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OCR for page 281
26
Morphologic, Neurochemical,
and Behavioral Responses to Toxic Agents
This chapter discusses the importance
of timing and dose for effects in develop-
ing organisms. Then, specific biologic
markers of neurodevelopment are dis-
cussed, ranging from the association of
minor physical anomalies with behavioral
effects, to measurement of neurochemical
concentrations, to behavioral assessment
of complex processes.
EFFECTS OF TIME OF EXPOSURE
AND DOSE: IRRADIATION AS A
PARADIGM
The extensive use of ionizing radiation
as an embryotoxic agent has been of para-
mount importance in the delineation of
several important concepts that have par-
ticular relevance to current efforts in
developmental toxicology. Studies in
radiation teratology are unique, in that
the physics of ionizing radiation has al-
lowed scientists to produce effects in
the embryo directly, with no concern for
the moderating influence of the so-called
placental barrier.
Work from four laboratories established
two basic concepts: stage specificity and
the relation of dosage to response (Hicks,
1953; Rugh, 1953; Russell and Russell,
1954; Wilson, 1954~. All the early studies
provided convincing evidence that the
production of specific congenital malfor-
281
mations depended on the stage of develop-
ment at which radiation was administered
and that the severity of the effect pro-
duced was a function of the dosage. The
Reimplantation stages of development
were usually shown to be relatively radio-
resistant when neonatal death was as-
sessed, although Rugh (1959) reported that
low doses of radiation during this period
produced a low incidence of exencephaly
in survivors.
Wilson (1954) showed the influence of
dose, especially on the production of eye
defects in rat fetuses. Irradiation with
25 reds on ED 9 produced eye defects in 6%
of the fetuses. The magnitude of response
increased with increasing dose; exposure
to 200 reds produced eye defects in 72% of
the fetuses. The embryo, under the experi-
mental conditions, became radioresistant
with time. Irradiation on ED 11 with either
25, 50, or 100 reds produced no eye defects
in survivors, although all survivors were
affected when the dose was 200 reds. Hicks
( 1953) also showed time-dependent re-
sponses to ionizing radiation in studies
of specific CNS defects. Hicks noted that
irradiation of the embryo early in develop-
ment produced forebrain defects, whereas
irradiation during fetal and neonatal
life resulted primarily in cerebellar
malformations.
As summarized by Rugh (1953), "the inert
OCR for page 282
282 ,
p'rimordium or the totally differentiated
cell will be relatively resistant in terms
of morphologic change. The actively dif-
ferentiating intermediate stage or
stages will be highly radiosensitive
since they are in the process of transfor-
mation Differentiation. Hicks devel-
oped a mechanistic framework to validate
the concept (Hicks, 1959; Hicks and D,Ama-
to, 1966~. He hypothesized that the radio-
sensitive cells (neuroblasts3 were local-
ized to the region of the neural epithelium
that was active in DNA synthesis and that
the immediate consequence of ionizing
radiation was massive and extensive death
of cells of that type. Cell death was a
transient phenomenon, and the embryo was
capable of repairing the damage quickly.
He postulated that the malformations ob-
served at term were the consequence of a
balance between the initial damage and
the degree of regulation and regeneration
inherent in that region of the embryo at
the time of injury.
Kallen (1965) documented that the devel-
oping nervous system is capable of stage-
dependent regeneration. The idea could
be validated under conditions in which
regenerative capacity was absent and radi-
ation would produce specific cell death
and specific neuronal deficits. As docu-
mented below, the microneurons of the cere-
bellum and the dentate gyrus, Which
develop postnatally, satisfy the neces-
sary conditions, and selective radiation
of these regions at appropriate periods
of development produces specific struc-
tural and functional deficits.
