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OCR for page 289
~7
Methodologic Issues of
Extrapolation from Animal Studies
to Human Toxicant Exposure
This chapter briefly reviews some of
the major approaches to relating animal
findings In functional teratology to the
assessment of potential human health haz-
ards. The approaches include:
· Investigation of underlying mechan-
isms of functional alterations observed
· .
In animals.
· Investigation in animals of normal
and abnormal development of functional
end points that are comparable in humans.
· Direct comparison of functional ef-
fects seen in animals and humans when
data are available on both.
Intrauterine exposures to some terato-
genic agents have been linked to gross
physical malformations in both humans and
animals. Structural abnormalities are
often profiled as syndromes, e.g., the
fetal alcohol syndrome (Clarren and Smith,
1978) and the fetal hydantoin syndrome
(Hanson et al., 1976~. Interest in people
with subtle functional effects after low-
dose exposures and people without overt
anomalies has increased. Since Wilson
( 1973) included functional alterations
in a list of possible effects of exposure
to developmental toxicants, research in
the subject has expanded greatly. All
functional systems are theoretically at
risk at some point in their development
289
and maturation. Only a few functional
systems have been studied, and that situa-
tion is changing (Kimmel and Buelke-Sam,
1981; Kavlock and Grabowski, 1983; Riley
and Vorhees, 1986~. Unlike studies that
evaluate multiple structural changes
after exposures, studies of postnatal
function typically evaluate effects in
a single organ system or on a single end
point, e.g., the CNS or immune deficiency.
Additional complications are encoun-
tered in cross-species comparisons of
postnatal functional alterations, be-
cause species often vary both in their
responsiveness or susceptibility to toxic
insult and in the manner in which they mani-
fest toxicity. Examples of research
aimed at overcoming those problems with
each approach are discussed.
INVESTIGATION OF UNDERLYING
MECHANISMS
One way of relating animal findings and
human hazard is to evaluate underlying
structural, biochemical, and physiologic
correlates of overt functional changes
seen in animals. The rationale is that the
determination of the target and degree
of toxicity produced by developmental
exposure will yield information relevant
to the human situation.
Mirmiran and colleagues (1985) recently
OCR for page 290
290
reviewed the relationships between be-
havioral alterations observed in humans
and animals and the underlying neurochemi-
cal and electrophysiologic disturbances
observed in rats that were exposed to phar-
maceutical agents during development.
Table 27-1 presents some of the findings.
The immaturity of the blood-brain bar-
rier and greater accumulation of many
of these compounds in the developing brain
make the fetal brain a major target of its
mother's medication. Mirmiran et al.
(1985) have shown that neonatal exposure
of rats to clonidine, an antihypertensive
agent, and clomipramine, an antidepres-
sant that acts on Norepinephrine and sero-
tonin neurotransmission, suppresses ra-
pid-eye-movement (REM) sleep in the devel-
oping rats. In adulthood, the offspring
rats showed hyperactivity, hyperanxiety,
reduced sexual behavior, disturbed sleep
patterns, and smaller cerebral cortex.
NEURODEVELOPMENTAL TOXICOLOaY
STUDY OF COMPARABLE
FUNCTIONAL END
A second approach to determining the
relationship of animal findings in post-
natal functional studies
situation is to select a
to the human
functional re-
sponse that is comparable across species.
A variety of potentially relevant end
points are available, e.g., sleep pat-
terns, neonatal vocalizations, and suck-
ling patterns. The development of these
end points and their sensitivity to toxic
insult could be compared directly across
species.
The startle reflex is valuable in such
an effort for several reasons:
· Startle can be elicited in all mam-
mals, including humans.
· The startle reflex is mediated via
simple neuronal circuits.
