VOLUME 114 | NUMBER 1 | January 2006 • Environmental Health Perspectives
Environmental tobacco smoke (ETS) exposure
is now recognized as a health risk for pregnant
women and children (Dunn and Zeise 1997;
Witschi et al. 1997), and it is increasingly evi-
dent that ETS affects the developing brain and
cardiovascular system (Eskenazi and Trupin
1995; Hutchison et al. 1998; Makin et al.
1991). The consequences of fetal or early
neonatal ETS exposure mimic those of active
maternal smoking, albeit with a lesser magni-
tude (Makin et al. 1991), and for both active
smoking and ETS, the dose–effect relation-
ships correlate well with the levels of nicotine
and its metabolites (Fried et al. 1995). ETS
generally achieves fetal nicotine metabolite
concentrations similar to those seen with light,
active maternal smoking (Eliopoulos et al.
1996; Jauniaux et al. 1999; Ostrea et al. 1994),
and young children exposed to ETS typically
display levels exceeding those seen in older
children (Fried et al. 1995; Kohler et al. 1999).
Animal studies demonstrate conclusively
that nicotine damages the developing brain
by altering the formation, survival, and
differentiation of brain cells, eliciting deficits
in structure, synaptic function, and behav-
ioral performance (Levin and Slotkin 1998;
Slotkin 1998, 2004; Walker et al. 1999).
This provides a mechanistic link between
maternal smoking during pregnancy and
adverse neurobehavioral consequences in the
offspring (Fried et al. 1992, 1998, 2003;
Wakschlag et al. 2002; Weitzman et al. 2002).
However, much less is known about the
mechanisms underlying comparable effects of
ETS. In a recent pair of studies, we found that
perinatal ETS exposure in rhesus monkeys
elicits alterations in cell signaling in the devel-
oping brain akin to those identified for nico-
tine administration in rodents, including the
up-regulation of nicotinic cholinergic recep-
tors, a characteristic of chronic nicotine-
induced neuronal stimulation (Slotkin et al.
2000, 2002). These findings were important
for two reasons: first, they provided the first
evidence that ETS supplies sufficient nicotine
to the developing brain to evoke inappropriate
activation of the pathways that lead to altered
cell development, and second, they demon-
strated these effects in primates. The latter
point is particularly important: the rat and
mouse are altricial species, so brain develop-
ment at birth corresponds to fetal stages of
human development (Rodier 1988), and thus
the concentrations or temporal factors for
nicotine or ETS may not reflect those experi-
enced in typical human exposure scenarios.
The present study, again using rhesus
monkeys, was undertaken for four distinct
purposes. First, we examined the relative
importance of continuous perinatal ETS expo-
sure compared with later exposure, determina-
tions that are essential to identify the critical
periods in which the developing brain is vul-
nerable to adverse effects of ETS. Our earlier
work in rats indicated an extended period of
vulnerability, lasting well into the postnatal
period, and therefore in the present study we
examined perinatal exposure up to 13 months
of age, compared with ETS administered only
during later postnatal stages, from 6 through
13 months. Second, we examined a variety of
cortical brain regions and the midbrain, areas
that, based on the known effects of nicotine,
are likely to be compromised by developmen-
tal ETS exposure (Levin and Slotkin 1998;
Slotkin 1998, 2004). Third, within each
region, we characterized the neural cell damage
caused by the different ETS regimens, using
strategies adapted from prior rodent studies of
nicotine or ETS (Gospe et al. 1996; Levin and
Slotkin 1998; Slotkin 1998, 2004). Each
neural cell contains only a single nucleus
(Winick and Noble 1965), so the DNA con-
centration (DNA per unit tissue weight)
reflects the cell packing density (Bell et al.
1987; Slotkin et al. 1984; Winick and Noble
1965). We also characterized the complement
of cell proteins that reflect indices of cell type
and size. The brain contains numerous glia,
which are considerably smaller than neurons
and thus possess less total protein per cell and
a higher surface-to-volume ratio, which can
be assessed by the proportions of total protein/
DNA and membrane/total protein. At the
same time, as neurons specialize, they enlarge
and develop axonal and neuritic projections,
which increases both the total protein/DNA
ratio and membrane/total protein ratio in par-
allel (Qiao et al. 2003, 2004; Slotkin et al.
2005). These indices thus provide insight into
the architectural alterations underlying the
Address correspondence to T.A. Slotkin, Box 3813
DUMC, Duke University Medical Center, Durham,
NC 27710 USA. Telephone: (919) 681-8015. Fax:
(919) 684-8197. E-mail: email@example.com.
We thank M.M. Cousins, C.A. Oliver, and
C.A. Tate for technical assistance.
Research was supported by grants from the Philip
Morris External Research Program and the National
Institutes of Health (ES011634, ES05707, and
The authors declare they have no competing
Received 5 May 2005; accepted 7 September 2005.
Perinatal Environmental Tobacco Smoke Exposure in Rhesus Monkeys:
Critical Periods and Regional Selectivity for Effects on Brain Cell
Development and Lipid Peroxidation
Theodore A. Slotkin,1Kent E. Pinkerton,2and Frederic J. Seidler1
1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA; 2Center for Health and
the Environment, and California National Primate Research Center, University of California, Davis, California, USA
Perinatal environmental tobacco smoke (ETS) exposure in humans elicits neurobehavioral deficits.
