brain of rabbit pups (Lorenzo and Gewirtz, 1977), it is possible that lead may alter turnover of serotonin without affecting endogenous levels. The evidence presented above indicates that both overt and asymptomatic CNS lead toxicity can be produced in rabbits when oral lead feeding by hand is initiated at birth. It is noteworthy that the effect of lead on the rabbit parallels, in many
148
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respects, that on the human offspring. In addition, the rabbit model presented here meets many of the other criteria advocated in a recent report by the National Academy of Sciences (Committee on Toxicology, 1976) with respect to (1) assessment of lead dosage, (2) route of administration, (3) effect of lead on organ systems, and (4) rate of brain growth. More practically, the use of the neonatal rabbit in such studies provides a readily available, larger species that permits experimental procedures, difficult or impossible to perform on smaller animals. Use of this model should aid in the elucidation of the etiology of lead-induced CNS dysfunction. ACKNOWLEDGMENTS This study was supported by the N.1.H Grant HD08945 and the Children’s Hospital Medical Center Mental Retardation and Human Developmental Research Program. We wish to thank the following Brookline High School volunteer students for their aid with caring, feeding, and testing of animals: Sandra Stoddard, Barbara Schnee, and Carole Pfieffer.
REFERENCES Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1968a). Developmental changes in mouse brain: Weight, water content and free amino acids. J. Neurochem. 15, 917-923. Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1968b). Water content of dog brain parts in relation to maturation of the brain. Amer. J. Physiol. 215, 846-848. Baloh, R., Sturm, R., Green, B., and Glesser, G. (1975). Neuropsychological effects of chronic asymptomatic increased lead absorption. Arch. Neural. 32, 326-330. Baemstein, H. D., and Grand, J. A. (1942). The relation of protein intake to lead poisoning in rats. J. Pharmacol.
Exp.
Ther.
74, 18-24.
Barlow, C. F., Domek, N. S., Goldberg, M. A., and Roth, L. J. (1961). Extracellular brain space measured by 35S-sulfate. Arch. Neurol. 5, 102- 110. Barry, P.S.I. (1975). A comparison of concentrations of lead in human tissues. &it. J. Znd. Med. 32, 119-139. Beattie, A. D., Moore, M. R., Goldberg, A., Finlayson, M. J. W., et al. (1975). Role of chronic lowlevel lead exposure in the aetiology of mental retardation. Lancer 1, 589-592. Bomschein. R. L., Fox, D. A., and Michaelson, I. A. (1977a). Estimation of daily exposure in neonatal rats receiving lead via dam’s milk. Toxicol. Appl. Pharmacol. 40, 577-587. Bomschein, R. L., Michaelson, I. A., Fox, D. A., and Loch, R. (1977b). Evaluation of animal models used to study effects of lead on neurochemistry and behavior. In “Biochemical Effects of Environmental Pollutants,” (S. D. Lee, Ed.) p. 44-460. Ann Arbor Science Publ., Ann Arbor, Mich. Browder, A. A., Joselow, M. M., and Louria. D. B. (1973). The problem of lead poisoning. Medicine 52, 121-139. Brown, D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharmacol. 32, 628-637. Brown, S., Dragann, N., and Vogel, W. H. (1971). Effects of lead acetate on learning and memory in rats. Arch, Environ. Health 22, 370-372. Byers, R., and Lord, E. E. (1943). Late effects of lead poisoning on mental development. Amer. J. Dis. Child. 66, 471-494. Chisolm, J., Jr. (1977). Is lead poisoning still a problem? Gin. Chem. 23, 252-253. Clasen, R. A., Hartman, J. F., Starr, A. J., Coogan, P. S., er al. (1974). Electron microscopic and chemical studies of the vascular changes and edema of lead encephalopathy. Amer. J. Pathol. 74, 215-234. Cole, M. A. (1977). Lead inhibition of enzyme synthesis in soil. Appl. Environ. Microbial. 33, 262-268. Colquhoun, D. (1971). “Lectures on Biostatistics,” pp. 167- 170. Clarendon Press, Oxford. Committee on Toxicology. (1976). Recommendations for the prevention of lead poisoning in children. Prepared for U.S. Consumer Product Safety Commission and the National Academy of Sciences, Washington, D.C.
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Conover, W. J. (1971). “Practical Nonparametric Statistics,” pp. 309-314. Wiley, New York. David, O., Hoffman, S., McGann, B., Sverd, J., and Clark, J. (1976). Low lead levels and mental retardation. Lancer 2, 1376- 1379. Davison, A. N., and Dabbing, J. (1968). The developing brain. In “Applied Neurochemistry” (A. N. Davison and J. Dobbing, Eds.), pp. 253-286. Blackwell, Oxford, England. de la Burde, B., and Choate, M. S. (1972). Does asymptomatic lead exposure in children have latent sequelae? J. Pediat. 81, 1088-1901. de la Burde, B., and Choate, M. S. (1975). Early asymptomatic lead exposure and development at school age. .I. Pediat. 87, 638-642. Femstrom, J. D., and Wurtman, R. J. (1971). Brain serotonin content; physiological dependence on plasma tryptophan levels. Science 173, 149-152. Forbes, G. B., and Reina, J. C. (1972). Effect of age on gastrointestinal absorption (Fe, Sr, Pb) in the rat. J. Nutr. 102, 647-652. Gilsinn, J. F. (1972). Estimates of the nature and extent of lead paint poisoning in the United States. U.S. Department of Commerce National Bureau of Standards. Tech. Note 746. Goldstein, G. W., Asbury, A. K., and Diamond, I. (1974). Pathogenesis of lead encephalopathy. Arch. Neural. 31, 382-389. Harel, S., Watanabe, K., Linke, I., and Schain, R. J. (1972). Growth and development of the rabbit brain. Biol. Neonat. 21, 381-399. Hass, G. M., Brown, D. V. L., Eisenstein, R., and Hemmens, A. (1964). Relations between lead poisoning in rabbit and man. Amer. J. Pathot. 65, 691-727. Henderson, D. A. (1955). Chronic nephritis in Queensland. Aust. Ann. Med. 4, 164. Hollister, A. S., Breese, G. R., Kuhn, C. M., Cooper, B. R., and Schanburg, S. M. (1976). An inhibitory role for brain serotonin-containing systems in the locomotor effects of d-amphetamine. J. Pharm. Exp. Ther. 198, 12-22. Joyce, C. R. B., Moore, H., and Weatherhall, M. (1954). The effect of Pb, Hg and Au on K+ turnover of rabbit blood cells. Brit. J. Pharmacol. 9, 463 -470. Jugo, S. (1976). Metabolism of toxic heavy metals in growing organisms. A review. Environ. Res. 13, 36-46. Krigman, M., Druse, M. Traylor, T., Wilson, M., et al. (1974a) Lead encephalopathy in the developing rat: effect upon myelination. J. Neuropathol. Exp. Neurol. 33, 58-73. Krigman, M. R., Dmse, M. J., Traylor, T. D., Wilson, M. H., et al. (1974b). Lead encephalopathy in the developing rat: Effect on cortical ontogenesis. J. Neuropathol. Exp. Neurol. 33, 671-686. Lampert, P., Garro, F., and Pentschew, A. (1967). Lead encephalopathy in suckling rats: An electron microscopic study. In “Brain Edema” (I. Klatzo and F. Seitelberger, Eds.), pp. 207-222. Springer-Verlag, New York. Levander, 0. A., Morris, V. C., Higgs, D. J., and Ferretti, R. J. (1975). Lead poisoning in vitamin Edeficient rats. J. Nutr. 105, 1481-1485. Lockeretz, W. (1975). Lead content of deciduous teeth of children in different environments. Arch. Environ. Health 30, 583-587. Lorenzo, A. V., and Gewirtz, M. (1977). Inhibition of ‘%tryptophan transport into brain of lead exposed neonatal rabbits. Brain Res. 132, 386-392. Mahaffey, K. R., Goyer, R., and Haseman, J. K. (1973). Dose-response to lead ingestion in rats fed low dietary calcium. J. Lab. Clin. Med. 82, 92- 100. Maker, H. S., Lehrer, G. M., and Silides, D. J. (1975). The effect of lead on mouse brain development. Environ. Res. 10, 76-91. Michaelson, I. A. (1973). Effects of inorganic lead on levels of RNA, DNA and protein of developing neonatal rat brain. Toxicol. Appl. Pharmacol. 26, 539-548. Michaelson, I. A., and Sauerhoff, M. W. (1974). An improved model of lead-induced brain dysfunction in the suckling rat. Toxicol. Appl. Pharmacol. 28, 88-96. Momcilovic, B. and Kostial, K. (1974). Kinetics of lead retention and distribution in suckling and adult rats. Environ. Res. 8, 214-220. Pentschew, A. (1965). Morphology and morphogenesis of lead encephalopathy. Acta Neuropathol. 5, 133-160. Pentschew, A., and Garro, F. (1966). Lead encephalo-myelopathy of the suckling rat and its implications on the porphyrinopathic nervous diseases. Acta Neuropathol. 6, 266-278.