TABLE 27-1 Sequelae of Developmental Exposure to Drugs in Humans and Animalsa
-
REM~ Sleep Relevant
Effects in Effects in Deprivation Transmitter
Drug Humans Animals Effects System
Clonidine Smaller head Hyperactivity, + + + Norepinephrine
circumference, delayed motor
questionable development
necrologic
status,
increased
myoclonic
jerks during
sleep
Diazepam Low Apgar score, Hyperactivity, + Gamma-amino-
reluctance to decreased male isobutyric acid
eat sexual behavior,
decreased
startle reflex
Imipramine- Poor sucking, Hyperactivity, + + + Norepinephrine,
lye agents irritability decreased male acetylcholine,
sexual behavior, serotonin
smaller brain
Reserpine Anorexia, Smaller brain, + Norepinephrine,
lethargy altered activity, dopami;ne,
altered startle seroton~n
reflex
aData from Mirmiran et al., 1985.
bREM=rapid eye movement.
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EXIRAPOLATIONFROMANIMAL STUDIES
· The startle reflex is modulated via
several neurotransmitter systems.
· The startle reflex can be measured
at early ages in many species.
· The startle reflex is quantifiable.
· Inhibitory or excitatory effects
can be determined.
· Startle displays different types
of plasticity.
Davis ( 1984) has reviewed aspects of
the mammalian startle reflex. It consists
of a characteristic, very rapid sequence
of muscular responses elicited by a
sudden, intense stimulus. Under compar-
able circumstances, the more intense the
stimulus, the greater the response. The
graded amplitude of the mammalian startle
response can be detected in direct muscle
recordings (e.g., electromyographic re-
cordings from a limb or muscles involved
in blinks) or in the output from transduc-
ers that measure cage movements when
whole-body startle is measured. A standard
feature of this reflex is its very short
latency; the response occurs only milli-
seconds after the onset of the eliciting
stimulus.
Although the neural circuitry that medi-
ates startle is at lower levels of the CNS,
higher neural networks can modulate it.
Nearly all defined neurotransmitter sys-
tems interact to modulate the startle re-
sponse (Fechter, 1974; Davis and Aghajani-
an, 1976; Davis and Sheard, 1976; Handley
and Thomas, 1979; Davis and Astrachan,
1981; Gallager et al., 1983; Holson et al.,
1985~. In the spinal cord and facial motor
nucleus, serotonin and norepinephrine
increase auditory startle and glycine
tonically inhibits it; it appears that
GABA can also inhibit the response at this
level. Supraspinally, dopamine and per-
haps GABA receptor stimulation increases
startle, and serotonin activation de-
presses it. Startle is also modulated in
several brain regions distant from the
primary startle pathway itself. There-
fore, the reflex can provide a sensitive
indicator of function after toxicant ex-
posure. Developmental insults that result
in changes in a neurotransmitter system
might be expressed as changes in the laten-
cy, amplitude, or modification of the re-
sponse. The type of change observed can
291
suggest which systems have been affected
by exposure.
Auditory startle has been used often
in studies of animal developmental toxi-
cology. Recently, automated procedures
have been applied in such studies, thus
allowing more specific characterization
of changes in this reflex. Automated pro-
cedures for stimulus presentation and data
collection have yielded useful informa-
tion for evaluating sensitization, habit-
uation, prepulse inhibition, and reflex
modification by prior associative learn-
ing after toxicant exposure (Hoffman,
1984~. Startle thus represents a poten-
tially powerful tool in developmental
toxicology for investigating sensorimotor
reactivity. The simplicity of the response
and the plasticity displayed within it
across animal species, including humans,
suggest that specific efforts to inves-
tigate the comparability of startle al-
terations in animals and humans after de-
velopmental insult are warranted.
DIRECT COMPARISONS BETWEEN
ANIMALS AND HUMANS
A third approach to determining the rela-
tionships among human and animal develop-
mental toxicity is to compare observed
effects when data are available for several
species. Few human behavioral-teratology
studies have been reported, and most ex-
perimental behavioral-teratology studies
have used rodents. The comparisons out-
lined here reflect that situation. In
addition, similarities and differences
in design and conduct between experimen-
tal and clinical research must be consid-
ered in any comparison of results. The
similarities between the two include the
following:
· Physical growth and development are
the most commonly measured end points.
· Several behavioral subsystems are
assessed with a battery of functional
tests.
· Experimental and control subjects
are matched for maternal and environmental
characteristics.