MICRONEURONAL RADIATION
Cerebellum
Hicks et al. (1962) reported that irradi-
ation of the cerebellum of the 6-day-old
rat with 200 R produced extensive damage
to the external granule cell layer. The
layer retained its regenerative capacity
and formed an ectopic granule cell layer
within 4 days. However, the initial damage
also interfered with Purkinje cells: their
form was altered, and the association of
Purkinje cells and granule cells formed
later was abnormal. Altman and coworkers
NEURODEVELOPMENTAL TOXICOLOGY
(Altman et al., 1969; Pellegrino and Alt-
man, 1979) confirmed that observation in
a series of experiments that effectively
verified the idea that cell killing without
substantial repair led to severe cellular
deficits within the cerebellum. They used
the knowledge that different microneuron-
al populations that arise in the external
granule layer do so at specific times after
birth; the basket cells were formed on PN
6-7, the stellate cells on PN 8-11, and the
granule cells on PN 8-21. They used sequen-
tial irradiation to monitor morphogenesis
and later performance in a variety of
tasks. In the first group, focal irradia-
tion of the cerebellar cortex with 200 R
on PN 4 and 5 produced cerebellar disor-
ganization similar to that observed by
Hicks et al. (1962~. In a second group of
animals, the focal irradiation consisted
of 200 R on PN 4-5 (as above) followed by
150 R on PN 7, 9, 11, 13, and 15. With the
fractionated regimen, all derivatives
of the external granule cell layer failed
to form-an effect that resulted in severe
motor deficits. Postponing the initial
irradiation to PN 8 and 12 produced selec-
tive neu renal deficits (stellate and late -
forming granule cells in one group, late-
forming granule cells in another group)
with corresponding selective behavioral
effects.
Initial observations failed to show
any postural or motor deficits in the sec-
ond group of rats that were irradiated
many times postnatally (Pellegrino and
Altman, 1979~. In fact, in experiments
with a motor-driven rotating rod, the ir-
radiated animals performed better than
controls; that is, they fell off the rod
less frequently. However, in open-field
tests, the irradiated rats were observed
to be significantly more active than con-
trol animals. Ambulation in the open
field is affected by agitation. It was
concluded that microneuronal hypoplasia
in the cerebellum that does not produce
demonstrable locomotor deficits can nev-
ertheless lead to hyperactivity at an age
when the animals tend to be the most active
(2 months). In adult animals, the differ-
ence disappeared.
The experiments documented a strong
correlation between the developmental
OCR for page 283
RESPONSES TO TOXIC AGENTS
history of a neuronal population and its
contribution to the behavioral hier-
archies within the animal. The studies
are a logical extension of the classical
studies reviewed earlier and confirm the
observation that the primary effect of
irradiation of sensitive cell populations
is cell death. It is clear, however, that
fractionated irradiation of developing
microneurons is not accompanied by exten-
sive regeneration. Hence, highly specific
and highly reproducible cell deficits can
be produced, and their behavioral conse-
quences can be monitored.
H.
Ippocampus
The same approach has been used to evalu-
ate the effects of microneuronal hypoplas-
ia on the hippocampus (Altman, 1986~.
Focal x irradiation of the hippocampus,
begun immediately after birth, prevents
the formation of nearly 85% of the granule
cells of the dentate gyrus.
The rats were then tested in the same
283
postulated that these, and other, selec-
tive effects on microneuronal populations
can provide the anatomic basis for mini-
mal brain dysfunction under the influence
of a broad spectrum of environmental
factors, such as alcohol, lead, and gluco-
corticoids.
APPLICATION TO OTHER TOXIC
SUBSTANCES
Several kinds of toxic agents can gener-
ate structural and behavioral alterations
in animals and humans that result from
fetal and perinatal exposure. Experimen-
tal studies of time and dose effects should
be done with these agents, as has been done
for irradiation.
Hicks et al. (1961), Hicks and D'Amato
(1963), and Berry and Eayrs (1966) showed
that one effect of irradiation during fetal
life was the alteration of migration pat-
terns in the layers of the cerebral cortex.