We exposed rhesus monkeys to ETS during gestation and through 13 months postnatally, or post-
natally only (6–13 months). At the conclusion of exposure, we examined cerebrocortical regions
and the midbrain for cell damage markers and lipid peroxidation. For perinatal ETS, two arche-
typal patterns were seen in the various regions, one characterized by cell loss (reduced DNA con-
centration) and corresponding increases in cell size (increased protein/DNA ratio), and a second
pattern suggesting replacement of larger neuronal cells with smaller and more numerous glia
(increased DNA concentration, decreased protein/DNA ratio). The membrane/total protein ratio,
a biomarker of neurite formation, also indicated potential damage to neuronal projections, accom-
panied by reactive sprouting. When ETS exposure was restricted to the postnatal period, the
effects were similar in regional selectivity, direction, and magnitude. These patterns resemble the
effects of prenatal nicotine exposure in rodent and primate models. Surprisingly, perinatal ETS
exposure reduced the level of lipid peroxidation as assessed by the concentration of thiobarbituric
acid reactive species, whereas postnatal ETS did not. The heart, a tissue that, like the brain, has
high oxygen demand, displayed a similar but earlier decrease (2–3 months) in lipid peroxidation
in the perinatal exposure model, whereas values were reduced at 13 months with the postnatal
exposure paradigm. Our results provide a mechanistic connection between perinatal ETS exposure
and neurobehavioral anomalies, reinforce the role of nicotine in these effects, and buttress the
importance of restricting or eliminating ETS exposure in young children. Key words: β-adrenergic
receptor, brain development, environmental tobacco smoke, heart development, lipid peroxida-
tion, muscarinic acetylcholine receptor, nicotine. Environ Health Perspect 114:34–39 (2006).
doi:10.1289/ehp.8286 available via http://dx.doi.org/ [Online 7 September 2005]
neurochemical effects of ETS. For example,
the typical response to neuronal injury, neu-
ronal replacement by smaller and more numer-
ous glia (O’Callaghan 1988, 1993), produces
an increase in cell packing density, a decrease
in total protein/DNA, and an increase in
membrane/total protein. In contrast, neuronal
loss accompanied by perikaryal swelling,
another archetypical injury response (Roy et al.
2005), elicits a decrease in cell packing density,
an increase in total protein/DNA, and a
decrease in the membrane/total protein ratio.
A third pattern, damage to neuritic projections,
produces a decrement in the membrane/total
protein ratio in the nerve terminal region but
an increase in areas where reactive sprouting
takes place (Kostrzewa and Jacobowitz 1974;
Navarro et al. 1988).
As a fourth objective, we made determina-
tions of lipid peroxidation. Nicotine induces
free radical generation and contributes a major
proportion of the net oxidative stress imposed
by tobacco use (Bhagwat et al. 1998; Newman
et al. 2002; Qiao et al. 2005; Yildiz et al.
1999). At the same time, many other products
in tobacco smoke similarly have the potential
to produce oxidative damage (Huang et al.
2005), and oxidative stress contributes to the
effects of many neurotoxicants (Gitto et al.
2002; Gupta 2004; Ohtsuka and Suzuki 2000;
Olanow and Arendash 1994). To evaluate the
role of oxidative damage in the effects of ETS
in our primate model of brain development,
we assessed the concentration of thiobarbituric
acid–reactive species (TBARS) (Guan et al.
2003), contrasting the effects in brain regions
with those in the heart. Both the brain and
heart are highly vulnerable because of their
high oxygen consumption, but the brain is
especially sensitive for two reasons: first,
neural cell membrane lipids are high in oxi-
dizable polyunsaturated fatty acids (Gupta
2004); second, the developing brain has an
increased metabolic demand associated with
its perinatal growth spurt, during which it has
lower reserves of protective enzymes and
antioxidants (James et al. 2005) and is defi-
cient in glia, which ordinarily protect neurons
from oxidative molecules (Tanaka et al. 1999).
In addition, we conducted studies to charac-
terize the temporal appearance of alterations in
cardiac TBARS as well as the potential
that underlie developmental vulnerability or
protection from oxidative stress.
Materials and Methods
Materials. We purchased standardized 1R4F
research cigarettes from the University of
Kentucky (Louisville, KY). [3H]AFDX384
(specific activity, 133 Ci/mmol) and [125I]iodo-
pindolol (specific activity, 2,200 Ci/mmol)
were obtained from PerkinElmer Life Sciences
(Boston, MA). All other chemicals were
purchased from Sigma Chemical Co.
(St. Louis, MO).
Animal treatments. All studies were car-
ried out in accordance with the declaration of
Helsinki and with the Guide for the Care and
Use of Laboratory Animals as adopted and pro-
mulgated by the National Institutes of Health
(National Research Council 1996). We
obtained 15 pregnant rhesus macaque mon-
keys from the California National Primate
Research Center breeding colony and assigned
them to three different treatment groups: ani-
mals to be exposed to filtered air, those to
receive both prenatal and postnatal ETS expo-
sure, and those to receive postnatal exposure
only. The estimated gestational age for each
dam was established by sonography performed
before gestation day (GD) 40. Animals were
selected based on a history of successful vagi-
nal delivery and previous infant rearing experi-
ence, with estimated delivery dates separated
by approximately 1 week per animal to facili-
tate experimental procedures. In addition to
the present study of indices of brain develop-
ment, these animals were used for evaluations
of ETS effects on perinatal lung development,
immune function, airway hyperresponsiveness
through autonomic regulation, endothelial
markers of mitochondrial DNA damage rele-
vant to cardiovascular disease, and other deter-
minations involving bone marrow, kidneys,
eyes, heart, aorta, gastrointestinal tract, and
To deliver ETS, we used two inhalation
chambers, each with an air capacity of 3.5 m3,
with each housing two monkeys. Aged and
diluted sidestream smoke was used as a surro-
gate for ETS. Standardized 1R4F research
cigarettes were smoked simultaneously with a
single puff volume of 35 mL per cigarette and
a duration of 2 sec, once per minute.
Sidestream smoke from the smoldering end of
each cigarette was collected and aged, and
then diluted with filtered air to achieve a final
particulate concentration of 1 mg/m3. Airflow
through the system was set for 30 changes per
hour, and samples were collected daily to
determine the concentrations of total sus-
pended particulates, nicotine (average,
162 µg/m3), and carbon monoxide (average,
4.3 ppm). These concentrations represent the
high end of field measurements reported for
household ETS but are within the range of
what a child would experience if the caretaker
is a smoker; the cloud of ETS generated
around a smoker contains particulates up
to 2 mg/m3, twice the exposure used here
(Jenkins et al. 2000; U.S. Environmental
Protection Agency 1992). Exposure to ETS
occurred for 6 hr/day, 5 days/week, beginning
at about GD50; pregnant animals in the con-
trol and postnatal exposure groups received
filtered air in the same apparatus on the same
schedule. All dams were allowed to give birth
spontaneously, and then ETS or filtered air
exposures were continued through 13 months
postnatally, with the chamber containing both
the mother and infant until removal of the
mother at weaning (5 months of age). The
group with ETS exposure limited to the post-
natal period was switched from filtered air to
ETS at 6 months of age and continued
through 13 months. At 13 months, the off-
spring were anesthetized with ketamine
(10 mg/kg intramuscular) and euthanized with
pentobarbital (80 mg/kg intravenous). The
heart was dissected and brain samples were
taken from the three regions of the cerebral
cortex (frontal, temporal, and occipital cortex)
as well as the midbrain, using anatomical land-
marks to ensure sampling of the same area
from each monkey. Tissues were flash-frozen
and stored at –80°C until assayed.