· The majority of studies are designed
to provide descriptive information.
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292
In human studies, weight and motor devel-
opment usually are measured for 1-2 years
after birth. In rodent studies, weight
is monitored repeatedly, most often
throughout the duration of the study, and
assessments of preweaning reflex develop-
ment are often carried out as well.
A battery of functional tests usually
are used for neurobehavioral evaluation
in both human and animal studies. The use
of a single assessment technique that in-
corporates multiple evaluations is most
common in human research. The Apgar test
(Apgar, 1953) is used routinely 1 and 5
minutes after birth; it consists of a 10-
point scale based on five components:
appearance (skin color), heart rate, la-
tency of the cry reflex, muscle tone, and
respiration. The Bayley scale of infant
development (Bayley, 1969) is used common-
ly for evaluation of older infants; it
contains sensory, motor, verbal, and cog-
nitive items, and results are summarized
in motor and mental development scores.
Neurobehavioral function in rodents is
evaluated with a test battery that often
includes assessment of reflex and sensori-
motor development, activity level, and
some evaluation of learning ability. Each
category of function is evaluated with
separate tests.
Another similarity in study designs
is the use of experimental and control
subjects matched for maternal and environ-
mental characteristics. In human studies,
mothers are matched as closely as possible
for age, parity, and nutritional and socio-
economic status. In animal studies, mater-
nal weight, parity, diet, and housing con-
ditions routinely are controlled across
groups.
Both clinical and experimental studies
designed to evaluate neurobehavioral
outcomes after prenatal drug or chemical
exposures provide primarily descriptive
information. The methods permit a descrip-
tion of functional deficits after insult,
but not of underlying physiologic or neuro-
chemical mechanisms responsible for the
observed behavioral alterations. As noted
above,-that situation is changing in animal
studies. Multidisciplinary efforts can
provide information on the mechanisms
involved and thus might suggest types of
intervention that could be effective in
NEURODEVELOPMEI`ITAL TOXICOLOGY
alleviating or improving clinical out-
comes.
Several basic differences in design
and conduct between human and animal
studies are common. They include differ-
ences in the relative age range, in timing
of administration of tests, in attempts
at standardization, and in methods of re-
porting results (Adams, 1986~.
The relative age range studied is
broader in much of the animal research
than in human studies. Practically, it
is very difficult to follow a prenatally
exposed person for more than a year or two
after birth. In the best of circumstances,
clinical investigators can extend evalua-
tions to 6 or 8 years of age. Funding,
time requirements, and population mobili-
ty and attrition all contribute to the
difficulty. In contrast, rodent studies
often include behavioral evaluations into
early adulthood. Such a longitudinal ap-
proach can be even more valuable if testing
is identical across the age span studied.
In human research, several functions
usually are evaluated at a single age.
Neurobehavioral function in rodents most
often is evaluated through separate test-
ing at different ages. Furthermore,
unlike the results of multifunctional
evaluations in humans, rodent performance
across tests is not integrated into a sin-
gle value or score.
A greater effort is made in human than
in animal studies to perform testing on
infants in a comparable behavioral state,
e.g., alert, drowsy, or asleep. Such con-
trol contributes to both absolute response
levels and decreased variability in be-
havioral data collected in infants and
children (Clifton and Nelson, 1976~. Ani-
mal researchers at best attempt to control
such factors by balancing time of day dur-
ing testing across experimental groups.
Developmental delays in physical, mo-
tor, and cognitive end points are consid-
ered more important in human than in animal
studies. Such delays can be assessed only
during particular periods of development.
Their biologic meaning in rodents after
prenatal exposures is not clear, and they
have been viewed as problematic (Tilson
and Wright, 1985~. One contributing factor
might be the time disparity in postnatal
developmental schedules between humans
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EXTRAPOLATIONFROMANIMAL STUDIES
and rodents, i.e., months and years versus
days.
Finally, a characteristic difference
between human and animal studies involves
the method of reporting results. Clinical
studies typically identify the incidence
of behavioral dysfunction in individual
control versus individual exposed sub-
jects. Animal data usually are presented
in terms of the presence or absence of group
mean differences. Thus, the incidence
of affected (and nonaffected) rodent
offspring in the exposed group is not
available.