A similar effect was observed in the mouse
fetus as a result of subjecting the dams
protocols as were the animals mentioned to hypervitaminosis A (Langman and Welch,
above with cerebellar microneuronal hype- 1967~.
plasia. They with hippocampal micro- Miller (1986) recently showed that ex-
neuronal hypoplasia were found to be ex- posure of pregnant rats to ethanol from
tremely hyperactive when tested in the ED 6 to ED 23 produced effects similar to
open field (Bayer et al., 1973~. They those seen after irradiation, i.e., a defi-
were also extremely active, compared with cit in cortical neurons and an alteration
control rats, in the running wheels (Peters
and Brunner, 1976~. The irradiated rats
displayed other behavioral changes usual-
ly associated with hippocampal damage,
including disappearance of spontaneous
alternation in a T maze and deficits in
passive avoidance learning (Bayer et al.,
1973~. The rats showed deficits at all
ages; the deficits differed in severity
with age and between tests. Further stud-
ies indicated that, as long as the learning
tasks ranged from very easy to moderately
difficult for normal rats, the irradiated
animals performed as well as normal rats
(Altman, 1987~. However, when the tasks
were more difficult, the irradiated ani-
mals were significantly impaired in tac-
tile and visual discrimination, in acqui-
sition learning, and in reversal learning.
Thus, selective microneuronal hypoplas-
ia in the hippocampus leads to selective
behavioral effects. Altman ( 1986) has
in their migration patterns. Although
not documented, the cytotoxicity of in-
gested ethanol on cortical neuroblasts
is a possible underlying mechanism of this
observation.
Similarly, the administration of lead
to rats immediately after birth results
in altered hippocampal cytodifferentia-
tion, including the presence of smaller
numbers of granule cells (Petit et al.,
1983; Kawamoto et al., 1984) and behavioral
changes. Observation of those effects
is confounded by the severe effects of lead
on brain endothelial cells (Winder et al.,
1983), so a specific effect on the granule
cell population cannot be ruled out.
RELATIONSHIP BETWEEN MINOR
PHYSICAL ANOMALIES AND
BEHAVIORAL EFFECTS
Agents that influence physical develop-
OCR for page 284
284
ment are likely to alter behavior. The
relationship between phenotypic and be-
havioral responses to pollutants is clear-
ly exemplified in the study of minor physi-
cal abnormalities (MPAs) and behavioral
pathology. These observations also sug-
gest a potentially useful set of markers
for CNS dysfunction.
The relationship between MPAs and be-
havioral aberrance was first observed
in schizophrenics (Waldrop and Halverson,
1972; Goldfarb and Botstein, in press).
The following were observed in increased
proportion in schizophrenics: excessive-
ly fine hair that stands on end, multiple
hair whorls, excessively large or small
head circumference, epicanthal folds,
hypertelorism. low-set ears. adherent
earlobes, high arched palate, curved fifth
finger, simian palmer crease, spaced toes,
and partial syndactyly. That most of those
changes are primarily ectodermal in origin
suggests that the timing and pathogenesis
of the events were shared by alterations
in the CNS.
The study of MPAs was extended to other
behavioral aberrations. In a study of
normal 2.5-year-olds, Waldrop et al.
(1968) found that, because the number of
MPAs was significantly correlated with
restless, aggressive impulsive behavior,
the MPAs might have been indicators of
hyperactivity. Behavior was stable when
the subjects were followed up to the age
of 5 years. The number of MPAs was found
to be negatively correlated with verbal
IQ (Rosenberg and Weller, 1973), with full-
scale IQ (Waldrop and Halverson, 1972;
Firestone and Prabhv, 1983), and with aca-
demic achievement (Halvorsen and Victor,
1976~. An apparent sexual dimorphism in
the relationship between MPAs and behavior
has been observed. Boys with high MPA
scores tend to be hyperactive, and girls
with high MPA scores seem to display more
inhibited, intractable behavior (Waldron
,
NEURODE~ELOPMENTAL TOXICOLOGY
MPAs are increased in autistic children
(Steg and Rapoport, 1974~. Quinn and Rapo-
port (1974) found an association between
increased MPAs and aggression and hyper-
activity, but not anxiety, in boys. In the
same sample, dopamine p-hydroxylase ac-
tivity in blood was correlated with MPA
score. MPA scores were higher in hyper-
active and retarded boys (Rapoport et al.,
1974) and in siblings who were considered
mentally normal.
Offspring with high MPA scores are
more likely to have been the products of
complicated pregnancies (e.g., with toxe-
mia or prematurity) than of uncomplicated
pregnancies (Simonds and Aston, 1981~.
