Transplacental Exposure to AZT Induces Adverse
Neurochemical and Behavioral Effects in a Mouse Model:
Protection by L-Acetylcarnitine
Anna Rita Zuena1., Chiara Giuli1., Aldina Venerosi Pesciolini2, Antonella Tramutola1, Maria
Antonietta Ajmone-Cat2, Carlo Cinque1, Giovanni Sebastiano Alema `1, Angela Giovine1,
Gianfranco Peluso5, Luisa Minghetti3, Raffaella Nicolai4, Gemma Calamandrei2*, Paola Casolini1
1Department of Physiology and Pharmacology ‘‘Vittorio Erspamer’’, I Faculty of Medicine, Sapienza University of Rome, Rome, Italy, 2Section of Neurotoxicology &
Neuroendocrinology, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita `, Rome, Italy, 3Section of Experimental Neurology, Department of Cell
Biology and Neurosciences, Istituto Superiore di Sanita `, Rome, Italy, 4Therapeutic Area Life Cycle Management, Corporate R&D Sigma-Tau S.p.A., Pomezia, Rome, Italy,
5Institute of Biochemistry of Proteins, CNR, Naples, Italy
Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other
antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-
saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of
antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of
mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical
evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting
in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT,
the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter
involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and
behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that
improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients.
We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired
in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased
brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of
AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective
in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the
offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and
strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-
effects of antiretroviral therapy in fetus/newborn.
Citation: Zuena AR, Giuli C, Venerosi Pesciolini A, Tramutola A, Ajmone-Cat MA, et al. (2013) Transplacental Exposure to AZT Induces Adverse Neurochemical and
Behavioral Effects in a Mouse Model: Protection by L-Acetylcarnitine. PLoS ONE 8(2): e55753. doi:10.1371/journal.pone.0055753
Editor: Michel Baudry, Western University of Health Sciences, United States of America
Received September 12, 2012; Accepted December 31, 2012; Published February 7, 2013
Copyright: ? 2013 Zuena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by funds to P.C. from University of Rome (faculty project 2009 prot. C26F09YEXM) and partly sponsored by Sigma-Tau I.F.R.
SpA (Rome) (D.S./2007/C.R./nu6). Raffaella Niccolai is an employee of the SIGMA TAU and participated in manuscript preparation and data analysis as an expert of
acetylcarnitine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Doctor Raffaella Nicolai is employed at Sigma-tau S.p.A. as Medical Research Senior Advisor; Raffaella Nicolai and Paola Casolini have been
designated among inventors in the following European patent application: 11137170.3-2112 ‘‘Compound useful for preventing cognitive deficit disorders in a
new born from HIV-seropositive pregnant female who is on treatment with azidothymidine’’. Part of this study has been financially supported by Sigma Tau-IFR
S.p.A., Italy that has synthesized and provided L-acetylcarnitine. Sigma-tau is an Italian pharmaceutical company involved in research on carnitine system which
produces and sells carnitine as drugs. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. There are no other
competing interests to declare.
* E-mail: email@example.com
. These authors contributed equally to this work.
Since 1994, effective reduction in maternal-fetal HIV-1
transmission has been achieved by administration of antiretroviral
(ARV) agents to HIV-1-infected women during pregnancy and to
their newborns during early neonatal period . The benefits
deriving from the introduction of prophylactic ARV therapy are
unquestionable but there is still considerable uncertainty on the
potential long-term adverse effects of ARV agents on exposed
children, in particular as for nucleoside reverse transcriptase
inhibitors (NRTIs) . In US and Europe, standard protocols of
care for pregnant seropositive women and their newborns always
include the NRTI zidovudine (AZT), alone or in combination with
another NRTI, a non-NRTI or a protease inhibitor [3,4].
Toxic effects of chronic treatment with AZT-based therapies
have been widely documented in HIV-1 positive adults, including
liver failure, lactic acidosis, myopathy, and neuropathy. These
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adverse effects have been mainly related to the ability of NRTIs,
secondary to their antiviral action , to interfere with mitochon-
drial function in different target organs [6–8]. Specifically AZT
induces alterations in mitochondrial structure and function by (a)
direct inhibition of mtDNA replication and repair via inhibition of
mtDNA polymerase c; (b) alterations in cellular metabolism
affecting oxidative phosphorylation enzyme activity and genera-
tion of reactive oxygen species; (c) mutations via incorporation of
the NRTI into mtDNA and replication blockage [9,10].
Results from clinical and cohort studies in offspring of women
exposed to NRTIs during pregnancy, though excluding major
adverse effects , have clearly shown the occurrence of
subclinical mitochondrial dysfunction [11,12], whose long-term
repercussion on high-requiring energy tissues, such as the heart
and the brain, is still a matter of concern. In a study carried out in
a small number of infants born to HIV-1-positive women,
quantification of mtDNA in cord blood evidenced mitochondrial
damage and mtDNA depletion in NRTI-exposed infants when
compared to unexposed infants . A prospective study carried
out in a large cohort of non-infected children within the French
Pediatric Cohort reported the occurrence of symptoms compatible
with mitochondrial dysfunction in a significant proportion of
NRTI-exposed infants . Such symptoms were primarily
neurologic and included cognitive delay, motor disturbances,
white-matter alteration in Magnetic Resonance Imaging and
increased risk to develop febrile seizures, associated to deficits in
one of the mitochondrial respiratory chain complexes [14–16].