In the light of these differences in
design, conduct, and reporting between
human and animal behavioral-teratology
studies, findings are discussed below if
appropriate data were available for com-
parison. The number of reported human
studies was the limiting factor in the
following brief overview; once those were
identified, the animal literature was
evaluated. In all cases, if human sub-
system dysfunction was reported, corro-
borative evidence was found in the animal
data if a comparable end point had been eval-
uated. In that manner, effects observed
after developmental exposures to lead,
mercury, PCBs, phenytoin, ethanol, and
methadone are compared. Articles review-
ing the spectrum of effects observed in
humans and animals are cited in the follow-
· · —
sing discussion.
293
Table 27-2 summarizes the comparability
of neuromotor effects after exposure to
particular toxicants. Delayed motor de-
velopment has been reported in both
humans and rodents exposed to lead (Rutter,
1980; Reiter, 1982), mercury (Reuhl and
Chang, 1979), alcohol (Abel, 1980), and
phenytoin (Hanson et al., 1976; Vorhees,
1983~. Cerebral palsy and seizure disor-
ders have been reported in humans develop-
mentally exposed to mercury, and motor
dysfunction and increased susceptibility
to seizure induction have been reported
in rats and mice. Developmental exposures
to PCBs have resulted in motor dysfunction
in both humans (Jacobson et al., 1984) and
mice (Tilson et al., 1979~. A prolonged
neonatal abstinence syndrome with neuro-
motor sequelae has been identified in human
infants and rodents prenatally exposed
to methadone (Hutchings, 1983~. The spe-
cific motor alterations observed in humans
and rodents were not always identical,
but the normal behavioral repertoires of
the two also are different. The data do
indicate that the motor systems of humans
and rodents are susceptible to disruption
after developmental exposures to the
agents in question.
The clinical relevance of experimental
data on cognitive functions is more dif-
ficult to evaluate. Tests of human and
animal cognitive abilities might evaluate
very different functions; i.e., a rodent
TABLE 27-2 Examples of Motor Dysfunction After Behavioral-Teratogen Exposuresa
Agent Effects in Humans Effects in Rodents
Lead
Mercury
PCBs
Phenytoin
Ethanol
Methadone
Delayed growth and motor devel-
opment, motor incoordination,
deficits in fine motor control
Delayed growth and motor devel-
opment, cerebral palsy, seizure
disorders
Depressed reflexes, delayed
motor development
Delayed growth and motor
development
Delayed growth and motor
development
Neonatal abstinence syndrome:
tremors, sleep disturbances,
hyporeflexia, irritability
aData from Adams 1986.
,
Delayed growth and motor development
Delayed growth and motor development,
ataxia, seizure susceptibility
Neuromotor weakness, poor balance
Delayed growth and motor development
Delayed growth and motor development
Neonatal abstinence syndrome:
hyperactivity, lability of state, sleep
disturbances
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294
brain is not capable of the many complex
functions evaluated in human assessments.
Techniques used to assess cognitive func-
tion measure responses that are modified
by sensory and motivational processes,
as well as motor capabilities. However,
performance on such tests can provide use-
ful information concerning the postex-
posure integrity of underlying systems
that contribute to an animal's or chills
ability to learn, process, store, and re-
trieve relevant information. Table 27-
3 summarizes some of the cognitive def-
icits noted after developmental insult.
Reduced general intelligence, as measured
on standardized tests, has been found in
some children who were exposed prenatally
to lead (Needleman et al., 1979), mercury
(Harada, 1976), ethanol (Streissguth et
al., 1984), and phenytoin (Hanson et al.,
1976~. The IQs of those children are often
less than 70. Mental retardation is one
of the most serious results of exposure
to the agents in question. Attentional
deficits have been reported in some chil-
dren prenatally exposed to lead, mercury,
and ethanol. Experimental studies have
indicated impairments in visual recogni-
tion memory in infants exposed to PCBs
(Jacobson et al., 1985) and increased reac-
tion times during a vigilance task in eth-
anol-exposed children.