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Portman, 0. W., Alexander, M., and Illingworth, D. R. (1972). Changes in brain and sciatic nerve composition with development of the rhesus monkey (Macaca mulatta), Brain J&s. 43, 197-213. Roscoe, D. E., Nielsen, S. W., Eaton, H. D., and Rousseau, J. E. (1975). Chronic plumbism in rabbits: A comparison of three diagnostic tests. Amer. J. Vef. Res. 36, 1225- 1229. Rosen, J. F., and Trinidad, E. E. (1974). Significance of plasma lead levels in normal and leadintoxicated children. Environ. Health Perspect. 7, 139- 144. Rosenblum, W. I., and Johnson, M. (1968). Neuropathologic changes produced in suckling mice by adding lead to the maternal diet. Arch. Pathol. 85, 640-648. Searle, B., Chan, W., and Davidow, B. (1972). Determination of lead in blood and urine by anodic stripping voltammetry. Clin. Chem. 47, 76-80. Silbergeld, E. K., and Goldberg, A. M. (1973). A lead-induced behavioral disorder. Life Sci. 13, 1375- 1383. Six, K. M., and Goyer, R. A. (1972). The influence of iron deficiency on tissue content and toxicity of ingested lead in the rat. J. Lab. C/in. Med. 79, 128-136. Sobotka, T. J., Brodie, R. E., and Cook, M. P. (1975). Psychophysiologic effects of early lead exposure. Toxicology 5, 175- 191. Stowe, H. D., Goyer, R. A., Krigman, M. R., Wilson, M., et al. (1973). Experimental oral lead toxicity in young dogs. Arch. Pathol. 9S, 106- 116. Thomas, J. A., Dallenbach, F. D., and Thomas, M. (1971). Considerations on the development of experimental lead encephalopathy. Virchows Arch. A. 352, 61-74. Urban, W. D. (1976). Statistical analysis of blood lead levels of children surveyed in Pittsburgh, Pennsylvania: Analytical methodology and summary results. National Bureau of Standards N. B. S. I. R. 76-1024. Vemadakis, A., and Woodbury, D. M. (1965). Cellular and extracellular spaces in developing rat brain. Arch. Neural. 12, 284-293. Von Studnitz, W. and Haeger-Aronsen, B. (1%2). Urinary excretion of amino acids in lead-poisoned rabbits. Acta Pharmacol. Toxicol. 19, 36-42. Waldron, M. A., and Stofen, D. (1974). “Sub-clinical Lead Poisoning,” pp. 101-107. Academic Press, London and New York. Whitney, J. C., Blackmore, D. K., Townsend, G. H., et af. (1976). Rabbit mortality survey. Lab. Anim. 10, 203-207. Winneke, G., Brockhaus, A., and Baltissen, R. (1977). Neurobehavioral and systemic effects of longterm blood lead elevation in rats. I. Discrimination learning and open field behavior. Arch. Toxicol. 37, 247-263.
RESEARCH
17, 13l-
1% (1978)
CNS Lead Toxicity
in Rabbit Offspring
A. V. LORENZO,~ MYRNA GEWIRTZ, AND DAMON AVERILL with technical assistance of M. Mauer Department
of Neurology and Pharmacology, Harvard Medical and the Division of Neuroscience and Neuropathology, Children’s Hospital Medical Center. Boston, Massachusetts
School, 02115
Received September 8, 1977 An animal model for asymptomatic and overt lead toxicity is presented in which rabbit pups, 1 to 30 days of age, were fed a milk supplement containing Pb(NO& Reduced body growth, increased mortality, hematologic abnormalities, kidney and liver histopathology, as well as encephalopathy, were observed in rabbits 2 to 30 days of age ingesting more than 2.8 mg of Pb/day. At the highest dose, brain lesions were widespread, involving the frontal and occipital cortex, striatum, hippocampus, and cerebellum. At moderate doses, the region most affected was the cerebellum. At the lowest dose no alteration in body, brain growth, mortality, or tissue histology was evident: however, basophilic stippling was observed. AS a group, these asymptomatic rabbits generally exhibited higher activity than non-lead-fed littermates. The rabbit model presented here should provide many advantages in the study of lead toxicity on the developing nervous system, since (a) maximum brain development in the rabbit, like that in rhesus monkey and man, occurs perinatally, and(b) the effects of lead toxicity in the rabbit closely parallel those in man.