Since AZT is central to highly active ARV therapy for reducing
mother-to-child transmission of HIV-1 [4,17], it is important to
determine the nature and magnitude of the long-term effects of in
utero AZT exposure as well as the mechanisms underlying the
toxicities of this compound. However, in epidemiological studies
the methodological issues related the potential effects of antiret-
roviral medications on neurobehavioral development are very
complex, given the numerous co-morbidity factors that may
influence neurobehavioral outcome in this specific group of
children. These factors include, among the others, in utero exposure
to maternal pro-inflammatory cytokines released during HIV
infection, prenatal exposure to drug of abuse, family disruption,
mental health problems in parents and birth complications .
Animal models represent a useful tool to overcome the method-
ological and ethical constraints implicated in human studies, and
to investigate the etiology of antiretroviral drugs’ toxicities in the
absence of other confounding factors. A number of animal studies
have been devoted to address this issue. These studies have clearly
shown that developmental exposure to AZT produces both early
and delayed behavioral changes in offspring. The behavioral
endpoints affected by transplacental exposure to AZT include
sensor and motor maturation, learning abilities, social/aggressive
behavior, exploration levels [19–30]. Knowledge of the neural
bases of behavioral AZT toxicity is so far very limited, but it has
been clearly shown that at doses comparable to those used in
clinical practice, AZT acts as a mitochondrial toxin in rodents and
non-human primates [31–36]. Extensive evidence links dysfunc-
tions of mitochondrial energy supply and the resulting oxidative
stress to excessive release of glutamate  the major excitatory
neurotransmitter in the mammalian central nervous system (CNS).
By acting on ionotropic (iGlu) and metabotropic (mGlu) receptors,
glutamate has a wide array of effects, ranging from modulation of
learning and memory capacities [38–40], neuroendocrine secre-
tion of glucocorticoids  and regulation of synaptic transmission
and plasticity in the CNS [42,43]. On these bases, we hypothesize
that the behavioral alterations found in rodents exposed to AZT in
utero are linked to oxidative stress resulting from AZT mitochon-
drial toxicity through the involvement of the glutamate system.
In order to assess this hypothesis we first evaluated in adult mice
exposed in utero to AZT at clinical relevant doses spatial learning
capacities, corticosterone response to acute stress, hippocampal
glutamate receptors expression and oxidative stress, the latter by
means of isoprostane and oxidized proteins level measurement.
Furthermore, considering the implication of early oxidative stress
induced by AZT in development of delayed neurobehavioral
alterations, we evaluated whether the administration during
pregnancy of a neuroprotective agent such as L-acetylcarnitine
(LAC), capable of protecting mitochondria from damage induced
by different noxa, was able to protect the transplacental AZT-
exposed offspring from the CNS toxicity of this drug and,
consequently, from the resulting behavioral impairment.
LAC, the acetyl ester of L-carnitine is a naturally occurring
endogenous compound in all mammalian species present in
relatively high levels in the brain, in particular in hypothalamus
and hippocampus. When systemically administered, LAC readily
crosses the blood-brain barrier influencing brain metabolism. LAC
neuroprotective effects rely on improved mitochondrial energetic
function, potentiation of antioxidant activities, stabilization of
membranes, modulation of protein and gene expression .
Clinical studies have shown that treatment with LAC improved
NRTI-induced polyneuropathy symptoms including pain, paraes-
thesia and numbness in HIV-1-positive patients [45–48]. Howev-
er, the potential efficacy of LAC in protecting from the side effects
of the prophylactic ARV therapy in pregnancy has never been
studied so far.
Materials and Methods
This study was carried out in accordance with the Italian
Animal Welfare legislation (art 4 and 5 of D.L. 116/92) that
implemented the European Committee Council 106 Directive
(86/609/EEC). The Italian Ministry of Health specifically
approved the protocol of this study on 12/21/2009, Authorization
nu 224/2009-b to G.C.
Animals and housing conditions
Adult male (n=30) and virgin female (n=60) mice of the out
bred Swiss-derived strain (CD-1) were purchased from Charles
River. Upon arrival at the laboratory, the animals were housed in
an air-conditioned room (temperature 2161uC, relative humidity
5565%) with a reversed 12:12 light cycle (light on at 19.00 h, light
off at 07.00 h). Water and food were available ad libitum. Females
were group-housed (4 per cage) for 7 days to coordinate their estral
cycle. After that, pairs of female mice were housed with a single
sexually experienced male mouse. Females were daily inspected
for the presence of a vaginal plug (gestational day 0; GD0) and
then individually housed in Plexiglas cages (33613614 cm).
Pregnancy rate was about 75%. The litters were culled at birth
to four females and four males, to maintain adequate litter
composition. Only male offsprings were used in this study.