They are also more likely to have siblings
and parents with high MPA scores; that
suggests that both genetic and nongenetic
mechanisms were involved in the pathogene-
sis (Smalley et al., 1988~.
Those observations link a class of rela-
tively easily identified and measurable
changes in physical structure with abnor-
mal CNS development with behavioral defi-
cits. Both the physical structures and
the CNS are ectodermal in origin. Thus,
the two classes of tissue might have re-
sponded in an analogous way to a given nox-
ious agent. The inference to be drawn is
that each class of observations (physical
and behavioral) could serve as a marker
of the other. MPAs in newborns have also
been found in dose-dependent relationship
with umbilical cord blood lead concentra-
tions (Needleman et al., 1984~.
NEUROCHEMICAL EFFECTS
For the assessment of nervous system
status with chemical methods, samples
of various bodily fluids are taken and
constituent materials are analyzed (see
Table 25-1~. The methods have required
the development of high-performance as-
says, because the amounts of sample
et al., 1976; O'Donnell and Van Tuinan, usually available and the concentrations
1979~. Quinn et al. (1977) classified present in the relevant compartments are
infants according to MPA numberintolow, small. The fluids are blood, urine, and
middle, and high groups. At 2 years of age, cerebrospinal fluid (CSF). Blood and urine
high-MPA boys were more irritable and had are of less utility, because of their re-
a higher incidence of night awakening, moteness from the nervous system and the
and high-MPA girls were less active and contribution of nonneuronal sources to
more withdrawn. the amounts of most substances in these
OCR for page 285
RESPONSES TO TOX[CAGE[JTS
compartments. CSF is not routinely avail-
able, inasmuch as its sampling requires
medical oversight and involves danger.
Advanced techniques of imaging have re-
cently enlarged the ways in which biochemi-
cal reactions and events in the brain can
be measured; these are discussed in the
Chapter 30, because they present the most
important new opportunities for obtaining
markers of neuropsychiatric function.
Neurochemical characteristics have
been studied in only two major intoxication
states: lead poisoning and brain damage
induced by ~V-methyl-4-phenyltetrahydro-
pyridine (MPTP). The neurochemistry of
lead poisoning has been extensively stud-
ied in animals (Silbergeld and Hruska,
1980; Winder et al., 1983~. Only recently
have attempts been made to extrapolate
from the results of those studies to the
development of biologic markers in humans.
Silbergeld and Chisolm ( 1976) studied
monoamine metabolites in urine of lead-
exposed children. As shown in Figure 26-
1, there is a correlation between blood
lead content and 24-hour urinary excretion
of the dopamine metabolite homovanillic
acid (HVA) in those children. HVA was meas-
ured before initiation of chelation thera-
py, within a week after the children were
removed from lead-contaminated environ-
ments (in all cases, lead paint). Over the
long term, urinary HVA content was reduced,
18
16 -
. _
14 -
10 -
-
o
-
E 6
>
.
.
4 ~
285
as was blood lead content, although both
blood lead and urinary HVA remained higher
in treated lead-exposed children than in
age-matched controls. Other neurochemi-
cals reported to be altered by lead in ani-
mal models - s uch as GAB A and enkephalins—
are less available to clinical measure-
ment, because they require CSF, and have
not been investigated in humans.
MPTP is a contaminant in some Designer
drugs or meperidine derivatives with
opiatelike characteristics. After the
remarkable finding that some addicts had
acute-onset necrologic disorders that
were indistinguishable from so-called
idiopathic Parkinson's disease (Langston
et al., 1983), attention focused on MPTP
as the active pathologic agent. MPTP was
found to be a specific basal ganglia toxin
that damages the same nigrostriatal dopa-
minergic pathways that are affected in
parkinsonism (Kopin and Markey, 1988~.
The mechanism of action of MPTP involves
uptake into dopaminergic neurons, inter-
action with oxidases within the neurons,
and selective cell killing, possibly by
the generation of free-radical oxygen or
hydroxyl radicals. As a dopaminergic tox-
in, MPTP could be expected to reduce output
of dopamine metabolites from brain into
CSF; this has been demonstrated in primate
models of intoxication (Kopin and Markey,
1988~.