INTRODUCTION Accidental lead poisoning in children and lead toxicity in industrial workers have been, in the past, the primary concern of health officials dealing with the problem of lead intoxication. At present, as a result of increased contamination of the environment with heavy metals, it is the opinion of some (Cole, 1977; Lockeretz, 1975; Urban, 1976; Waldron and Stofen, 1974), but not others (Barry, 1975), that continuous exposure to low levels of lead in the environment may constitute the more serious problem. Indeed, numerous investigators have expressed grave concern regarding the possible impairment of mental development of children exposed to environmental concentrations of lead during infancy or early childhood (Baloh et al., 1975; Beattie et al., 1975; Chisolm, 1977; David et al., 1976; de la Burde and Choate, 1972, 1975). Childhood lead poisoning is recognized as one of the more serious health hazards in the United States. It is estimated that some 5 to 10% of all children in the U.S. have abnormally high blood lead levels (Gilsinn, 1972). Of the many children exposed to lead each year, only a small proportion (0.5%) exhibit overt clinical symptoms; the majority remain clinically asymptomatic (Browder et al., 1973). Many of the clinically asymptomatic children develop subtle neurological and behavioral defects (Browder et al., 1973; Byers and Lord, 1943; David et al., 1973; de la Burde and Choate, 1975). Unfortunately, other factors such as I Address reprint requests to: A. V. Lorenzo, Children’s Hospital Medical Center, Neuroscience Research Department, 300 Longwood Avenue, Boston, Massachusetts, 02115. 131 0013-9351/78/0171-0131$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
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nutritional and socioeconomic status confound the relationship of lead to mental development (Committee on Toxicology, 1976; Waldron and Stofen, 1974). It is difftcult, therefore, to ascribe the increased frequency of intellectual impairment and behavioral aberrations observed in these clinically asymptomatic children to low-level lead exposure only (Beattie et al., 1975; Committee on Toxicology, 1976). For this reason investigators have attempted to develop animal models that parallel the effects of lead toxicity on the developing human central nervous system (CNS) (Bornschein et al., 1977b; Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966; Silbergeld and Goldberg, 1973). In 1966, Pentschew and Garro introduced an animal model for lead encephalopathy which has become the prototype for subsequent experiments. In this model, lactating rats exposed to 4% lead carbonate in their diet transmit lead via the maternal milk to the suckling rat. The lead-exposed suckling animal has proven to be a valuable experimental model to study the effects of overt lead toxicity on the CNS (Goldstein et al., 1974; Krigman et al., 1974a,b; Rosenblum and Johnson, 1968; Thomas et al., 1971). Its use to investigate cognitive as well as behavioral abnormalities arising from asymptomatic lead poisoning is not devoid, however, of serious drawbacks (Sobotka et al., 1975). The ingestion of 1 to 5% lead salts by the dam leads to diminished food intake (Bornschein et al., 1977b), weight loss (Krigman et al; 1974a; Lampert et al., 1967; Michaelson and Sauerhoff, 1974), and alteration in the nutritional quality of the milk in the dam (Bornschein et al., 1977b). Since the dam’s milk constitutes the only source of nutriment available to the offspring before weaning, the pronounced growth retardation exhibited by the offspring (Bornschein et al., 1977a, b; Krigman et al., 1974a; Michaelson, 1973; Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966; Silbergeld and Goldberg, 1973) can, in part, be attributed to malnutrition (Baernstein and Grand, 1942), specific nutritional deficiencies, or both. Nutritional deficiencies are known to potentiate the toxic effects of lead (Jugo, 1976; Levander et al., 1975; Mahaffey et al., 1973; Six and Goyer, 1972). When they occur during the critical period of brain development, long-lasting neuropsychological abnormalities result (Davison and Dobbing, 1968). These secondary effects arising from lead-induced changes in the dam apparently can be circumvented if either the concentration of lead in the diet is drastically reduced (Brown, 1975), or if lead is fed directly to the neonatal animal (Sobotka et al., 1975). Direct administration of lead to young rats (Brown et al., 1971), dogs (Stowe et al., 1973), or rabbits (Hass et al., 1964), however, does not necessarily result in CNS dysfunction even when, in the case of the rabbit, lead exposure is extended for 9 months (Hass et al., 1964). Abnormal behavior in weanling rats that are otherwise asymptomatic can be produced, if lead administration is started at birth (Brown, 1975; Sobotka et al., 1975; Winneke et al., 1977). In the present study in which rabbit pups are fed lead directly, we present a model for overt and asymptomatic lead toxicity that (a) provides a more accurate assessment of lead dosage to the offspring and (b) avoids confounding variables arising from the effect of lead on the dam. METHODS
Sixty-five New Zealand female rabbits, weighing 3.5 to 5.0 kg, impregnated in our animal facility, were housed in individual cages provided with a nesting box
LEAD
TOXICITY
IN
RABBIT
133
OFFSPRING
containing straw. They were fed normal laboratory chow (Blue Seal Lab Pels, H.K. Webster Co., Lawrence, Mass.) and tap water ad libitum. Twelve to 24 hours after parturition, the progeny were weighed, and the litter size was reduced to six pups. Each day, from 1 to 30 days of age, offspring were examined, weighed, and fed 1 ml of milk formula consisting of 100 mg of “Unilac” (Upjohn Co., Kalamazoo, Mich.) powder dissolved in 1 ml of water warmed to 37°C to which was added 0 (controls), 1.0, 4.5, 9.0, or 18.0 mg/ml of Pb (NO,),. Spontaneous locomotor activity was measured daily for a total of 20 min in an activity cage (Lehigh Valley Electronics, Model 1487) which registered, during four consecutive 5 min periods, the number of times a photoelectric light beam path was interrupted. Blood, brain, and peripheral tissues were obtained from animals killed at 2, 10, 15, 20, and 30 days of age or at autopsy. Tissue samples were divided and were either fixed in 10% buffered formalin for histology, dried to constant weight in a vacuum oven at 100°C to determine water content, or stored for lead assay. Samples of blood were obtained by cardiac puncture using heparinized disposable syringes. Possible lead contamination of syringes and heparin was assessed by drawing aliquots from a lo-ml heparin vial into five individual syringes (Table 1). Other tissues were dissected free using acidwashed, stainless-steel surgical instruments. Blood samples were transferred to lead-free, loo-p1 capillary tubes. [Environmental Sciences Associates (ESA), Burlington, Mass.]. Blood and tissue samples were transferred to lead-free tubes provided by ESA, for digestion in a mixture of nitric, sulfuric, and perchloric acids. Concentrations of lead in tissues and in sources of lead other than the oral dose administered, which could potentially increase the total lead ingested or contaminate tissue samples, were assayed with an ESA anodic stripping voltammeter (Model 3010 for blood and Model 2014 for tissues) according to the method of Searle er al. (1972) (Table 1). A matrix of distilled HZ0 and perchloric acid (pH 1.8), containing various concentrations of lead, was used to calibrate the instruments daily. Analysis of 15 aliquots of blood drawn from a single rabbit 18 hours after iv injection of 5 mg of lead acetate/kg, by the above procedure, indicated a blood-lead concentration of 86.6 + 1.2 pg%. Blank level variability differed from day to day, ranging from 0.007 (n=4) to 0.018 (n=5) pg of lead. RESULTS
Lead Concentration in Tissues Concentration of lead in blood of dams and offspring fed 0, 1.O, and 4.5 mg of Pb TABLE POSSIBLE
SOURCES
Source
n*
Unilac Water Rabbit chow (pellets) Heparin
4 5 4 5
n n = number of samples assayed, b Values are means 2 SEM.