The experimental design of this study is depicted in Fig. 1. AZT
was purchased from Sigma-Aldrich and LAC was provided by
Sigma-Tau S.p.A. AZT was dissolved in bidistilled water and LAC
Following finding of the vaginal plugs 45 females were
randomly assigned to one of the two subcutaneous (s.c.) treatments
with either LAC (100 mg/kg, in a volume of 1.25 ml/kg) or
LAC Protects from AZT Neurobehavioral Effects
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vehicle (saline, same volume as LAC) that were administered daily
from GD0 to the day of delivery. Starting on GD10, pregnant
mice in each vehicle and LAC group were also administered by
oral gavage (p.os) with either water (3.3 ml/kg) or AZT (150 mg/
kg, same volume) in the morning and evening till the day of
delivery. The resulting treatment groups were as follows: Control
(saline s.c.+water p.os), LAC (LAC s.c.+water p.os), AZT (saline
s.c.+AZT p.os) and AZT+LAC (AZT p.os.+LAC s.c.)
The AZT dose administered has been selected after multi-dose
studies and taking into account the results of other groups’ and
ours studies in rodents, which identified doses producing
behavioral effects and/or mitochondrial dysfunction in absence
of significant reproductive or morphological effects in the mother
and offspring [22,29].
In order to measure the AZT levels in blood and brain, blood
was withdrawn from the orbital plexus after delivery in five
mothers of AZT group at the end of pregnancy, in a period
between 8 and 12 hours after the last AZT treatment. Four pups
from each of the same litters were sacrificed at birth, and their
plasma and brain hemispheres collected and pooled (n=5).
Females’ body weight was monitored daily during pregnancy.
Proportion of term pregnancies, gestation length, litter size, sex
ratio and neonatal mortality were also measured to exclude
potential effects of the treatment on reproductive performances.
The day of birth was defined as postnatal day 0 (PND0). At this
age, before litter culling, eight pups from each treatment group
were sacrificed for F2-isoprostane measurement in the total brain
homogenate. Weaning took place at 21 days of age (PND21) and
male offspring of each group housed in two per cage. They were
maintained under controlled environmental conditions until 2
months of age (PND60) when they were assigned to water maze
spatial learning test, plasma corticosterone assay, protein oxidation
and glutamate receptor analysis. Body weights were recorded at
birth (PND0), after weaning (PND24) and before performance of
the spatial learning task (PND60).
Determination of AZT Concentrations in Plasma and
Plasma and brain homogenate samples were analyzed sepa-
rately using spiked standards in blank plasma and brain
homogenate. Commercial available AZT (Sigma-Aldrich) was
used as internal standard. For plasma, 100 ml samples were spiked
with 100 ng of internal standard. The samples were subjected to
liquid-liquid extraction using 8 parts of ethyl acetate for 1 part of
sample and vortexed vigorously for 5 min. The mixture was
centrifuged at 14,000 rpm at room temperature for 10 min. The
supernatant was transferred to clean glass tubes, dried under a
flow of liquid nitrogen, and reconstituted in 100 ml of mobile
phase, and 30 ml was injected onto the HPLC column. Whole
brain was homogenized in 3 volumes of 5% bovine serum albumin
in PBS using a Dounce homogenizer (7 ml; Kontes Glass); 500 ml
of brain homogenate was spiked with 50 ng of internal standard,
and sample preparation was similar to that of plasma. The mobile
phase for AZT analysis consisted of buffer (50 mM ammonium
phosphate, 50 mM sodium citrate buffer, and 10 ppm sodium
azide, pH 6.5) and methanol (82:18) at a flow rate of 0.2 ml/min,
and UV absorbance was measured at 266 nm. The plasma and
brain concentrations are reported as nanograms per milliliter and
nanograms per gram of brain tissue, respectively.
AZT determination was performed by HPLC analysis using a
Hypersil-BDS column (C-18, 2.1 mm6150 mm, 5 mM; Thermo
Electron Corporation) maintained at 40uC using a Shimadzu
column oven (CTO-10Avp). The HPLC system consisted of a
Shimadzu pump (LC-10ATvp), flow control valve (FCV-10ALvp),
degasser (DGU-20A5), auto injector (SIL-10ADvp), system con-
troller (SCL-10Avp), and detector (SPD-10Avp).
On PND0 the levels of the F2-isoprostane (F2-IsoP) were
measured in brain homogenates, as previously described .
Briefly, brains were weighed and homogenized in 50 mM Tris
buffer, pH 7.5 (1 mg/0.1 ml), containing the anti-oxidant 10 mM
BHT to block spontaneous oxidation. Homogenates were
vigorously vortexed and incubated for 5 min on ice before
centrifuging at 14,000 rpm for 45 min at 4uC. Supernatants were
collected and stored at 280uC until required. 15-F2t-IsoP, the
major member of F2-IsoP family, was measured by a specific
enzyme immunoassay (Cayman Chemical), according to the
manufacturer’s instructions. Detection limit was 2 pg/ml; anti-
15-F2t-IsoP antibody cross-reactivity with other iso-prostaglandins
was less than 0.15%.
Detection of Oxidized Proteins
Protein oxidation was measured in the same homogenates
utilized for western blot of glutamate receptors in 2-month-old
mice. Oxidized protein was detected by using an oxidized protein
detection kit (OxyBlot, Chemicon International). The OxyBlot
provides reagents for sensitive immunodetection of carbonyl
groups, which is a hallmark of the oxidation status of proteins.
The procedure was performed according to manufacturer’s
recommendation. Briefly, 30 mg of hippocampal homogenates
were derivatized with or without 2,4-dinitrophenylhydrazine
(DNPH) and samples loaded onto a 12% SDS-PAGE gel. After
separation, proteins were electrotransferred to a nitrocellulose
Figure 1. Experimental design of the study.