Each group contained both male and
female offspring: four males and one female in
the control group, three males and two females
in the group receiving continuous ETS expo-
sure, and three males and two females in the
group receiving only postnatal ETS exposure.
In an additional set of monkeys (five
males and three females in the controls, three
males and five females in the ETS group), we
evaluated cardiac effects elicited by continu-
ous perinatal ETS exposure at an earlier time
point (postnatal days 70–80).
Biomarkers of neural cell development.
Tissues were thawed in 19 volumes of ice-cold
10 mM sodium–potassium phosphate buffer
(pH 7.4) and homogenized with a Polytron
(Brinkmann Instruments, Westbury, NY).
DNA was assessed with a modified (Trauth
et al. 2000) fluorescent dye-binding method
(Labarca and Piagen 1980). Aliquots were
diluted in 50 mM sodium phosphate, 2 M
NaCl, 2 mM EDTA (pH 7.4), and sonicated
briefly (Virsonic Cell Disrupter, Virtis,
Gardiner, NY). Hoechst 33258 was added to a
final concentration of 1 µg/mL. Samples were
then read in a spectrofluorometer using an
excitation wavelength of 356 nm and an emis-
sion wavelength of 458 nm and were quanti-
tated using standards of purified DNA. The
total concentration of tissue proteins was
assayed from the original homogenate spectro-
photometrically with bicinchoninic acid
(Smith et al. 1985); in addition, we assessed
the concentration of membrane proteins from
the membrane preparations used for radio-
ligand binding. For calculation of the ratio of
membrane/total protein, the membrane pro-
tein value was averaged across the different
Thiobarbituric acid reactive species. Lipid
peroxidation was evaluated by assessment of
TBARS using established techniques (Ohkawa
et al. 1979). Triplicate aliquots of the same
homogenate used for determination of DNA
and proteins were added to an equal volume of
Perinatal exposure to ETS
Environmental Health Perspectives • VOLUME 114 | NUMBER 1 | January 2006
10% trichloroacetic acid, followed by addition
of 1 volume of thiobarbituric acid reagent:
0.75% 2-thiobarbituric acid dissolved in 1 M
NaOH, followed by addition of acetic acid to a
final concentration of 20%. Samples were
incubated for 1 hr at 95–100°C, cooled to
ambient temperature, and sedimented at
3,500 × g for 10 min. The pellet was discarded,
the supernatant solution was resedimented,
and the absorbance of the final supernatant
solution was determined at 532 nm. Standard
curves were constructed with known concen-
trations of malondialdehyde that had been run
through the same reaction. Values were deter-
mined relative to total protein.
Receptor binding assays. Cardiac receptor
binding capabilities were determined by meth-
ods described previously (McMillian et al.
1983; Slotkin et al. 1987a; Song et al. 1997;
Zahalka et al. 1993). Aliquots of the origi-
nal tissue homogenate were sedimented at
40,000 × g for 15 min and were then prepared
in two different ways, one for β-adrenergic
receptor (βAR) binding and the other for
m2-acetylcholine receptor (m2AChR) binding.
For βARs, the membrane pellets were resus-
pended and resedimented in a buffer consisting
of 125 mM sucrose, 6 mM MgCl2, 50 mM
Tris-HCl (pH 7.5), whereas for m2AChR
binding, we maintained the same sodium-
phosphate buffer used for the original homo-
genization. To evaluate βAR binding, aliquots
of membrane preparation were incubated with
[125I]iodopindolol (final concentration, 67
pM), in 145 mM NaCl, 2 mM MgCl2, 1 mM
Na ascorbate, 20 mM Tris (pH 7.5), for
20 min at room temperature in a total volume
of 250 µL. Displacement of nonspecific bind-
ing was evaluated with 100 µM d,l-isopro-
terenol. Binding to m2AChRs was evaluated
with 1 nM [3H]AFDX384 incubated for
60 min at room temperature in 10 mM
sodium phosphate (pH 7.4), and nonspecific
binding was evaluated with 1 µM atropine.
Data analysis. Data are presented as means
and SEs. The effects of ETS exposure were first
evaluated by global analysis of variance
(ANOVA; data log-transformed because of het-
erogeneous variance) incorporating the three
different treatments (control, continuous peri-
natal ETS, postnatal ETS 6–13 months), the
various regions, and the repeated measures rep-
resenting the biomarkers of neural cell develop-
ment: DNA concentration, total protein/DNA
ratio, and membrane/total protein ratio.
Because this initial test indicated a significant
difference of treatment effects according to the
type of measurement, we used lower order
ANOVAs (treatment, region) to assess the
effects separately for each measure. Finally,
where the lower order test indicated an interac-
tion of treatment with region, separate post hoc
analyses (Fisher’s protected least significant dif-
ference) were undertaken to determine the
effects of ETS exposure on each individual
region; in the absence of an interaction, only
the main effect of ETS was reported. Similarly,
TBARS were assessed initially with a two-factor
ANOVA (treatment, region), and cardiac
receptor binding studies were first evaluated by
ANOVA incorporating treatment and receptor
type (βAR, m2AChR). Significance for all tests
was assumed at p < 0.05.
Prepartum ultrasonography performed at
GD40, GD90, GD120, and GD150 revealed
no significant differences in fetal growth
between those exposed to ETS or those
exposed to filtered air. Similarly, the ETS
group showed normal weights and other
somatic indices of gestational age at birth, and
there were no effects on growth through 13
months postnatal age (not shown).