1 OF LEAD
Lead concentration (mhl or g) 0.30 0.01 0.33 0.01
f 2 f f
0.04b 0.00 0.10 0.00
134
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AND
AVERILL
(NO&/day is shown in Table 2. Even though dosage, based on kilogram body weight, decreased with age, blood lead concentrations kept increasing from 2 to 20 days. By 30 days, the lead concentration in blood was lower. Controls kept separate had lower lead values than those housed with lead-fed littermates. Presumably, this may be the result of eating contaminated straw, preening and licking behavior, or ingesting excreted lead transmitted via the dam’s milk or a combination thereof. The latter, however, does not appear likely, since the concentration of lead in the dam’s blood was relatively low (Table 2). The percentage of blood lead associated with plasma was highest at 2 days and decreased thereafter (Table 2). Nevertheless, the level at 20 and 30 days was somewhat higher than that reported for older animals and children (Rosen and Trinidad, 1974). The concentration and pattern of lead accumulation from 2 to 30 days in tissues of animals exposed to 0, 1.0, and 4.5 mg of Pb (NO&/day is shown in Fig. 1. At all doses and ages the concentration of lead in bone (ulna) exceeded the levels found in other tissues. Generally, lead accumulation in bone increased with continuing exposure to lead, even when concentrations in blood remained constant (Fig. 1A) or decreased (Figs. 1B and C). Lead concentration in liver was next highest. With TABLE CONCENTRATION
OF LEAD
(pglml)
2
IN BLOOD
OF DAMS
AND
NEWBORN
RABBITS~
Daily lead dose (mg of lead nitrate/day) Age (days) 2
0 0.56 k 0.13’ (7) 0.10 ‘- 0.01’
1 1.45 k 0.27
15
0.49 2. 0.01 (7) 0.08 +- 0.01’ (4) 0.56 f 0.05
(6) 20 30
0.75 -t 0.11 (10) 0.54 k 0.12
(8)
15.3d
(6)
(6)
10
4.5
1.54 2 0.25 (9)
6.1d
1.28 k 0.21 (5) 2.15 2 0.35 (10) 0.87 f 0.11
4.ld
(6)
3.ld l.8d
P/B x lOO*
2.00 2 0.62 (7)
76.2d
3.69 k 0.68 (5)
24.6d
3.99 f 1.24 (5) 6.60 + 0.73
18.6d
(6)
0.79 k 0.09 (7)
33.41
(20) 28.37
(21) 15.47
(16) 14.56 8.3d
8.01 (20) 9.09 (11)
0.14 2 0.05’ (3) Dams
0.06 f O.Olf
(8) n Values are means * SE; number of animals in parentheses. * P/B x 100, lead concentration in plasma as a percentage of blood lead concentration for all lead dosages. c Non-lead-fed animals housed with lead-fed littermates. d Average dose of lead nitrate (milligram per kilogram basis) fed orally to pups at designated age. p Non-lead-fed animals kept separate from lead-fed neonates. r Blood was obtained from the marginal ear vein of the dam on day last pup of litter died.
LEAD
TOXICITY
I A. w mg
B.
Pb mo&~w
In 0
5
18 lo
ls
11, 20
IN
26
m
0 0
a 6
1.0
RABBIT
135
OFFSPRING
maPbtNO&dw
8 lo
1s
c.
0 20
’ 22
II 20
0
4.Sm(1 PbO40&14
t 5
8 lo
t Is
“1 20
2s
20
ms
FIG. 1. Lead concentration in tissues of lead- and non-lead-fed rabbit neonates from 2 to 30 days of age receiving (A) 0 (B) 1.0, and (C) 4.5 mg of lead nitrate/day. Ordinate scale is logarithmic. Values represent means, and bars indicate SEM.
the exception of levels in 20- and 30-day-old control animals (Fig. IA), liver concentrations consistently exceeded concentrations in blood. In contrast to lead accumulation in bone, that in liver tended to decrease after 15 or 20 days more or less paralleling the fall observed in blood lead levels. The lead concentration in kidneys of controls was lower than that of other tissues assayed; however, with lead exposure [l.O and 4.5 mg of Pb (NO&/day] lead concentration in kidneys became markedly elevated, sometimes exceeding levels observed in blood and CNS of these animals. Interestingly, accumulation of lead in kidneys appeared to abruptly increase after 10 and plateau after 20 days, a pattern which may be related to initiation of lead excretion by the kidney. The concentration of lead in cerebellum and whole brain was usually lower or equal to that of blood. At all doses, and nearly at all ages, the concentration of lead in cerebellum exceeded that of whole brain. Even though accumulation in cerebellum and whole brain fluctuated with continuing lead exposure, in general, accumulation in cerebellum was highest during the first 15 days of lead exposure, while that in whole brain was highest from 15 to 30 days of age. Lead concentrations in selected tissues of pups receiving 4.5 mg of lead nitrate/day and which succumbed to the toxic effects
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of lead are shown in Table 3. Lead concentrations were considerably higher in tissues of autopsied noenates than in tissues of littermates fed the same daily doses but which were killed at 2, 10, or 15 days. Appearance
of Offspring
At all doses, lead-fed offspring could be distinguished from control littermates by the ruffled appearance of their pelts (Fig. 2). At the highest doses some of the lead-fed animals exhibited a grayish pallor and a splayed stance. The spontaneous motor activity (motility) of 11 lead-fed (1 mg of lead nitrate/day) and six non-lead-fed animals was generally highest during the first and second Sminute intervals (Fig. 3) (except in the 20- to 25day-old control group). During the second or third period, the activity declined sharply and thereafter tended to level off or increase slightly. In general, the average motility of the leadtreated animals was always greater than their non-lead-fed littermates, except at Day 10 when the activity was equal to or lower than that of controls. No significant differences were found between individual mean activities (Colquhoun, 1971) of the two groups; however, using the nonparametric test of Smimov (Conover, 1971), the activity during the first two Sminute periods of lead-fed pups was found to be significantly different (P
3
OF LEAD IN TISSUES OF NEONATES NITRATE PER DAY THAT DIED OF LEAD
Concentration Age
(days)
0 - 56
6 - 10 11 -15 Average for all ages (autopsied) Values for neonates killed’
FED 4.5 MC OF LEAD TOXICITY~
(&g)
Kidney
Liver
Brain
Cerebellum
42.3 (1) 67.5 k 17.1 (3) 35.1 r 11.7 (3) 50.6 k 11.2 (7)
306.2 (1) 419.5 2 146.0 (3) 96.5 2 28 (3) 264.9 f 84.1 (7)
0.5 (1) 4.2; 2.0
-
6.1 ? 1.0
19.9 2 2.1
(18) P <
0.001”
(18) P <
0.001
9.2; 9.9
4.5 k 2.2 (3) 3.4 k 1.2
9.5 2 -
(6)
(2)
1.1 + 0.1
5.7 T 1.1 (13)
(16) P < 0.05
-
a Values are means 2 SE; number of neonates in each group in parentheses. b Age span during which neonates died as a result of lead toxicity. c Values for littermates killed from 2 to 15 days of age which also were fed 4.5 mg of lead nitrate/day. d P values determined by Student’s I test.
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OFFSPRING
FIG. 2. Typical appearance of rabbit neonates fed 0 and 4.5 mg of lead nitrate/day for 15 days. The ruffled appearance of the pelt of rabbits receiving concentrations of lead higher than 1 mg of lead nitrate/day was usually accompanied by growth retardation and splayed stance. Similar observations have been reported for rats (Michaelson, 1973) and mice (Rosenblum and Johnson, 1968) exposed to lead via the dam’s milk. Young rabbits fed 1 mg of lead nitrate/day, however, exhibited a ruffled pelt but no other overt signs of lead exposure. Since it is known that in humans lead concentrates in hair, it is possible that the altered appearance of the pelt of neonatal rabbits may reflect an accumulation of lead.