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membrane and incubated with a primary antibody against the
derivatized carbonyl groups followed by incubation with a
horseradish peroxidase-conjugate goat anti-rabbit antibody. The
oxidized proteins were visualized by using an enhanced chemilu-
minescence system (Amersham Biosciences). The relative optical
densities were quantified using the NIH ImageJ medical imaging
software. No immunoreactivity was detected in the non-DNPH
derivatized brain homogenates.
Water Maze procedure
On PND60 mice from each treatment group were tested in the
Morris water maze for their spatial learning ability, applying a
one-day protocol that has been successfully applied to different
strains of mice . The apparatus consisted of a circular pool
(diameter 110 cm, height 60 cm) located in a test room with white
walls with several cues on them. The pool, with its inner surface
painted black, was filled to a depth of 40 cm with water
(maintained at 2561uC), covering an invisible (black) 10-cm
square platform. The platform was located approximately 0.5 cm
below the surface of the water. The pool was virtually divided into
4 quadrants (North, South, East, or West) and the platform placed
at a fixed position in the center of the North quadrant. A single-
day training procedure was carried out, and each subject
underwent four four-trial sessions of training, with each session
separated by 30 min. On each trial, the subject was gently released
in the water with its head facing the pool wall from one of
quadrants. The order of the starting quadrant was changed in
each session and trial. A maximum of 60 sec was allowed, during
which the mouse had to find the platform, climb onto it and
allowed to remain there for 10 sec. If the animal did not find the
platform, it was gently guided with a grid and allowed to stay for
A video camera above the center of the pool was connected to a
computerized tracking system that recorded and analyzed animal
behavior (San Diego Instruments). The time of escape onto the
platform and the swimming speed were measured. After the last
trial of the last session of training, animals were submitted to a
single 60 sec ‘‘probe trial’’ in which the platform was removed
from the pool. The animal started the probe trial in the south
quadrant and the time that it swam through the north quadrant,
where the platform had been previously located, provided a
measure of learning accuracy in recalling the former position of
the platform. A session with a visible platform was performed at
the end of the learning task to assess the swimming speed of the
different groups of animals.
Corticosterone secretion after acute restraint stress
A second group of mice from each treatment group was used for
assessment of plasma corticosterone levels in basal conditions and
following 15 min of acute restraint stress. The procedure was
performed in the animal facility at 9:00 AM. Restraint stress was
performed as follows: the mouse was removed from its cage and
placed in an adjustable Plexiglas restraint device for 15 min under
a very bright light. Blood samples were collected from the tail tip
in heparinized capillary tubes at the beginning (basal value) and at
the end of the restraint procedure (stress value). At the end of the
procedure the mice were immediately returned to their cages.
Blood samples were centrifuged at 19006g at 4uC for 20 min;
plasma was removed and kept frozen at 220uC until assay. Plasma
corticosterone concentrations were determined by radioimmuno-
assay (MP Biomedicals). The cross-reactivity of the polyclonal
corticosterone-antisera with respective related substances was
negligible. The inter- and intra-assay coefficients of variance were
7% and 4%, respectively, with a detection limit of 0.01 mg/
Western blot analysis
A third set of mice was sacrificed on PND60 to assess expression
of iGlu and mGlu receptors. For the iGlu receptors we assessed
AMPA Glu1 and Glu2 subunit, and NMDA NR1 subunit. For the
mGlu receptors we measured both group I mGluR1 and mGluR5
subtypes and group II mGlu2/3 subtypes. Mice were killed by
decapitation and brains rapidly removed; hippocampi were
dissected and stored at 280uC. On the day of the experiment,
tissue was homogenized at 4uC with a polytron in 500 ml of
100 mM Tris buffer, containing phenylmethylsulfonyl fluoride
1 mM, leupeptin 10 mg/ml and aprotinin 10 mg/ml pH 7.2.
Protein concentrations were determined using the Bradford
protein assay. Thirty micrograms of protein were re-suspended
in sodium dodecyl sulfate (SDS)-bromophenol blue loading buffer
with 0.5 M dithiothreitol. The samples were separated on 8%
SDS-polyacrylamide gels (Amersham Bioscience) and after elec-
trophoresis (Mini-PROTEAN 3 System, Bio-Rad), the proteins
were transferred to nitrocellulose membranes (Amersham Biosci-
ence) using a system of mini transblot cell (BioRad) overnight.
After transfer, blots were incubated in a solution (blocking
solution) containing Tris-buffered saline (TBS), 10% (w/v)
Tween-20, 1% (w/v) non-fat milk and 1% (w/v) bovine serum
albumin. Subsequently, blots were incubated overnight with rabbit
anti-mGluR1a (1:1000), anti-mGluR5 (1:1000), anti-mGluR2/3
(1:1000), anti-NR1 (1:1000), mouse antiGlu1 (1:500) (Upstate
Biotechnology) and mouse antiGlu2 (1:500; Chemicon Interna-
tional) in blocking solution at 4uC. After incubation with the
primary antibody, the blots were incubated with horseradish
peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies
(1:5000; Amersham Bioscience) for 1 h at room temperature
(21uC62). To ensure that each lane was loaded with an equivalent
amount of protein, the blots were probed with an anti-actin serum
(1:1000; Sigma) overnight at 4uC. Subsequently, blots were
incubated with horseradish peroxidase-conjugated goat anti-
mouse antibodies (1:5000; Amersham Bioscience) for 1 h at room
temperature. Immunoreactive bands were visualized with an
enhanced chemiluminescence system (Amersham Biosciences).