At 13 months of age, there were no differ-
ences in body weights among the three groups
(control, 2.3 ± 0.1 kg; continuous ETS, 2.2 ±
0.2 kg; ETS 6–13 months, 2.3 ± 0.2 kg), nor
were there differences in general health or
activity. Nevertheless, ANOVA across the
three biomarkers of neural cell development
indicated highly significant differences among
the three groups (p < 0.0008) that depended
both upon the specific measure and brain
region (treatment × measure × region,
p < 0.0002). Accordingly, we subdivided the
assessments into the three different develop-
mental indices and reevaluated the main treat-
ment effects and regional specificity. The DNA
concentration, an index of cell packing density,
showed regionally selective changes elicited by
ETS exposure (Figure 1A). Although values
were unaffected in the frontal cortex, both the
occipital cortex and midbrain displayed signifi-
cant increases after either continuous ETS
exposure or ETS exposure restricted to the
postnatal 6–13 month period. In contrast, val-
ues tended to be reduced in the temporal cor-
tex, achieving statistical significance with the
postnatal exposure group.
Both indices of cell size also displayed ETS-
induced differences. For the total protein/DNA
ratio, the values were reciprocally related to the
change in DNA concentration. Accordingly,
reductions were seen in the occipital cortex and
midbrain, whereas an increase was obtained in
the temporal cortex (Figure 1B). The mem-
brane/total protein ratio showed overall
increases that were not regionally selective but
that were statistically significant both for con-
tinuous ETS exposure and for the group receiv-
ing only postnatal exposure (Figure 1C).
In contrast to the similarity of effects of
continuous perinatal ETS exposure and post-
natal exposure on neural cell development bio-
markers, there were radically different effects
on TBARS (Figure 2). The continuous ETS
group showed marked reductions in TBARS in
the frontal cortex and temporal cortex, without
significant effects in the other regions or in the
heart. In contrast, when ETS exposure
occurred postnatally from 6–13 months, there
were no significant differences in TBARS. The
absence of effects in the heart, a tissue that, like
the brain, has high oxygen demand, could
imply that only the brain is targeted by ETS
exposure, or alternatively that the heart may
show similar effects but with a different tempo-
ral relationship. To distinguish these two possi-
bilities, we performed an additional study with
continuous ETS exposure, but conducting the
Slotkin et al.
VOLUME 114 | NUMBER 1 | January 2006 • Environmental Health Perspectives
Membrane/total protein (%)
p < 0.04
p < 0.005
p < 0.05
Total protein/DNA (µg/µg)
p < 0.005
p < 0.02
p < 0.02
DNA concentration (µg/g)
ETS 6–13 months
Figure 1. Effects of ETS exposure on biomarkers of neural cell development: (A) DNA concentration (ANOVA: treatment, p < 0.01; treatment × region, p < 0.0001).
(B) Total protein/DNA ratio (ANOVA: treatment × region, p < 0.004). (C) Membrane/total protein ratio (ANOVA: treatment, p < 0.008; there was no treatment ×
region interaction); the main effect of each ETS treatment in (C) is as follows: continuous ETS, p < 0.005; ETS 6–13 months, p < 0.008.
*Individual values for which the ETS groups differ from the corresponding control. These were not evaluated in (C) because of the absense of a treatment × region interaction.
evaluations earlier, on postnatal days 70–80.
Under these circumstances, we obtained the
same robust decrease in TBARS in the heart
that we observed later in the brain (control,
1.71 ± 0.4 nmol/mg protein; ETS, 0.88 ±
0.11 nmol/mg protein; p < 0.008).
Earlier studies in rodents, using either
nicotine or ETS exposure, indicated down-
regulation and/or desensitization of cardiac
autonomic receptors whose activity influences
oxidative demand (Joseph et al. 2002; Navarro
et al. 1990; Remondino et al. 2003; Slotkin
et al. 1999, 2001). Accordingly, we assessed
effects on both cardiac βAR and m2AChR
binding with both the continuous perinatal
exposure and postnatal exposure models
(Figure 3). Although continuous exposure had
no significant effect, both receptor types were
down-regulated in the group where ETS expo-
sure was restricted to the postnatal period of
Perinatal or postnatal ETS exposure elicited
two characteristic patterns of neural cellular
effects, both of which resemble earlier findings
for effects of prenatal nicotine exposure in
rodents (Levin and Slotkin 1998; Roy et al.
1998, 2002; Roy and Sabherwal 1994, 1998;
Slotkin 1998, 2004; Slotkin et al. 1987b). In
the occipital cortex and midbrain, there were
smaller cells (reduced total protein/DNA ratio)
and a corresponding increase in cell packing
density (DNA concentration), features that are
likely to reflect neuronal damage and “reactive
gliosis,” that is, replacement with smaller, glial
cells (O’Callaghan 1988, 1993; Roy et al.
1998, 2002; Roy and Sabherwal 1994, 1998).
In contrast, in the temporal cortex, we found a
reduction in the total number of cells (reduced
DNA) with hypertrophy of the remaining cells
(increased total protein/DNA ratio), changes
indicative of cell loss with perikaryal swelling
(Roy et al. 2005). Superimposed on these two
patterns, we also found an overall increase in
the membrane/total protein ratio, which is
compatible either with smaller cells (higher
surface-to-volume ratio) or with increased neu-
ritic sprouting. Given the disparate underlying
regional patterns for the other two markers, the
first explanation is likely to be true for the
occipital cortex and midbrain, whereas the lat-
ter is more probable for the temporal cortex:
reactive sprouting is typical after damage to
developing nerve terminals or projections
(Kostrzewa and Jacobowitz 1974) and, again,
has been found for the effects of prenatal nico-
tine exposure in rodents (Navarro et al. 1988).
These neurochemical inferences point to the
need for detailed, quantitative morphologic
investigations of ETS effects on primate devel-
opment paralleling those done for nicotine in
rodent models (Roy et al. 1998, 2002; Roy
and Sabherwal 1994, 1998), and the present
results provide the necessary guidance as to
which regions should be evaluated and what
types of changes are likely to be found.
In addition to regional selectivity, there
were two other notable features of ETS-
induced alterations in neurochemistry. First,
although the changes were statistically signifi-
cant, not surprisingly, the effects were smaller
in magnitude than those associated with direct
nicotine administration (Levin and Slotkin
1998; Slotkin 1998, 2004). Given that ETS
delivers higher levels of oxidative free radicals
than does just the administration of nicotine
(Huang et al. 2005), our results imply that the
role of nicotine in adverse neurobehavioral
outcomes is primary; indeed, as discussed
below, nicotine-induced damage may actually
limit the contributions of oxidative injury.