The mortality determined at 30 days of age for pups fed 0, 1.0,4.5,9.0, and 18.0 mg of Pb (NO,)$day was approximately 16, 17,50,85, and lOO%, respectively. At 4.5,9.0, and 18.0 mg of Pb (NO&day, 50% of the offspring had died by 11,7, and 5 days, respectively. While the mortality for pups fed 1 mg of Pb (NO&/day was
400 I
OL58T6%3 is-
4?36%eQ Tiicc-
-
5MY
10
IMvts-
s’w GG5
FIG. 3. Locomotor activity in counts per minute of rabbit pups was always measured prior to feeding 1 mg of lead nitrate/day.
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CONTROL
125
18 mg Pb/day
FIG. 4. Average weight gain as a function of age and lead exposure. Scale for animals treated with 18 mg of lead nitrate/day is different from others.
approximately 17%, a seemingly high rate, it should be noted that untreated, hutch-raised rabbit pups generally have a mortality which exceeds 11.2% (Whitney et al., 1976). The high mortality obtained in the asymptomatic [l mg of Pb (NO&/day] groups can probably be attributed to the daily handling of pups by the investigators, as reflected by the mortality of 16% observed for non-lead-fed offspring. Hematologic Studies Basophilic stippling of erythrocytes was observed in all lead-fed siblings, even those receiving 1 mg of lead nitrate/day. Stippling occurred as early as Day 2, but thereafter, the total number of stippled red blood cells declined with age so that by Day 30 stippling was absent. Anisocytosis and poikilocytosis were marked in lead-fed rabbits, though present too, to some extent, in controls. “Thornapple” forms of erythrocytes were noted only in lead-fed animals; Roscoe and associates (1975) reported, however, that these are common in normal rabbit blood smears as well. The mean hematocrit of rabbit pups fed 4.5 mg of Pb (NO&day fell conspicuously only at Day 20 (Fig. 5) when blood lead levels were maximum. With 1 mg of Pb (NO&/day, there was no dramatic change in the hematocrit
LEAD
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1
139
OFFSPRING
4.5
Fro 5. Mean hematocrit of neonatal rabbits fed 0, 1, and 4.5 mg of lead nitrate/day. Numbers appearing within bars represent age of groups while those appearing above the bars represent averages and SEM of blood lead concentrations for the respective group. A dagger (t) denotes statistically significant differences (P
values determined at 10, 15, 20, or 30 days. Furthermore, at 1 mg of Pb (NO&/day, the hematocrit values, determined at 10, 1.5, 20, and 30 days, did not differ significantly from those of control littermates. By Day 30, when blood lead levels of pups exposed to 1 and 4.5 mg of Pb (NO&day was approximately equal to that of the control littermates. Tissue Histology
The principle renal changes observed in rabbit neonates fed 4.5, 9.0, and 18.0 mg of Pb (NO&day were acute tubular necrosis, proteinuria, and intranuclear inclusion bodies. Mild to moderate hepatocellular changes, which included focal fatty degeneration and occasional intranuclear inclusion bodies, were present in animals fed 4.5, 9.0 and 18.0 mg of Pb (NO&/day. No histopathological changes in kidney or liver were present in controls or pups fed 1.0 mg of Pb (NO&day. Hematoxylinand eosin-stained sections of brains from rabbits exposed to 4.5,
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FIG. 6. Hematoxylin- and eosin-stained sections of (A) striatum, (B) hippocampus, (C) occipital cortex, and (D) cerebellum from a 6-day-old rabbit neonate exposed to 18 mg of lead nitrate/day. (A) Widespread petechiation and hemorrhagic necrosis as shown in the section of caudate nucleus was pronounced throughout the striatal area. (B) Disseminated petechiation and ecchymosis is prominent
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throughout the pyramidal cell layer and less so m the molecular and granular cell layer of the hippocampus. Clear fluid spaces and cavitation are observed throughout all layers. In (C) disseminated petechiae are observed in the deep cortical cell layers (polymorphous). In the cerebellum (D) cellular infiltration and hemorrhagic necrosis of molecular, Purkinje, and granular layers is extensive. Magnification, x 360.
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9.0, and 18.0 mg of Pb (NO&day contained disseminated lesions which were absent in brain sections from rabbits that received 0 or 1 mg of Pb (NO&day. At 4.5 and 9.0 mg of Pb (NO&/day, the cerebral cortex contained microhemorrhages and acute neuronal necrosis, while at 18.0 mg of Pb (NO&/day there were hemorrhagic necrosis, gliosis, and cellular depletion in striatal areas, olfactory lobe, hippocampus, and occipital cortex (Fig. 6). The telencephalic white matter manifested erythrophagocytosis and diffuse petechiation, while the medulla, pons, midbrain, thalamus, and hippocampus exhibited mild to moderate disseminated petechiae and ecchymosis. Extensive lesions were also noted in the cerebella of animals fed 4.5, 9.0, and 18.0 mg of Pb (NO&/day (Fig. 7). At 4.5 mg Pb (NO&/day, multifocal petechiae were observed in the molecular, Purkinje, and internal granular cell layers. Following 9.0 mg of Pb (NO&/day, Purkinje cell loss, cellular depletion in the internal granular layer, focal gliosis within the molecular cell layer, as well as cystic degeneration of the white matter and focal Purkinje cell calcification were observed in one animal surviving to 24 days of age. At 18.0 mg of Pb (NO&day, there was hemorrhagic necrosis of the white matter with neuronal loss and gliosis in animals surviving more than 1 week. No significant lesions were present in brains of animals fed 1 mg of Pb (NO&day. Brain Water Content The mean water content of six brain areas in all lead (n=36)- and non-lead (n =36)-treated pups was highest at 2 days of age and generally declined thereafter (Fig. 8). In most brain areas the decrease was greatest between 10 and 20 days and minimal or absent between 20 and 30 days. At all ages, the mean water content of the medulla and the cervical spinal cord was significantly lower [P
In order to assess the relative vulnerability of organ systems in developing animals to lead toxicity, the dose, the distribution, and tissue concentrations of lead should be known. In the present study, we have attempted to provide simultaneous measurements of blood and tissue lead concentrations of rabbit
LEAD
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80-
75I I
2
I
10
20
30 AGE
1
I,
2
10
20
30
(days)
FIG. 8. The mean water content of six brain areas in as a percentage of the wet tissue weight. Because of the dose groups and the independence of the percentage [means + SEM (bars)] for all lead-treated rabbits were 18 mg of Pb (NO&/day. P
lead (0)- and non-lead (.)-fed pups expressed relatively small number of rabbits in the higher water content relative to lead dosage, values combined; (i.e., those receiving 1, 4.5, 9, and
pups at various ages of development. Lead concentration in blood and tissues of rabbits exposed to lead increased up to 20 days. Thereafter, with exception of bone and kidney, tissue levels declined, possibly signaling the development of a more efficient process of excretion, deposition into bone, or both. The fact that lead levels in bone can be markedly increased at 2 and 10 days of age by feeding higher lead concentrations (Fig. 1) suggests that, in the rabbit as in the rat (Momcilovic and Kostial, 1974), an efficient process for lead deposition is present very early in development. Consequently, such a process would not be expected to cause an abrupt decrease in tissue lead levels after 20 days of age. Severe lead toxicity, resulting in early mortality, progressively eliminating from consideration at later ages animals with the highest tissue lead levels (Table 3), may be another factor. This process as hypothesized by Bornschein ef al. (1977b) would be important in the high lead dose groups experiencing significant mortality rates. In the low dose lead group, therefore, the development onset of a more efficient excretory process appears to be the more plausible explanation for the decrease