After immunoblotting, digitized images of bands immunoreactive
for target (mGluR1, mGluR5, mGluR2/3, NR1 or Glu1) and
control (actin) molecules were acquired and the area of
immunoreactivity corresponding to each band was measured
using the NIH ImageJ medical imaging software. A ratio of target
to actin was then determined, and these values were compared for
Body weight data were analyzed separately for each age point
by one-way ANOVA. Latencies to reach the platform in the
Morris water maze were analyzed by three-way ANOVA for
repeated measures (treatment6trial6session with repeated mea-
sures on trials and sessions). Each litter in each final treatment
group contributed with a single subject to spatial learning task,
corticosterone assessment and measurement of glutamate recep-
tors/protein carbonyl content. The ANOVA analyses were always
followed by Fisher’s LSD post-hoc comparisons. Probe trial,
swimming speed, plasma corticosterone concentrations and
immunoblotting data were analyzed by Student’s t-test. The level
of significance was set at p,0.05.
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AZT administered during gestation reaches the brain of
In female mice after delivery, mean AZT plasma concentration
was 8.661.0 ng/ml, a value comparable to that found in women
treated during pregnancy following clinical protocols . Plasma
concentration of AZT in pups was undetectable, but significant
AZT levels were found in the brain (70.266.1 ng/mg of brain
tissue), indicating the transplacental passage of AZT to the brain of
fetuses (AZT vs Control, p,0.05 Fisher’s LSD post-hoc).
AZT induced decrease of body weight at birth is not
prevented by LAC
Proportion of term pregnancies, gestation length, litter size, sex
ratio and neonatal mortality was not affected by prenatal
treatment with AZT or LAC. The mean body weight of the
offspring at different ages is shown in Table 1. At birth (PND0)
AZT treatment significantly decreased body weight (main
treatment effect F3,36=11.58, p,0.05; AZT vs Control, p,0.05
Fisher’s LSD post-hoc), an effect that was not prevented by
administration of LAC in the AZT+LAC group (AZT+LAC vs
Control, p,0.05 Fisher’s LSD post-hoc). LAC per se did not modify
pups’ body weight at birth, but increased it on PND24 (LAC vs all
other groups, p,0.05 Fisher’s LSD post-hoc). At PND60, mean
body weight was comparable in the four experimental groups.
AZT- induced impairment of spatial learning and
memory is counteracted by LAC treatment
Figure 2A shows the latencies to reach the hidden platform
throughout the four sessions of the Morris water maze. ANOVA
for repeated measures evidenced a significant main effect of the
treatment received (F3,26=5.95, p,0.05) and a significant three
way interaction treatment6session6trial (F3,27=1.89, p,0.05).
Post-hoc analysis showed that subjects receiving prenatal AZT
treatment displayed a significant increase in escape latency in
comparison to the Control group in all the four sessions. The
AZT+LAC group had escape latencies comparable to the Control
group and significantly different from those of the AZT group in
the 3rdand 4thsession, suggesting that pre-treatment with LAC
significantly improved the AZT-induced acquisition deficit.
Finally, LAC treatment alone did not modify learning abilities.
During the probe trial (Fig. 2B) AZT subjects spent significantly
lower time than Control mice in the quadrant where the hidden
platform was located during the acquisition trials (t=22.05,
p,0.05), indicating that they were impaired in recalling the
former position of the platform. Notably, performances of
AZT+LAC mice in probe trial did not differ from those of
No treatment-induced difference was recorded in the swimming
speed of the different groups of mice (Fig. 2C).
LAC treatment protects from AZT-induced enhancement
of stress response
In basal conditions, no significant differences were found in
LAC=3.4560.65 mg/dl;AZT+LAC=3.3460.29 mg/dl).
dent’s t-test of plasma corticosterone stress response, calculated
as differences (delta) between restraint stress values and basal
values, revealed that mice in the AZT group had higher plasma
corticosterone concentration in respect to the Control group
(t=2.76, p,0.05). Of note, the AZT+LAC animals showed levels
of corticosterone similar to the control mice suggesting that the
prenatal treatment with LAC was able to protect the development
of hypothalamus pituitary adrenal (HPA) axis from the effects of
AZT (Fig. 3).
Reduced expression of hippocampal metabotropic and
ionotropic Glu receptors caused by AZT is counteracted
by LAC treatment
The AZT prenatal treatment caused a significant reduction of
mGlu1a receptor expression in respect to Control mice (p,0.05,
Student’s t-test, t=22.32; Fig. 4). Interestingly, the mGlu1a
receptors in AZT+LAC group showed a trend to increase
(although not statistical significant) in respect to AZT animals
and their expression did not differ from that found in the Control
group. Therefore, LAC treatment appeared to counteract the
AZT-induced reduction of mGlu1a receptor expression. This
phenomenon, although less evident, was also observed for mGlu5
receptor expression: the AZT group showed reduced receptor
expression compared to Control group (p,0.05, Student’s t-test,
t=23.17), while the AZT+LAC group showed a trend to
increase. For what concerns the mGlu2/3 receptor no differences
between the four experimental groups were found.