Nevertheless, as seen here for different cortical
regions, heterogeneity of the effects is likely to
reduce measured differences by diluting highly
affected nuclei or neuron types with larger
amounts of unaffected subregions. Con-
sequently, biochemical examinations of even
broader regional groupings may give false nega-
tive results because of opposing changes in
different subregions (Gospe et al. 1996). Even
here, with subregional dissection into the
frontal, temporal, and occipital cortex, we are
still incorporating heterogeneous layers and
nuclei, which means that significant, small
changes imply much larger focal effects that are
likely to be identified by quantitative morphol-
ogy. Indeed, with prenatal nicotine exposure in
rats, we have already shown distinct targeting
of different types of neurons even within a sin-
gle layer of the somatosensory cortex or in spe-
cific zones of the hippocampus (Roy and
Sabherwal 1994, 1998; Roy et al. 2002).
The second unexpected feature of the
effects of ETS was that both continuous and
postnatal exposure produced neurochemical
changes that were similar in regional selectivity
and magnitude, despite the obvious, major dif-
ferences in exposure period and duration.
Translated to human ETS exposure, this find-
ing points out the importance of reducing the
exposure of young children to tobacco smoke
in the home or in child care settings. However,
our results also pose a conundrum: how can
continuous perinatal exposure give the same
net effect as exposure restricted to the postnatal
period of 6–13 months of age? It is highly
unlikely that damage to the developing brain
occurs only with postnatal exposure, given the
known effects of prenatal nicotine on brain
development (Levin and Slotkin 1998; Slotkin
1998, 2004; Slotkin et al. 2005). Alternatively,
the effects of continuous perinatal exposure
may be greater than those of postnatal ETS,
with the differences masked by the limits of
resolution imposed by regional heterogeneity;
in that case, detailed morphologic studies will
again reveal the disparities between the two
exposure paradigms. However, our results for
lipid peroxidation also point to the possibility
that some factors operate to constrain the
degree of these specific types of cellular dam-
age. Surprisingly, perinatal ETS exposure
reduced TBARS in cortical subregions, rather
than evoking the expected increase, thus sug-
gesting an enhancement of antioxidant
defenses in the exposed offspring. This result is
in keeping with a recent study of human
maternal and cord blood, which similarly
found an increase in antioxidant molecules
with active smoking during pregnancy and
smaller changes in the same direction with
ETS exposure (Fayol et al. 2005). Here, we
found evidence that prenatal ETS exposure
programs antioxidant responses that limit the
additional effects of postnatal ETS: TBARS
were reduced with the perinatal exposure
model but not with postnatal exposure, despite
the fact that both groups received equivalent
ETS for the 7 months preceding the tissue
sampling at 13 months of age. It is likely that
programming of defense mechanisms is still
going on in the neonatal period, albeit at a
much lower level than with prenatal exposure,
Perinatal exposure to ETS
Environmental Health Perspectives • VOLUME 114 | NUMBER 1 | January 2006
p < 0.006
p < 0.002
Brain regions (nmol/mg protein)
ETS 6–13 months
Heart (nmol/mg protein)
Figure 2. Effects of ETS exposure on TBARS in brain
regions and heart (note different scales). ANOVA
across all treatments and tissues: treatment, p <
0.0001; treatment × tissue, p < 0.004. Lower order
ANOVAs for each tissue are shown within the figure.
*Individual values for which the ETS groups differ from the
βAR binding (fmol/mg protein)
ETS 6–13 months
m2AChR binding (fmol/mg protein)
Figure 3. Effects of ETS exposure on cardiac βAR
and m2AChR binding (note different scales);
ANOVA: treatment, p < 0.004. There was no inter-
action of treatment × receptor type; the only main
treatment effect is ETS 6–13 months (p < 0.003).
There were no significant differences in the con-
centration of membrane proteins.
because we did not find an elevation in
TBARS in the postnatal ETS group. In con-
trast, nicotine administered by itself to older
animals produces an increase in TBARS in a
variety of brain regions, even at nicotine doses
simulating ETS exposure (Qiao et al. 2005),
whereas much higher doses in the fetus do not
(Slotkin et al. 2005). Although we did not
evaluate which specific mechanisms contribute
to the net reductions in TBARS, it is impor-
tant to note that some of the factors may actu-
ally not be beneficial. During development, a
mild degree of oxidative stress is required for
the appropriate timing of neuronal cell differ-
entiation (Katoh et al. 1997), so oxidative
stress from ETS exposure and the adaptive
changes in defense mechanisms are both likely
to preempt this natural signal. Furthermore, a
number of the known, neurotoxic effects of
nicotine on brain development are themselves
liable to reduce oxidative damage. Nicotine
actually protects developing neurons from the
effects of other oxidative molecules (Guan
et al. 2003; Qiao et al. 2005). In addition, the
developmental neurotoxicity of nicotine pro-
duces changes that promote resistance to
oxidative stress, including marked reductions
in synaptic development and activity, and the
replacement of damaged neurons with glia,
cells that possess major antioxidant pathways
(Levin and Slotkin 1998; Roy et al. 2002;
Slotkin 1998, 2004; Slotkin et al. 2005;
Tanaka et al. 1999). Specifically, prenatal nico-
tine exposure grossly reduces neonatal activity
of nerve pathways using catecholamine neuro-
transmitters (Levin and Slotkin 1998; Slotkin
1998, 2004), which are strongly oxidative
(Olanow and Arendash 1994). Accordingly,
the primary neurotoxic effects of nicotine may
limit the apparent contribution of oxidative
damage to the net neurobehavioral effects of
ETS, so looking at lipid peroxidation alone
may be misleading without considering the
Unlike the effects of ETS on TBARS in
the brain, we did not find significant reduc-
tions in the heart after perinatal ETS expo-
sure, nor did postnatal ETS produce an effect.
These results indicate either that the heart
displays a different critical period for the pro-
gramming of antioxidant defenses, or alterna-
tively that the timetable for appearance and
disappearance of the effect might be different.