LEAD
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in tissue lead levels observed after 20 days. The declining tissue lead concentrations could also be attributed to decreasing dose per kilogram with age (Table 2). It should be noted, however, that the greatest decrease in dosage (64%) occurs from 2 to 10 days of age when tissue lead levels are increasing, and not from 20 to 30 days (42%) when levels are falling. The high plasma to blood lead ratios observed in the younger rabbits, which subsequently declined with age (Table 2), cannot be attributed to the relatively higher lead dosage fed to the younger offspring, since a similar plasma to blood lead distribution was observed in control littermates. If we assume that lead in plasma, as opposed to that in erythrocytes, is more readily available to soft tissues, then high plasma lead concentrations may constitute an important factor in the greater susceptibility of the young neonatal animal to the toxic effects of lead. Indeed, plasma lead concentrations have been reported to be higher in younger than in older rats injected iv with lead citrate (Jugo, 1976). No age-related alterations in plasma lead concentrations, however, have been observed in infants or children (Rosen and Trinidad, 1974). The relative distribution of lead in immature rabbit tissues parallels, in some respects, the lead tissue distribution observed in other animals, as well as in humans (Barry, 1975; Krigman et al., 1974a; Stowe et al., 1973). Thus, with lead exposure, the highest concentrations of lead were found in bone, followed by liver, kidney, blood, and brain. Lead concentration in the cerebellum was, for the most part, always greater than in whole brain and could account for the marked susceptibility of the cerebellum to the toxic effects of lead. A similar relative lead distribution in brain has been reported for rats (Michaelson, 1973) and dogs (Stowe et al., 1973) by some investigators, but not by others (Goldstein et al., 1974). It is of interest, in this respect, that in our study, the difference in lead concentration between cerebellum and brain was greatest at 2 and 10 days but, thereafter, decreased with age. Possibly, the disparity in results reported in the literature may, in part, be due to sacrificing animals and assaying their tissue levels at different ages. Most manifestations of blood dyscrasia associated with lead toxicity in man are closely modeled in the rabbit (Hass et al., 1964; Roscoe et al., 1975). Rabbit and man are among the few species in which lead interferes with porphyrin metabolism (Hass et al., 1964) and produces punctate basophilic stippling (Hass et al., 1964; Roscoe et al., 1975) as well as K+ loss from erythrocytes (Joyce et al., 1954). The recognized relationship between the hematocrit and lead concentrations in blood is demonstrated by the decrease in hematocrit seen at 20 days in rabbits fed 4.5 mg of Pb (NO&/day. The conspicuous renal changes in human lead poisoning, such as the presence of intranuclear inclusion bodies in the tubular lining cells, tubular necrosis (Henderson, 1955), and aminoaciduria paralleled findings in young rabbits (Hass et al., 1964; von Studnitz and Haeger-Aronsen, 1962). In humans, pathologic changes, with regard to their location and sequence of development, are closely modeled by rabbits (Hass et al., 1964). The absence of statistically significant increases in water content in most brain areas of rabbits fed 1.0 or 4.5 mg of lead nitrate/day at 2, 10, 15, 20, or 30 days of
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age (Fig. 8) confirms the results of some (Krigman et al., 1974b; Michaelson, 1973), but not others (Clasen et al., 1974). In white matter, medulla, and cerebellum of 20-day-old, lead-fed rabbits, the water content was greater than that of littermate controls, confirming the results of Goldstein et al. (1974) and Michaelson (1973). The decline of water content with age in the non-lead-fed rabbit is reminiscent of developmental changes in brain water content of other species (Agrawal et al., 1968 a,b; Vernadakis and Woodbury, 1965; Barlow et al., 1961). The slower decrease in water content observed in lead-fed littermates, also noted by Michaelson in rats (1973), suggests that lead retards the maturation of this process. Since the fall in brain water content in animals during the period of rapid brain development is thought to reflect the most active phase of myelination (Agrawal et al. 1968a; Barlow et al., 1961), and lead exposure is known to retard myelination (Krigman et al., 1974a), it is likely that these results (Fig. 8) represent the effect of lead on myelination. The inability to detect a difference in water content between lead and control 30-day-old rabbit offspring may be attributable (as described above) to the early mortality of those offspring most susceptible to lead. It is interesting that marked increases in water volume, indicating possible brain edema, were noted only in a small percentage of lead-exposed animals. The lack of frank edema in most of our lead-intoxicated rabbits parallels the data of Pentschew (1965), who noted an ostensible increase in brain volume in only 6 out of 20 cases of acute lead encephalopathy in infants and small children. Many of the histopathologic CNS lesions observed by us in the lead-exposed rabbit were similar to those found in brains of other species, including man. These results stand in contradistinction to those of others who failed to produce encephalopathy ‘in rabbits, even when lead exposure was from 3 to 12 months of age (Hass et al., 1964). The inability to produce lead encephalopathy in young rabbits possibly is related to the fact that rabbit brain, like that of man (Davison and Dobbing, 1968) and rhesus monkey (Portman et al., 1972), undergoes its maximal development around parturition (Hare1 et al., 1972). Since the brain is most vulnerable to perinatal stress during the period of maximum brain development (Davison and Dobbing, 1968), the threshold to lead toxicity also could be lower during this period. Other factors predisposing to greater CNS lead toxicity in the neonatal animal are the higher gastrointestinal absorption of lead (Forbes and Reina, 1972; Jugo, 1976), the lower overall excretion (Forbes and Reina, 1972), and the greater CNS uptake and retention of lead (Jugo, 1976; Momcilovic and Kostial, 1974). The critical dependency on age in the ability to produce lead encephalopathy is apparent also in the rat. Suckling rats exposed to lead via dams whose diet contains 4% lead carbonate, develop fatal encephalopathy by 4 weeks of age (Goldstein et al., 1974; Krigman et al., 1974a; Pentschew and Garro, 1966; Thomas et al., 1971). The encephalopathy can be avoided if at the time of weaning offspring are prevented from eating the chow containing 4% lead salt (Michaelson and Sauerhoff, 1974; Pentschew and Garro, 1966), or if the concentration of lead in the chow is reduced lOOO-fold, or to that found in the dam’s milk (Michaelson and Sauerhoff, 1974). Paradoxically, encephalopathy also is avoided if weanlings are started on a diet of 4% Pb (NO&/day beginning at 3 weeks and continuing to