The animal literature indicates im-
paired learning and memory abilities in
rodents after developmental exposures
to the agents. Performance deficits on
avoidance tasks have been reported after
NEURODEVELOPMENTAL TOXICOLOGY
exposures to lead (Kimmel et al., 1978),
mercury (Spyker et al., 1972), PCBs (Tilson
et al., 1979), ethanol (Abel, 1980), and
phenytoin (Vorhees, 1983~. Results of
water-maze tasks have indicated impaired
function in rodents exposed to mercury
(Spyker et al., 1972), ethanol (Abel,
1980), and phenytoin (Vorhees, 1983~.
Hughes and Sparber (1979) found that pre-
natal mercury exposure disrupted operant
performance.
It is interesting that prenatal metha-
done exposure does not appear to alter
cognitive performance in either humans
or animals (Hutchings, 1983~. The fact
that both humans and animals showed no
effects on cognitive performance supports
the utility of experimental-teratology
data in assessing toxic effects.
Sensory/perceptual processes have
not been carefully evaluated in most be-
havioral-teratology studies (Adams and
Buelke-Sam, 1981; Ison, 1984~. As shown
in Table 27-4, clinical case reports
have suggested that some persons exposed
in utero to ethanol (Clarren and Smith,
1978) or to phenytoin (Hill et al., 1974)
have unspecified hearing defects. Visual
impairments have been reported in some
children exposed in utero to phenytoin
(Wilson et al., 1978~. However, specific
sensory functions have not been evaluated
in animals that have been exposed prenatal-
ly to alcohol or phenytoin. Vorhees (1983)
reported delayed development of auditory
responsiveness in rats treated with pheny-
toin prenatally.
TABLE 27-3 Examples of Cognitive Dysfunction After Behavioral-Teratogen Exposuresa
Agent Effects in Humans Effects in Rodents
Lead Decreased general intelligence,
decreased attention span, impaired
verbal ability
PCBs
Phenytoin
Ethanol
Mercury Decreased general intelligence,
decreased attention span
Impaired visual recognition memory
Decreased general intelligence
Decreased general intelligence,
decreased attention span, delayed
reaction time
Methadone None reported
Impaired learning ability on passive
avoidance and T-maze tasks
Learning deficits on many tasks
Impaired learning on avoidance tasks
Impaired spatial learning on water-maze tasks
Learning deficits on many tasks
None reported
aData from Adams, 1986.
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EXIRAPOLATIONFROMANIM41L STUDIES
TABLE 274 Examples of Sensory/Perceptual Processing Dysfunction After Behavioral-Teratogen Exposuresa
Agent Effects in Humans Effects in Rodents
295
Lead Decreased visual acuity, altered brain Decreased visual acuity, altered brain
electrophysiologic response to visual electrophysiologic response to visual
stimulation stimulation
Mercury Unspecified hearing defects, altered Increased reactivity to auditory startle stimuli
reactivity to visual and auditory stimuli
PCBs None specifically evaluated None specifically evaluated
Phenytoin Unspecified hearing defects and visual Delayed development of auditory startle
impairments in some cases response
Ethanol Unspecified hearing defects in some None specifically evaluated
cases
Methadone Deficits in visual, auditory, and Hyperreactivity to aversive stimuli
tactile perception, but no specific
sensory deficits
aData from Adams, 1986.
Decreases in visual acuity have been
reported to occur in children (Rummo et
al., 1979), rats (Fox et al., 1977, 1979;
Fox and Wright, 1982), and monkeys
(Bushnell et al., 1977) after exposure
to inorganic lead. Altered electrophysio-
logic brain activity in response to visual
stimulation has also been found in lead-
exposed children (Otto et al., 1981, 1982,
1985; Otto and Reiter, 1983) and rats (Fox
etal.,1979~.
Fetal exposure to methylmercury has
been reported to produce postnatal altera-
tions in reactivity to visual and auditory
stimulation in humans (Harada, 1976,
1977~. Studies done on prenatally exposed
rats have shown increased reactivity to
auditory stimuli (Buelke-Sam et al.,
1985~.