.
r = .729
p <.001
· /
1.0 1.5 2 0 2 5 3 0 3 5 4 0
PbB(pM/L)
FIGURE 26 1 Correlation between blood lead and 24
hour urinary excretion in children. Source: Silbergeld
and Chisolm, 1976.
OCR for page 286
286
BEHAVIORAL EFFECTS
Behavior is the observable response
of an organism to changes in the external
or internal environment. That definition
includes actions ranging from reflex re-
sponses to the solving of complex problems
and performance on psychometric tests and
in social situations. Behavioral analysis
can proceed at any of those levels.
The investigator of neurobehavioral
function is confronted with a choice be-
tween attempting to characterize a pos-
sible deficit by measuring a single, iso-
lated function and scaling more complex
integrative behaviors. The choice might
present a trade-off between the precision
of a test that measures a single function
(or as close to a single function as pos-
sible) and the relative imprecision of
a test instrument that measures the sub-
ject's integrative capacity in a more com-
plex single-demand task or in a number
of tasks. The use of measures of single
functions can be more effective in detect-
ing neurotoxic deficits than gross-per-
formance tests, because they are more fo-
cused and demanding (Smith, 1985~. Tests
of integrative capacity might yield in-
creased sensitivity, but lead to less pre-
cision as to the location and degree of a
deficit.
A number of outcomes are presented below,
roughly in increasing order of functional
complexity. In each instance, their utili-
ty when testing for lead exposures is no-
ted, not because that represents their
sole utility, but because the effects of
lead exposure is the most widely studied
toxicity in humans.
· —
· Nerve conduction time. Measurements
of nerve conduction time have been useful
in assessing neurotoxicants that act on
myelin development or on Schwann cells.
Occupational exposures to lead at doses
that did not produce symptoms were associ-
ated with impairments in nerve conduction
(Seppalainen and Hernberg, 1980~. Chil-
dren exposed to lead from industrial
sources were found to have dose-dependent
nerve-conduction slowing (Landrigan et
al., 1976~. Because normal variation is
large, assessment of nerve conduction time
NEURODE~ELOPMENTAL TOXICOLOGY
is not well suited as a screening device
in neurotoxicant exposure.
· Sensory psychophysical functions and
scotopic vision. Measures of visual acuity
in bright light were not related to lead
exposure in primates. Visual acuity in
the dark was diminished in primates exposed
to lead (Bushnell et al., 1977~. The re-
sults suggest specific impairment of
rod function.
· Evoked potentials. Because evoked po-
tentials recorded from the scalp or
spinal cord reflect activity in multisyn-
aptic pathways, evoked potentials can
provide useful information in assessing
sensory transmission from the periphery
to the cerebral cortex. Three types of
evoked potentials have been recorded:
auditory evoked potentials for which the
stimuli are generally pure tones or clicks,
but can be phonemes or words; visual evoked
potentials for which the stimuli are stro-
boscopic light flashes or checkerboard
patterns on computer monitors; and somato-
sensory evoked potentials for which the
stimuli are brief electric impulses deliv-
ered to the skin. Auditory evoked poten-
tials have been found to be altered in ex-
posure to lead (Otto et al., 1981), carbon
monoxide (Groll-Knoff et al., 1978), and
trichloroethylene (Winneke et al., 1978~.
Visual evoked potentials have been found
to be affected in exposure to xylene
(Seppalainen et al., 1981), methyl mercury
(Iwata, 1980), and n-hexane (Seppalainen
et al., 1979~. Somatosensory evoked poten-
tials have been reported to be altered in
exposure to lead (Seppalainen, 1978), but
not in exposure to n-hexane severe enough
to be symptomatic (Zappoli et al., 1978~.
· Auditory discrimination. Auditorydis-
crimination can be tested against various
masking backgrounds, such as taped sounds
containing a signal against background
noises of increasing loudness (e.g., elec-
tric fan sounds or noise from a cafe) (Gold-
man et al., 1970~. In studies of asympto-
matic children, those with the higher con-
centrations of lead in their teeth had
lower scores on this measure (Needleman
etal.,1979~.