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11 weeks of age (Goldstein et al., 1974). The above results suggest that exposure to lead, even at extremely low concentrations, must occur shortly after birth, if the neonatal CNS is to become vulnerable to concentrations of lead which produce encephalopathy. This critical dependency on age, during which time the neonatal animal must first be exposed to lead, also appears to be an important factor in developing cognitive and behavioral deficits following exposure to low concentrations of lead (Brown, 1975; de la Burde and Choate, 1972, 1975; Sobotka er al., 1975). Increased locomotor activity over age-matched controls also has been reported in mice following exposure to lead from birth to 40 days of age (Silbergeld and Goldberg, 1973). In these studies, however, growth retardation was evident in the lead-treated offspring. When weight-matched controls were used, the sustained hyperactivity was found to be attributable to early undernutrition (Bomschein et al., 1977b). Nutritional deficiency arising from the effect of lead on the dam, however, is not a necessary factor in the induction of behavioral deficits in the offspring. This is indicated by the occurrence of behavioral alterations in adult and weanling rats which were initially exposed, from birth to 3 weeks of age, to lead directly (Brown, 1975; Sobotka et al., 1975). Increases in activity, ranging from 40 to 93% greater than pair-fed controls, have been noted in neonatal rats exposed to 25 ppm of dietary lead (Michaelson and Sauerhoff, 1974). In our studies, in which low concentrations of lead were fed directly to rabbit pups, activity generally was higher in lead than in non-lead-fed littermates (except at IO days of age). In agreement with Winneke et al. (1976), we have interpreted the generally higher activity exhibited by the lead-fed offspring to be a hyperresponsiveness to a novel situation. Indeed, the response of lead-fed asymptomatic pups to handling by investigators always appeared to be more exaggerated and of longer duration than that of littermate controls. This was most apparent when animals retreated from the approaching hand of the investigators. Hyperactivity, also exhibited by mice exposed to lead has been attributed to retardation of brain development (Maker et al., 1975) and not to specific neurological effects of lead. Even though serotonin levels in the CNS of lead-exposed rats have been found to be unaltered (Sobotka et al., 1975), such behavior would be consistent with alteration in serotonergic function. Increased locomotor activity following administration of d-amphetamine is potentiated by reducing the availability of tryptophan and/or interfering with serotonin synthesis (Hollister et al., 1976). It is known that serotonin turnover, dependent on tryptophan availability from blood, as well as other factors (Femstrom and Wurtman, 1971), may change without concurrent alterations in serotonin steady-state levels. Since low concentrations of lead [l mg of Pb (NO&day] are known to interfere with the passage of tryptophan into the brain of rabbit pups (Lorenzo and Gewirtz, 1977), it is possible that lead may alter turnover of serotonin without affecting endogenous levels. The evidence presented above indicates that both overt and asymptomatic CNS lead toxicity can be produced in rabbits when oral lead feeding by hand is initiated at birth. It is noteworthy that the effect of lead on the rabbit parallels, in many
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respects, that on the human offspring. In addition, the rabbit model presented here meets many of the other criteria advocated in a recent report by the National Academy of Sciences (Committee on Toxicology, 1976) with respect to (1) assessment of lead dosage, (2) route of administration, (3) effect of lead on organ systems, and (4) rate of brain growth. More practically, the use of the neonatal rabbit in such studies provides a readily available, larger species that permits experimental procedures, difficult or impossible to perform on smaller animals. Use of this model should aid in the elucidation of the etiology of lead-induced CNS dysfunction. ACKNOWLEDGMENTS This study was supported by the N.1.H Grant HD08945 and the Children’s Hospital Medical Center Mental Retardation and Human Developmental Research Program. We wish to thank the following Brookline High School volunteer students for their aid with caring, feeding, and testing of animals: Sandra Stoddard, Barbara Schnee, and Carole Pfieffer.
REFERENCES Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1968a). Developmental changes in mouse brain: Weight, water content and free amino acids. J. Neurochem. 15, 917-923. Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1968b). Water content of dog brain parts in relation to maturation of the brain. Amer. J. Physiol. 215, 846-848. Baloh, R., Sturm, R., Green, B., and Glesser, G. (1975). Neuropsychological effects of chronic asymptomatic increased lead absorption. Arch. Neural. 32, 326-330. Baemstein, H. D., and Grand, J. A. (1942). The relation of protein intake to lead poisoning in rats. J. Pharmacol.
Exp.
Ther.
74, 18-24.
Barlow, C. F., Domek, N. S., Goldberg, M. A., and Roth, L. J. (1961). Extracellular brain space measured by 35S-sulfate. Arch. Neurol. 5, 102- 110. Barry, P.S.I. (1975). A comparison of concentrations of lead in human tissues. &it. J. Znd. Med. 32, 119-139. Beattie, A. D., Moore, M. R., Goldberg, A., Finlayson, M. J. W., et al. (1975). Role of chronic lowlevel lead exposure in the aetiology of mental retardation. Lancer 1, 589-592. Bomschein. R. L., Fox, D. A., and Michaelson, I. A. (1977a). Estimation of daily exposure in neonatal rats receiving lead via dam’s milk. Toxicol. Appl. Pharmacol. 40, 577-587. Bomschein, R. L., Michaelson, I. A., Fox, D. A., and Loch, R. (1977b). Evaluation of animal models used to study effects of lead on neurochemistry and behavior. In “Biochemical Effects of Environmental Pollutants,” (S. D. Lee, Ed.) p. 44-460. Ann Arbor Science Publ., Ann Arbor, Mich. Browder, A. A., Joselow, M. M., and Louria. D. B. (1973). The problem of lead poisoning. Medicine 52, 121-139. Brown, D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharmacol. 32, 628-637. Brown, S., Dragann, N., and Vogel, W. H. (1971). Effects of lead acetate on learning and memory in rats. Arch, Environ. Health 22, 370-372. Byers, R., and Lord, E. E. (1943). Late effects of lead poisoning on mental development. Amer. J. Dis. Child. 66, 471-494. Chisolm, J., Jr. (1977). Is lead poisoning still a problem? Gin. Chem. 23, 252-253. Clasen, R. A., Hartman, J. F., Starr, A. J., Coogan, P. S., er al. (1974). Electron microscopic and chemical studies of the vascular changes and edema of lead encephalopathy. Amer. J. Pathol. 74, 215-234. Cole, M. A. (1977). Lead inhibition of enzyme synthesis in soil. Appl. Environ. Microbial. 33, 262-268. Colquhoun, D. (1971). “Lectures on Biostatistics,” pp. 167- 170. Clarendon Press, Oxford. Committee on Toxicology. (1976). Recommendations for the prevention of lead poisoning in children. Prepared for U.S. Consumer Product Safety Commission and the National Academy of Sciences, Washington, D.C.