In fact, when we examined lipid peroxidation
at an earlier time point 2–3 months after birth,
we were able to demonstrate a significant
reduction in cardiac TBARS in the perinatal
exposure group, implying that the effects were
present but disappeared by the later sampling
at 13 months of age. Similarly, then, brain
regions that did not display a significant
decrease at 13 months may not in fact be
spared from the effects but may simply show a
more rapid return to normal oxidative status.
The temporal dichotomy is a reflection of
the fact that TBARS measurements take a
momentary “snapshot” of lipid peroxidation
rather than representing long-term damage,
whereas neural cell biomarkers provide a much
longer integrative time frame.
The results in the heart also provide con-
firmation that the protection from oxidative
stress comprises alterations that actually reflect
functional loss, evidenced by the reductions in
βARs and m2AChRs. Cardiac βAR overstimu-
lation evokes oxidative stress, leading to
myocyte apoptosis (Remondino et al. 2003),
whereas βARs protect neurons (Sarker et al.
2000) and show no down-regulation by devel-
opmental ETS exposure (Slotkin et al. 2005).
In turn, cardiac m2AChRs may be reduced as
a compensation to maintain the balance of
autonomic input or, alternatively, may be
specifically down-regulated because of their
similar involvement in oxidative stress (Joseph
et al. 2002). Indeed, in rats with ETS expo-
sure, the degree of cardiac m2AChR down-
regulation exceeds that of βARs (Slotkin et al.
2001). Again, there may be a specific role for
nicotine in these potentially maladaptive
responses: by itself, prenatal nicotine exposure
leads to decrements in cardiac βAR function
(Navarro et al. 1990).
In summary, our findings show that peri-
natal or postnatal ETS exposure in primates
elicits changes in brain cell development akin
to those found for either prenatal nicotine
exposure or perinatal ETS exposure in rodents
(Gospe et al. 1996; Levin and Slotkin 1998;
Navarro et al. 1988; Slotkin 1998, 2004) as
well as for prenatal nicotine in monkeys
(Slotkin et al. 2005). This reinforces a mecha-
nistic connection between nicotine as a specific
contributor to the adverse neurobehavioral
effects of developmental ETS exposure and
supports the use of nicotine metabolite mea-
surements in fetuses and children as an appro-
priate predictor of outcome (Eliopoulos et al.
1996; Fried et al. 1995; Jauniaux et al. 1999;
Kohler et al. 1999; Ostrea et al. 1994). Equally
significant, we found that postnatal ETS pro-
duces effects very similar to those achieved
with continuous prenatal and postnatal expo-
sure, buttressing the importance of restricting
or eliminating exposure in young children.
Finally, although ETS exposure also elicits
signs of chronic oxidative stress, demonstration
of a specific role of this mechanism in brain
damage remains elusive, confounded by adap-
tive mechanisms and perhaps most of all by the
underlying damage caused by nicotine. Indeed,
for prenatal exposure, attempts to offset oxida-
tive damage by dietary supplementation with
antioxidants may actually worsen nicotine-
related neurodevelopmental damage by sec-
ondary pharmacokinetic effects that increase
nicotine concentrations in the fetal compart-
ment (Slotkin et al. 2005), indicating the
danger of focusing on oxidative damage as a
primary mechanism rather than on the net
neurotoxic outcome of all ETS components.
Bell JM, Whitmore WL, Queen KL, Orband-Miller L, Slotkin TA.
1987. Biochemical determinants of growth sparing during
neonatal nutritional deprivation or enhancement: ornithine
decarboxylase, polyamines, and macromolecules in brain
regions and heart. Pediatr Res 22:599–604.
Bhagwat SV, Vijayasarathy C, Raza H, Mullick J, Avadhani NG.
1998. Preferential effects of nicotine and 4-(N-methyl-N-
nitrosamino)-1-(3-pyridyl)-1-butanone on mitochondrial
glutathione S-transferase A4-4 induction and increased
oxidative stress in the rat brain. Biochem Pharmacol
Dunn A, Zeise L. 1997. Health Effects of Exposure to
Environmental Tobacco Smoke. Sacramento, CA:California
Environmental Protection Agency.
Eliopoulos C, Klein J, Chitayat D, Greenwald M, Koren G. 1996.
Nicotine and cotinine in maternal and neonatal hair as
markers of gestational smoking. Clin Invest Med 19:231–242.
Eskenazi B, Trupin LS. 1995. Passive and active maternal smok-
ing during pregnancy, as measured by serum cotinine, and
postnatal smoke exposure. 2. effect on neurodevelopment
at age 5 years. Am J Epidemiol 142:S19–S29.
Fayol L, Gulian JM, Dalmasso C, Calaf R, Simeoni U, Millet V.
2005. Antioxidant status of neonates exposed in utero to
tobacco smoke. Biol Neonate 87:121–126.
Fried PA, O’Connell CM, Watkinson B. 1992. 60- and 72-month
follow-up of children prenatally exposed to marijuana, ciga-
rettes, and alcohol: cognitive and language assessment.
J Dev Behav Pediatr 13:383–391.
Fried PA, Perkins SL, Watkinson B, McCartney JS. 1995.
Association between creatinine-adjusted and unadjusted
urine cotinine values in children and the mother’s report of
exposure to environmental tobacco smoke. Clin Biochem
Fried PA, Watkinson B, Gray R. 1998. Differential effects on cog-
nitive functioning in 9- to 12-year olds prenatally exposed to
cigarettes and marihuana. Neurotoxicol Teratol 20:293–306.
Fried PA, Watkinson B, Gray R. 2003. Differential effects on cogni-
tive functioning in 13- to 16-year-olds prenatally exposed to
cigarettes and marihuana. Neurotoxicol Teratol 25:427–436.
Gitto E, Reiter RJ, Karbownik M, Tan DX, Gitto P, Barberi S,
et al. 2002. Causes of oxidative stress in the pre- and peri-
natal period. Biol Neonate 81:146–157.
Gospe SM, Zhou SS, Pinkerton KE. 1996. Effects of environmen-
tal tobacco smoke exposure in utero and/or postnatally on
brain development. Pediatr Res 39:494–498.