In Fig. 5 protein expression relative to ionotropic receptors is
shown. The analysis by Student’s t-test confirmed that AZT
prenatal treatment yielded a significant reduction of Glu1 subunit
of AMPA receptor expression in respect to Control mice (p,0.05,
Student’s t-test, t=23,43). Notably, the Glu1 subunit in
AZT+LAC group showed a significant increase (p,0.05, Stu-
dent’s t-test, t=22.59) in comparison with AZT mice and did not
differ from Controls suggesting that LAC treatment prevented the
reduction of receptor expression due to AZT. As for the AMPA
GluR2 and NMDA NR1 subunits, no differences between groups
Increased levels of lipid peroxidation and oxidative stress
caused by AZT are prevented by LAC treatment
Occurrence of free radical generation and oxidative stress was
monitored at birth and at PND60. On PND0, we tested the levels
of 15-F2t-IsoP, a reliable and sensitive marker of oxidative stress
, suitable for oxidative stress evaluation in small size samples.
Consistent with the oxidative stress hypothesis of AZT toxicity, 15-
F2t-IsoP levels in whole brain homogenates (Fig. 6A) showed a
trend to increase in samples from AZT prenatally-treated pups,
but not in those from LAC or LAC+AZT groups. Protein carbonyl
levels, which are considered a measure of protein oxidation and an
index of oxidative stress [53–55], were measured by OxyBlot
Table 1. Effect of transplacental exposure to AZT and LAC on
body weight recorded at different ages in male offspring.
Control AZTLAC AZT+ +LAC
PND2415.0160.81 14.4960.68 17.0460.77# 14.4360.51
AZT treatment affected body weight at birth irrespectively of LAC
administration, but this effect disappeared by PND 24. LAC treated mice was
generally heavier than Control group. (Fisher’s LSD post-hoc after one way
*p,0.05 vs Control;
#p,0.05 vs all other groups). Values are means 6 S.E.M (n=9–11 mice per
LAC Protects from AZT Neurobehavioral Effects
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analysis of homogenates prepared from hippocampi of mice
sacrificed at PND60, in parallel with glutamate receptor expres-
sion. In agreement with the tendency observed at PND0, a
significant increase in carbonyls was found in the hippocampal
homogenates from AZT prenatally-treated mice (45% of Control,
p,0.05, Student’s t-test, t=2,15; Fig. 6). Significant decreases of
protein carbonyls were detected in the hippocampus of mice
treated with LAC and with the combination of AZT+LAC, as
expected due to the antioxidant effect of LAC (p,0.05, Student’s
t-test, t=23,45 for LAC, t=25,07 for AZT+LAC; Fig. 6).
In the past years, reports of clinical signs suggestive of
mitochondrial dysfunction in non-infected children exposed to
NRTIs have prompted studies addressing the issue of develop-
mental toxicity of such drugs and, in particular, of AZT.
Occurrence of subclinical mitochondrial dysfunction has been
consistently shown by either clinical and experimental studies in
different tissues and organs, including the brain [11–14,36,56]. In
particular, a recent study provided evidence that in utero exposure
to the NRTI, AZT and Lamivudine, is associated with brain
mitochondrial impairment, which progresses over time and might
possibly end up in delayed neurobehavioral effects . In
laboratory rodents, developmental exposure to AZT produces
both early and delayed behavioral changes in offspring including
alteration in sensorimotor maturation, social/aggressive behavior,
responses to environmental stressors and learning abilities [19–30].
Altogether, these data pointed to the potential risk of sub-clinical
side effects of ARV therapy on the developing brain that may go
undetected in clinical and epidemiological studies. Thus, current
recommendations call for long-term clinical follow-up for any
child with in utero exposure to ARV drugs  to monitor
significant side effect of ARV drugs that may augment risk in
children and adolescents born to HIV-1-positive women, who
represent a vulnerable group as for psychiatric health [18,57–59].
The main results of the present preclinical study contribute to
clarify this controversial issue, as they show that 1) transplacental
exposure to AZT enhances oxidative stress and causes a consistent
and permanent alteration of hippocampal glutammatergic neuro-
transmission functionally associated to a deficit in spatial learning
and memory, 2) LAC administration from the beginning of
Figure 2. Effect of AZT and LAC treatments on spatial learning and memory assessed in the Morris Water Maze. A) Training
sessions: Latencies to reach the hidden platform throughout the four sessions of the task. The higher latencies shown by AZT-treated mice in
comparison to Control mice throughout the four sessions clearly indicate a learning impairment (* p,0.05, Fisher’s LSD post-hoc performed on
significant three-way ANOVA interaction), counteracted by LAC treatment, as the AZT+LAC group had latencies comparable to the Control group in
the 3rd and 4th session, and significantly different from those of the AZT group (# p,0.05). LAC treatment alone did not modify learning abilities. B)
Probe trial: AZT-treated mice spent less time than Control mice in the quadrant where the platform was located during training (* p,0.05 AZT vs
Control after t test), indicating a deficit in memory recall. AZT+LAC mice did not differ from Controls. C) No treatment-induced difference was
recorded in the swimming speed assessed in a trial with a visible platform. Values are expressed as means 6 S.E.M (n=7–8 mice per group).