Wilson et al. (1979) reported that chil-
dren prenatally exposed to methadone
had deficits in visual, auditory, and tac-
tile perception, but these were inter-
preted as resulting from poor attentional
and strategic processing abilities, rath-
er than from specific sensory deficits.
Lodge (1976) reported alterations in brain
electrophysiologic responses to visual
stimuli and hypersensitivity to auditory
stimulation in children exposed to metha-
done before birth. The integrity of sen-
sory functioning has not been specifically
evaluated in studies carried out in ro-
dents, but hypersensitivity to aversive
stimulation has been reported by Hutchings
(1983~.
In nearly all the cases outlined above,
the clinical problem was identified before
the development of animal models to explore
such toxicity. The literature of animal
behavioral teratology has expanded great-
ly in recent years and suggests that a num-
ber of additional drugs and chemicals war-
rant clinical investigation. However,
the potential value of animal data in pre-
dicting human hazard cannot be determined
fully until clinical studies to look for
behavioral dysfunctions identified in
experimental animals are designed and
conducted.
DATA INTERPRETATION
We have discussed many problems that
contribute to the difficulty of evaluating
the relevance of animal data in identifying
potential health hazards in developing
humans. The first three issues that follow
bear on the interpretation of both human
and animal data; the last two are related
to problems in cross-species extrapola-
tion of results.
The first problem centers on determining
whether behavioral-teratology findings
are a result of primary developmental toxi-
city or are secondary to maternal toxicity
or to primary toxicity produced in other
organ systems, e.g., liver or kidney. The
heart of the issue is the relative suscep-
tibility of the developing organism
(whether human or animal) to toxic insult.
If postnatal dysfunction only accompanies
maternal toxicity, such information might
be of value in alerting clinicians to moni-
OCR for page 296
296
tor such pregnancies more closely. If
postnatal functional deficits are ob-
tained in the absence of overt toxic signs,
the potential for selective developmental
toxicity must be considered in human risk
assessment.
Genetics might play a large role in sus-
ceptibility or expression of postnatal
dysfunction. Genetic makeup could predis-
pose the parents or offspring to greater
sensitivity to a toxicant. Toxicants could
produce postnatal dysfunction via genetic
mechanisms, or such dysfunction could be
transmitted to later generations (cf.
Fujii et al., 1987; Stoetzer et al., 1987~.
Thus, whether developmental-toxicology
studies are performed with inbred strains
of mice or in the highly diverse human and
whether one or both parents have been ex-
posed to a toxicant are important consider-
ations in evaluating and interpreting
postnatal functional data.
The postnatal environment can have an
impact on developmental toxicity, maxi-
mizina or minimizing the expression of
NEURODEVELOPMENTAL TOXICOLOGY
enrichment might contribute to the mani-
festations of toxicity.
Once those issues are considered, two
additional aspects must be dealt with in
determining the relevance of animal data
to the human situation. The first concerns
the disparity in timing of organ-system
development across species. The rat gesta-
tion period covers approximately 3 weeks,
and CNS development, including cell dif-
ferentiation and migration, continues
into the immediate neonatal period. More
CNS development occurs in humans during
the 9-month gestation period, although
completion of histogenesis does not occur
until well after birth. Agent exposure
is timed specifically in animal studies,
and care is taken to standardize doses
within treatment groups. Such control
usually is not available in human studies,
and retrospective investigations often
rely on maternal reporting of exposure
to drugs and when it occurred.
Postnatal development schedules also
differ between humans and animals. Post-
damage. In human studies, the role of ma- natal development through puberty in a
ternal socioeconomic status is great in rat requires approximately 6 weeks. It
that regard, as well as contributing to takes years to reach that stage in humans.
Overall prenatal and perinatal status. Thus, prenatal and postnatal disparities
In animal research, controlling litter in timing must be accounted for, as well
size is one means of standardizing this as the variations in agent exposures during
factor. In both types of research, the the comparative process.
degree of environmental experience and
Representative terms from entire chapter:
animal studies