Guan ZZ, Yu WF, Nordberg A. 2003. Dual effects of nicotine on
oxidative stress and neuroprotection in PC12 cells.
Neurochem Intl 43:243–249.
Gupta RC. 2004. Brain regional heterogeneity and toxicological
mechanisms of organophosphates and carbamates. Toxicol
Mech Methods 14:103–143.
Huang MF, Lin WL, Ma YC. 2005. A study of reactive oxygen
species in mainstream of cigarette. Indoor Air 15:135–140.
Hutchison SJ, Glantz SA, Zhu BQ, Sun YP, Chou TM, Chatterjee
K, et al. 1998. In-utero and neonatal exposure to second-
hand smoke causes vascular dysfunction in newborn rats.
J Am Coll Cardiol 32:1463–1467.
James SJ, Slikker W, Melnyk S, New E, Pogribna M, Jernigan S.
2005. Thimerosal neurotoxicity is associated with gluta-
thione depletion: protection with glutathione precursors.
Jauniaux E, Gulbis B, Acharya G, Thiry P, Rodeck C. 1999.
Maternal tobacco exposure and cotinine levels in fetal fluids
in the first half of pregnancy. Obstet Gynecol 83:25–29.
Jenkins RA, Guerin MR, Tomkins BA. 2000. The Chemistry of
Environmental Tobacco Smoke: Composition and
Measurement. 2nd ed. Boca Raton, FL:Lewis Publishers.
Joseph JA, Fisher DR, Strain J. 2002. Muscarinic receptor sub-
type determines vulnerability to oxidative stress in COS-7
cells. Free Radic Biol Med 32:153–161.
Katoh S, Mitsui Y, Kitani K, Suzuki T. 1997. Hyperoxia induces
the differentiated neuronal phenotype of PC12 cells by
producing reactive oxygen species. Biochem Biophys Res
Kohler E, Sollich V, Schuster R, Thal W. 1999. Passive smoke
exposure in infants and children with respiratory tract
diseases. Human Exp Toxicol 18:212–217.
Slotkin et al.
VOLUME 114 | NUMBER 1 | January 2006 • Environmental Health Perspectives
Perinatal exposure to ETS
Environmental Health Perspectives • VOLUME 114 | NUMBER 1 | January 2006
Kostrzewa R, Jacobowitz DM. 1974. Pharmacological actions
of 6-hydroxydopamine. Pharmacol Rev 26:199–288.
Labarca C, Piagen K. 1980. A simple, rapid, and sensitive DNA
assay procedure. Anal Biochem 102:344–352.
Levin ED, Slotkin TA. 1998. Developmental neurotoxicity of nico-
tine. In: Handbook of Developmental Neurotoxicology
(Slikker W, Chang LW, ed). San Diego:Academic Press,
Makin J, Fried PA, Watkinson B. 1991. A comparison of active
and passive smoking during pregnancy: long-term effects.
Neurotoxicol Teratol 13:5–12.
McMillian MK, Schanberg SM, Kuhn CM. 1983. Ontogeny of rat
hepatic adrenoceptors. J Pharmacol Exp Ther 227:181–186.
National Research Council. 1996. Guide for the Care and Use of
Laboratory Animals. Washington, DC:National Academy
Navarro HA, Mills E, Seidler FJ, Baker FE, Lappi SE, Tayyeb MI,
et al. 1990. Prenatal nicotine exposure impairs β-adrenergic
function: persistent chronotropic subsensitivity despite
recovery from deficits in receptor binding. Brain Res Bull
Navarro HA, Seidler FJ, Whitmore WL, Slotkin TA. 1988. Prenatal
exposure to nicotine via maternal infusions: effects on
development of catecholamine systems. J Pharmacol Exp
Newman MB, Arendash GW, Shytle RD, Bickford PC, Tighe T,
Sanberg PR. 2002. Nicotine’s oxidative and antioxidant
properties in CNS. Life Sciences 71:2807–2820.
O’Callaghan JP. 1988. Neurotypic and gliotypic proteins as bio-
chemical markers of neurotoxicity. Neurotoxicol Teratol
O’Callaghan JP. 1993. Quantitative features of reactive gliosis
following toxicant-induced damage of the CNS. Ann NY
Acad Sci 679:195–210.
Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal
Ohtsuka K, Suzuki T. 2000. Roles of molecular chaperones in
the nervous system. Brain Res Bull 53:141–146.
Olanow CW, Arendash GW. 1994. Metals and free radicals in
neurodegeneration. Curr Opin Neurol 7:548–558.
Ostrea EM, Knapp DK, Romero A, Montes M, Ostrea AR. 1994.
Meconium analysis to assess fetal exposure to nicotine by
active and passive maternal smoking. J Pediatr 124:471–476.
Qiao D, Seidler FJ, Abreu-Villaça Y, Tate CA, Cousins MM,
Slotkin TA. 2004. Chlorpyrifos exposure during neurulation:
cholinergic synaptic dysfunction and cellular alterations in
brain regions at adolescence and adulthood. Dev Brain Res
Qiao D, Seidler FJ, Slotkin TA. 2005. Oxidative mechanisms con-
tributing to the developmental neurotoxicity of nicotine and
chlorpyrifos. Toxicol Appl Pharmacol 206:17–26;
Qiao D, Seidler FJ, Tate CA, Cousins MM, Slotkin TA. 2003.
Fetal chlorpyrifos exposure: adverse effects on brain cell
development and cholinergic biomarkers emerge post-
natally and continue into adolescence and adulthood.
Environ Health Perspect 111:536–544.
Remondino A, Kwon SH, Communal C, Pimentel DR, Sawyer DB,
Singh K, et al. 2003. β-Adrenergic receptor-stimulated
apoptosis in cardiac myocytes is mediated by reactive oxy-
gen species/c-Jun NH2-terminal kinase-dependent activa-
tion of the mitochondrial pathway. Circ Res 92:136–138.
Rodier PM. 1988. Structural-functional relationships in experi-
mentally induced brain damage. Prog Brain Res 73:335–348.
Roy TS, Andrews JE, Seidler FJ, Slotkin TA. 1998. Nicotine
evokes cell death in embryonic rat brain during neurulation.
J Pharmacol Exp Ther 287:1135–1144.