LAC Protects from AZT Neurobehavioral Effects
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pregnancy prevents/ameliorates the adverse effects of AZT on
both neurochemical and behavioral parameters.
In line with previous findings in rodent models, we show that
transplacental AZT exposure has significant long-term effects on
behavior of exposed offspring. In particular, AZT treatment has
detrimental effects on performances in the water maze task, one of
the most widely used tests to measure hippocampal-dependent
spatial-based learning and memory in rodent models with high
translational value with respect to humans . We observed a
marked deficit in acquisition and memory recall, as both
acquisition of the platform location and retrieval of the acquired
information in the probe trial were impaired in AZT-treated mice.
Since swimming speed was not affected by AZT treatment, we can
exclude motor impairment possibly associated with the well-
known effects of this drug on muscle tissue .
Different neurotransmitter systems have been implicated in the
effects exerted by AZT and other ARV drugs on behavior but a
correlation between specific neural mechanisms and the behav-
ioral changes observed has not been attempted yet. Extensive
evidence indicates that learning and memory components of
spatial navigation in rodents rely on hippocampal synaptic
plasticity [62–64] that is largely modulated by hippocampal mGlu
[65,66] and by AMPA receptors . Our findings indicate that
hippocampal glutamate neurotransmission is permanently affected
by early AZT exposure and support a role of this important
neurochemical pathway in the behavioral effects here reported.
The reduced expression of metabotropic group I and ionotropic
Glu1 subunit of AMPA receptors in the hippocampus is well in
agreement with the specific impairment of spatial learning and
memory performances observed in AZT treated mice. Recent data
strongly support a role for the different glutamate receptor
subtypes in spatial cognition: administration to rats of mGluR1
antagonists affects synaptic plasticity and impairs acquisition in the
Morris water maze , while blockade of mGlu5 receptors
interferes with long-term potentiation and has detrimental effects
on spatial learning . As well as for metabotropic group I
receptors, there is ample evidence that the dynamic regulation of
AMPA receptors - which mediate most of the fast excitatory
synaptic transmission - can change synaptic function and regulate
storage of information [70,71]. Based on our behavioral and
neurochemical findings, we hypothesize that the lower expression
of different subgroups of glutamate receptors in AZT mice results
in reduced plasticity of hippocampal glutamatergic synapses and,
as an ultimate consequence, in diminished capacity to encode
relevant information during the spatial learning task.
Such effect could be further amplified by the enhanced
corticosterone responsiveness to stress in AZT exposed mice.
Our findings show that administration of AZT during pregnancy
caused increased release of plasma corticosterone following acute
restraint stress at adulthood. As water maze training induces
release of high corticosterone levels shortly after completion of the
task in mice , an abnormal physiological reaction to stress
could interfere with both acquisition and consolidation of
information in AZT mice. An inverse relationship between spatial
learning and memory and elevated corticosterone levels has been
widely demonstrated in rodents: exposure to mild stressor or
moderate glucocorticoid levels facilitates spatial memory [73–76]
while chronic stress or higher glucocorticoid levels exert
detrimental effects on neuronal plasticity and spatial memory
[77–79]. The increased corticosterone release after acute stress
observed in AZT treated mice deserves further consideration, as it
is suggestive of altered HPA axis response caused by early AZT
exposure. A recent study indicates that chronic AZT is able to
increase corticosterone release in adult rats possibly by a central
action . In a developmental perspective, AZT treatment could
interfere with the physiological setting of HPA axis activity, thus
reducing the capability to respond adaptively to environmental
challenges. Previous studies from our laboratories indicate that
mice exposed to AZT during development present abnormal
reaction to novelty, and that pre- and/or postnatally AZT exposed
adult male mice show maladaptive intraspecific social/aggressive
behavior depending on the length of exposure [23,24].
Overall, our findings confirm the behavioral effects of transpla-
cental AZT in rodent models. More importantly, they point for the
first time to a specific and permanent action of this drug on
glutamate neurotransmission. We propose that such effect is linked
to the well-knownmitochondrial
[11,14,31,32,34,81–83]. NRTI-induced alterations in mitochon-
drial structure and function result from interference with various
mechanisms involved in the normal maintenance of mitochondrial
function. In particular, it has been suggested that AZT directly
impairs the electron transport chain (ETC) thus increasing
production of reactive oxygen species (ROS) and oxidative stress,
which will eventually lead to a loss of the mtDNA integrity .
Our data confirm that transplacental exposure to AZT induces
oxidative stress in the fetal brain and determines long-term effects
on the oxidative status of brain tissue as two different oxidative
stress markers, 15-F2t-isoP and protein carbonyl levels at birth and
at adulthood, respectively, were enhanced in AZT-treated
F2- Isoprostanes are lipid peroxidation products; they are
among the most sensitive in vivo biomarkers of oxidative stress and
are the marker of choice for the evaluation of oxidative damage in
small size samples such as newborn mouse brain . Similarly,
immunodetection of carbonyl groups provides a measure of
oxidation status of proteins, and in turn, of oxidant injury. The
increased levels of 15-F2t-isoP and protein oxidation are indicative
of increased levels of ROS, likely deriving from AZT-dependent
Figure 3. Effect of AZT and LAC treatments on plasma
corticosterone secretion measured after 15-min restraint
stress. In basal conditions, no significant differences were observed
in plasma corticosterone levels between groups (see text for details),
while following acute stress AZT-treated mice had markedly enhanced
corticosterone release in comparison to Control mice (* p,0.05 AZT vs
Control, Student’s t test). LAC administration protected from AZT-
induced increase of corticosterone. Corticosterone levels were calcu-
lated as delta between restraint stress values and basal values. Values
are expressed as means 6 S.E.M. (n=7 mice per group).