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TOXICITY
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Conover, W. J. (1971). “Practical Nonparametric Statistics,” pp. 309-314. Wiley, New York. David, O., Hoffman, S., McGann, B., Sverd, J., and Clark, J. (1976). Low lead levels and mental retardation. Lancer 2, 1376- 1379. Davison, A. N., and Dabbing, J. (1968). The developing brain. In “Applied Neurochemistry” (A. N. Davison and J. Dobbing, Eds.), pp. 253-286. Blackwell, Oxford, England. de la Burde, B., and Choate, M. S. (1972). Does asymptomatic lead exposure in children have latent sequelae? J. Pediat. 81, 1088-1901. de la Burde, B., and Choate, M. S. (1975). Early asymptomatic lead exposure and development at school age. .I. Pediat. 87, 638-642. Femstrom, J. D., and Wurtman, R. J. (1971). Brain serotonin content; physiological dependence on plasma tryptophan levels. Science 173, 149-152. Forbes, G. B., and Reina, J. C. (1972). Effect of age on gastrointestinal absorption (Fe, Sr, Pb) in the rat. J. Nutr. 102, 647-652. Gilsinn, J. F. (1972). Estimates of the nature and extent of lead paint poisoning in the United States. U.S. Department of Commerce National Bureau of Standards. Tech. Note 746. Goldstein, G. W., Asbury, A. K., and Diamond, I. (1974). Pathogenesis of lead encephalopathy. Arch. Neural. 31, 382-389. Harel, S., Watanabe, K., Linke, I., and Schain, R. J. (1972). Growth and development of the rabbit brain. Biol. Neonat. 21, 381-399. Hass, G. M., Brown, D. V. L., Eisenstein, R., and Hemmens, A. (1964). Relations between lead poisoning in rabbit and man. Amer. J. Pathot. 65, 691-727. Henderson, D. A. (1955). Chronic nephritis in Queensland. Aust. Ann. Med. 4, 164. Hollister, A. S., Breese, G. R., Kuhn, C. M., Cooper, B. R., and Schanburg, S. M. (1976). An inhibitory role for brain serotonin-containing systems in the locomotor effects of d-amphetamine. J. Pharm. Exp. Ther. 198, 12-22. Joyce, C. R. B., Moore, H., and Weatherhall, M. (1954). The effect of Pb, Hg and Au on K+ turnover of rabbit blood cells. Brit. J. Pharmacol. 9, 463 -470. Jugo, S. (1976). Metabolism of toxic heavy metals in growing organisms. A review. Environ. Res. 13, 36-46. Krigman, M., Druse, M. Traylor, T., Wilson, M., et al. (1974a) Lead encephalopathy in the developing rat: effect upon myelination. J. Neuropathol. Exp. Neurol. 33, 58-73. Krigman, M. R., Dmse, M. J., Traylor, T. D., Wilson, M. H., et al. (1974b). Lead encephalopathy in the developing rat: Effect on cortical ontogenesis. J. Neuropathol. Exp. Neurol. 33, 671-686. Lampert, P., Garro, F., and Pentschew, A. (1967). Lead encephalopathy in suckling rats: An electron microscopic study. In “Brain Edema” (I. Klatzo and F. Seitelberger, Eds.), pp. 207-222. Springer-Verlag, New York. Levander, 0. A., Morris, V. C., Higgs, D. J., and Ferretti, R. J. (1975). Lead poisoning in vitamin Edeficient rats. J. Nutr. 105, 1481-1485. Lockeretz, W. (1975). Lead content of deciduous teeth of children in different environments. Arch. Environ. Health 30, 583-587. Lorenzo, A. V., and Gewirtz, M. (1977). Inhibition of ‘%tryptophan transport into brain of lead exposed neonatal rabbits. Brain Res. 132, 386-392. Mahaffey, K. R., Goyer, R., and Haseman, J. K. (1973). Dose-response to lead ingestion in rats fed low dietary calcium. J. Lab. Clin. Med. 82, 92- 100. Maker, H. S., Lehrer, G. M., and Silides, D. J. (1975). The effect of lead on mouse brain development. Environ. Res. 10, 76-91. Michaelson, I. A. (1973). Effects of inorganic lead on levels of RNA, DNA and protein of developing neonatal rat brain. Toxicol. Appl. Pharmacol. 26, 539-548. Michaelson, I. A., and Sauerhoff, M. W. (1974). An improved model of lead-induced brain dysfunction in the suckling rat. Toxicol. Appl. Pharmacol. 28, 88-96. Momcilovic, B. and Kostial, K. (1974). Kinetics of lead retention and distribution in suckling and adult rats. Environ. Res. 8, 214-220. Pentschew, A. (1965). Morphology and morphogenesis of lead encephalopathy. Acta Neuropathol. 5, 133-160. Pentschew, A., and Garro, F. (1966). Lead encephalo-myelopathy of the suckling rat and its implications on the porphyrinopathic nervous diseases. Acta Neuropathol. 6, 266-278.
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Portman, 0. W., Alexander, M., and Illingworth, D. R. (1972). Changes in brain and sciatic nerve composition with development of the rhesus monkey (Macaca mulatta), Brain J&s. 43, 197-213. Roscoe, D. E., Nielsen, S. W., Eaton, H. D., and Rousseau, J. E. (1975). Chronic plumbism in rabbits: A comparison of three diagnostic tests. Amer. J. Vef. Res. 36, 1225- 1229. Rosen, J. F., and Trinidad, E. E. (1974). Significance of plasma lead levels in normal and leadintoxicated children. Environ. Health Perspect. 7, 139- 144. Rosenblum, W. I., and Johnson, M. (1968). Neuropathologic changes produced in suckling mice by adding lead to the maternal diet. Arch. Pathol. 85, 640-648. Searle, B., Chan, W., and Davidow, B. (1972). Determination of lead in blood and urine by anodic stripping voltammetry. Clin. Chem. 47, 76-80. Silbergeld, E. K., and Goldberg, A. M. (1973). A lead-induced behavioral disorder. Life Sci. 13, 1375- 1383. Six, K. M., and Goyer, R. A. (1972). The influence of iron deficiency on tissue content and toxicity of ingested lead in the rat. J. Lab. C/in. Med. 79, 128-136. Sobotka, T. J., Brodie, R. E., and Cook, M. P. (1975). Psychophysiologic effects of early lead exposure. Toxicology 5, 175- 191. Stowe, H. D., Goyer, R. A., Krigman, M. R., Wilson, M., et al. (1973). Experimental oral lead toxicity in young dogs. Arch. Pathol. 9S, 106- 116. Thomas, J. A., Dallenbach, F. D., and Thomas, M. (1971). Considerations on the development of experimental lead encephalopathy. Virchows Arch. A. 352, 61-74. Urban, W. D. (1976). Statistical analysis of blood lead levels of children surveyed in Pittsburgh, Pennsylvania: Analytical methodology and summary results. National Bureau of Standards N. B. S. I. R. 76-1024. Vemadakis, A., and Woodbury, D. M. (1965). Cellular and extracellular spaces in developing rat brain. Arch. Neural. 12, 284-293. Von Studnitz, W. and Haeger-Aronsen, B. (1%2). Urinary excretion of amino acids in lead-poisoned rabbits. Acta Pharmacol. Toxicol. 19, 36-42. Waldron, M. A., and Stofen, D. (1974). “Sub-clinical Lead Poisoning,” pp. 101-107. Academic Press, London and New York. Whitney, J. C., Blackmore, D. K., Townsend, G. H., et af. (1976). Rabbit mortality survey. Lab. Anim. 10, 203-207. Winneke, G., Brockhaus, A., and Baltissen, R. (1977). Neurobehavioral and systemic effects of longterm blood lead elevation in rats. I. Discrimination learning and open field behavior. Arch. Toxicol. 37, 247-263.