Roy TS, Sabherwal U. 1994. Effects of prenatal nicotine expo-
sure on the morphogenesis of somatosensory cortex.
Neurotoxicol Teratol 16:411–421.
Roy TS, Sabherwal U. 1998. Effects of gestational nicotine expo-
sure on hippocampal morphology. Neurotoxicol Teratol
Roy TS, Seidler FJ, Slotkin TA. 2002. Prenatal nicotine exposure
evokes alterations of cell structure in hippocampus and
somatosensory cortex. J Pharmacol Exp Ther 300:124–133.
Roy TS, Sharma V, Seidler FJ, Slotkin TA. 2005. Quantitative
morphological assessment reveals neuronal and glial
deficits in hippocampus after a brief subtoxic exposure to
chlorpyrifos in neonatal rats. Dev Brain Res 155:71–80.
Sarker KP, Uchimura T, Nakajima T, Sorimachi M, Kitajima I,
Maruyama I. 2000. Epinephrine prevents nitric oxide/per-
oxynitrite induced apoptosis of neuronal cells through
β-adrenergic receptor activation. Neurosci Res Commun
Slotkin TA. 1998. Fetal nicotine or cocaine exposure: which one
is worse? J Pharmacol Exp Ther 285:931–945.
Slotkin TA. 2004. Cholinergic systems in brain development and
disruption by neurotoxicants: nicotine, environmental
tobacco smoke, organophosphates. Toxicol Appl Pharmacol
Slotkin TA, Epps TA, Stenger ML, Sawyer KJ, Seidler FJ. 1999.
Cholinergic receptors in heart and brainstem of rats
exposed to nicotine during development: implications for
hypoxia tolerance and perinatal mortality. Dev Brain Res
Slotkin TA, Orband-Miller L, Queen KL. 1987a. Development of
[3H]nicotine binding sites in brain regions of rats exposed
to nicotine prenatally via maternal injections or infusions.
J Pharmacol Exp Ther 242:232–237.
Slotkin TA, Orband-Miller L, Queen KL, Whitmore WL, Seidler FJ.
1987b. Effects of prenatal nicotine exposure on biochemical
development of rat brain regions: maternal drug infusions
via osmotic minipumps. J Pharmacol Exp Ther 240:602–611.
Slotkin TA, Persons D, Slepetis RJ, Taylor D, Bartolome J. 1984.
Control of nucleic acid and protein synthesis in developing
brain, kidney, and heart of the neonatal rat: effects of
α-difluoromethylornithine, a specific, irreversible inhibitor
of ornithine decarboxylase. Teratology 30:211–224.
Slotkin TA, Pinkerton KE, Auman JT, Qiao D, Seidler FJ. 2002.
Perinatal exposure to environmental tobacco smoke
upregulates nicotinic cholinergic receptors in monkey
brain. Dev Brain Res 133:175–179.
Slotkin TA, Pinkerton KE, Garofolo MC, Auman JT, McCook EC,
Seidler FJ. 2001. Perinatal exposure to environmental
tobacco smoke induces adenylyl cyclase and alters
receptor-mediated signaling in brain and heart of neonatal
rats. Brain Res 898:73–81.
Slotkin TA, Pinkerton KE, Seidler FJ. 2000. Perinatal exposure to
environmental tobacco smoke alters cell signaling in a pri-
mate model: autonomic receptors and the control of
adenylyl cyclase activity in heart and lung. Dev Brain Res
Slotkin TA, Seidler FJ, Qiao D, Aldridge JE, Tate CA, Cousins MM,
et al. 2005. Effects of prenatal nicotine exposure on primate
brain development and attempted amelioration with supple-
mental choline or vitamin C: neurotransmitter receptors, cell
signaling and cell development biomarkers in fetal brain
regions of rhesus monkeys. Neuropsychopharmacology
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,
Provenzano MD, et al. 1985. Measurement of protein using
bicinchoninic acid. Anal Biochem 150:76–85.
Song X, Seidler FJ, Saleh JL, Zhang J, Padilla S, Slotkin TA. 1997.
Cellular mechanisms for developmental toxicity of chlor-
pyrifos: targeting the adenylyl cyclase signaling cascade.
Toxicol Appl Pharmacol 145:158–174.
Tanaka J, Toku K, Zhang B, Isihara K, Sakanaka M, Maeda N.
1999. Astrocytes prevent neuronal death induced by reac-
tive oxygen and nitrogen species. Glia 28:85–96.
Trauth JA, Seidler FJ, Slotkin TA. 2000. An animal model of
adolescent nicotine exposure: effects on gene expression
and macromolecular constituents in rat brain regions.
Brain Res 867:29–39.
U.S. Environmental Protection Agency. 1992. Respiratory
Health Effects of Passive Smoking: Lung Cancer and Other
Disorders. Washington, DC:Office of Research and
Development, U.S. Environmental Protection Agency.
Wakschlag LS, Pickett KE, Cook E, Benowitz NL, Leventhal BL.
2002. Maternal smoking during pregnancy and severe anti-
social behavior in offspring: a review. Am J Public Health
Walker A, Rosenberg M, Balaban-Gil K. 1999. Neuro-
developmental and neurobehavioral sequelae of selected
substances of abuse and psychiatric medications in utero.
Child Adolesc Psychiat Clin North Am 8:845–867.
Weitzman M, Byrd RS, Aligne CA, Moss M. 2002. The effects of
tobacco exposure on children’s behavioral and cognitive
functioning: implications for clinical and public health policy
and future research. Neurotoxicol Teratol 24:397–406.
Winick M, Noble A. 1965. Quantitative changes in DNA, RNA
and protein during prenatal and postnatal growth in the
rat. Dev Biol 12:451–466.
Witschi H, Joad JP, Pinkerton KE. 1997. The toxicology of
environmental tobacco smoke. Annu Rev Pharmacol
Yildiz D, Liu YS, Ercal N, Armstrong DW. 1999. Comparison of
pure nicotine- and smokeless tobacco extract-induced
toxicities and oxidative stress. Arch Environ Contam
Zahalka EA, Seidler FJ, Yanai J, Slotkin TA. 1993. Fetal nicotine
exposure alters ontogeny of M1-receptors and their link to
G-proteins. Neurotoxicol Teratol 15:107–115.