OCR for page 287
RESPONSES TO TOXIC AGENTS
· Vibration sense. Vibration sense can
be measured with a number of methods; the
most simple (and least sensitive) uses
a tuning fork. Computerized methods with
increased sensitivity and precision are
available (Maurissen and Weiss, 1980~.
Decreased vibratory sensitivity is ob-
served in many conditions that involve
the central and peripheral nervous sys-
tems. Among them are systemic disease,
such as diabetes, chronic liver failure,
pernicious anemia, peripheral neuropath-
ies, syphilis, spinal-cord lesions, and
uremia; exposure to pharmaceuticals, such
as isoniazid, phenytoin, vincristine,
and glutethimide; and exposure to chemi-
cals, such as acrylamide, arsenic, n-hex-
ane, mercury, and methylbutylketone
(Maurissen, 1985~.
· Motor function. Quantitative measures
of tremor have been evaluated in methyl
mercury exposure. Quantitatively in-
creased tremors were shown when clinical
studies were noninformative. Studies of
motor patterns of children classified as
to lead exposure are under way. One study,
which measured on-task behavior of chil-
dren differentially exposed to lead, found
that high-lead children spent more time
in off-task behavior in the classroom-
e.g., out-of-chair activity, staring out
of the window, and talking to classmates
(Needleman and Bellinger, 1981~.
· Attention. Attention is a complex func-
tion of arousal, vigilance, and resistance
to distraction. A number of measures of
it are available. In the Continuous Per-
formance Test, a measure of vigilance
(Rosvold et al., 1956), the subject is
presented with a set of stimuli (letters
of the alphabet) on a screen for short in-
tervals in rapid succession. The critical
stimulus (the letter X) is presented at
a predetermined probability. The demand
task is to press the response key when the
critical stimulus appears. The number
~ ~ · e · -
ot errors ot omission, commission, and
latency to response can be determined.
Reaction time with various intervals of
delay has been used to discriminate among
children who differed as to lead exposure
287
(Needleman et al., 1979; Yule et al., 1981;
Hunter et al., 1985~.
· Visual-motor integration. Measures
of visual-motor integration, such as
the Bender-Gestalt test (Trillingsgaard
et al., 1985), have found wide application
in the study of brain damage in children.
A number of skills are called on: spatial
v~sua~za~on, eye - hand coordination,
and visual-spatial memory.
· Speech and language function. Speech
and language function is the sum of many
competences and can be perturbed at many
levels. At the perceptual level, auditory
acuity for pure tones and the ability to
screen out background distractions can
be measured (Goldman et al., 1970~. Short-
term memory and the ability to discriminate
patterns are testable (Seashore Rhythm
Test). The ability to comprehend language
is testable with many instruments, such
as the Token Test, the verbal subtests of
the WISC-R, and the Illinois Test of Psy-
cholinguistic Abilities.
· Psychometric intelligence. Studies of
psychometric intelligence in children
have been widely used in recent years to
study lead toxicity, exposure to polybro-
minated biphenyls (PBBs), and fetal al-
cohol exposure. Three tests have been used
generally: the Bayley Scales of Infant
Development for children between 6 months
and 3 years old, the McCarthy Scales for
children more than 3 years old but less than
5 years old, and the Wechsler Intelligence
Scales revised for children over 5 years
old. The most sensitive subscales of these
instruments appear to be the verbal and
general cognitive index. Numerous studies
of effects of lead exposure at low dose in
children have shown IQ deficits after con-
trol of relevant covariates (U.S. EPA,
1986~.
· Social behavior. Attention to cogni-
tive, perceptual, and motor competences
should not direct attention away from so-
cial behavior of children. Three groups
of investigators have rated classroom
behavior in lead-exposed children with
structured questionnaires (Needleman
et al., 1979; Yule et al., 1981; Hatzakis
OCR for page 288
288
et al., 1987~. Teachers blind to a sub
ject's lead exposure reported a dose
dependent increase in nonadaptive class-
room behavior, such as distractibility,
-
NEURODEKELOPMENTAL TOXICOLOGY
inability to work independently, dis-
organization, hyperactivity, impulsivi-
ty, and inability to follow directions
(Needleman et al., 1979~.
Representative terms from entire chapter:
granule cells