LAC Protects from AZT Neurobehavioral Effects
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mitochondrial impairment, as described in several previous studies
The evidence of early oxidative stress in AZT exposed offspring
allows us to advance a mechanistic explanation for the endocrine
and neurobehavioral effects here reported. Impairment of
mitochondrial function and production of increased levels of
ROS may reduce the capacity of the cell to generate ATP, and
induce excess glutamate release at the synapse and, eventually,
excitotoxicity . In turn, an excess of glutamate release in the
early stage of life results in overstimulation of glutamatergic
receptors that can be possibly permanently modified in their
expression and functionality. Besides, it should be considered that
a significant portion of the control of HPA axis activity is mediated
by glutamate [85–88] thus enhanced glutamate release during
Figure 4. Effect of AZT and LAC treatments on hippocampal mGlu receptors expression. (A) Representatives of immunoblots with
mGlu1a and mGlu5 receptor antibodies showed a 142 kDa and 130 kDa bands, corresponding to receptor monomers, respectively. Blots with
antibodies recognize an epitope common to mGlu2 receptors monomer(s) (100 kDa) and mGlu3 receptors dimers (206 kDa). (B) Results are
expressed as the ratio of the optical density (OD) of the mGluR1a, mGluR5 or mGlu2/3 band and the b-actin band. The AZT prenatal treatment caused
a significant reduction of mGlu1a and mGlu5 receptors expression in respect to Control mice (* p,0.05, Student’s t-test) counteracted by LAC
treatment as AZT+LAC mice differ from Control. No differences between groups were found in the mGluR2/3 (summary of OD monomers and
dimers). Values are expressed as means 6 S.E.M. (n=6 mice per group).
LAC Protects from AZT Neurobehavioral Effects
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early development could result in lifelong alteration of HPA axis
responsiveness. The link between AZT-induced oxidative stress in
the fetal stage and the long-term consequences on neurobehavioral
functions at the adult stage is strongly supported by the protective
effect exerted by LAC. Indeed, we show that most of the
alterations induced by AZT are prevented or reduced by
administration of LAC from the beginning of pregnancy.
Several lines of evidence indicate that administration of
antioxidant agents, including vitamin C, E and coenzyme Q10
prevents or alleviates oxidative stress and symptoms of AZT-
induced myopathy in both rats and humans (see 61 for a review).
More relevant to our study, LAC is known to decrease oxidative
stress by 1) exerting a protective effect on mitochondrial structure
and function, making the ETC less prone for electron leak and
superoxide production, and promoting mitochondrial biogenesis,
as indicated by increased mitochondrial size and number ; 2)
stimulating the endogenous cellular antioxidant defense mecha-
nisms. These effects possibly rely on the ability of LAC to act as a
donor of acetyl groups. In a recent work, a proteomic survey of
protein acetylation identified that 20% of mitochondrial proteins
presented with acetylation sites and these proteins included those
involved in the tricarboxylic acid cycle, fatty acid b-oxidation,
Figure 5. Effect of AZT and LAC treatments on hippocampal AMPA and NMDA receptors expression. (A) Representatives of
immunoblots with GluR1 and GluR2 receptor antibodies showed a 110 kDa and 100 kDa bands corresponding to AMPA receptor subunits,
respectively. Blot with NR1 receptor antibody showed a 130 kDa band, corresponding to NMDA receptor subunit. (B) Results are expressed as the
ratio of the optical density (OD) of the GluR1, GluR2 and NR1 band and the b-actin band. AZT prenatal treatment caused a significant reduction of the
GluR1 subunit of AMPA receptors expression in respect to Control mice (* p,0.05 AZT vs Control, Student’s t-test) that was fully prevented by LAC
treatment: in AZT+LAC mice the Glu1 subunit was increased in respect to AZT mice (# p,0.05 vs AZT, Student’s t-test) and did not differ from
Controls. Expression of the GluR2 subunit of AMPA and NR1 subunit of NMDA receptors was not affected by either treatment. Values are expressed as
means 6 S.E.M. (n=6 mice per group).
LAC Protects from AZT Neurobehavioral Effects
PLOS ONE | www.plosone.org9 February 2013 | Volume 8 | Issue 2 | e55753
amino acid and carbohydrate metabolism, membrane transport,
and ETC . These data clearly show that acetylation can
control the activity of mitochondrial enzymes and, since acetyl-
CoA is the acetylation donor for all known acetyltransferases, the
concentration of mitochondrial acetyl-CoA could be a limiting
factor in the acetylation reaction. By increasing the acetyl-CoA
content, supplemented LAC should increase the acetylation status
of mitochondrial proteins thus improving mitochondrial function
and integrity. Interestingly, low levels of circulating LAC have
been found in HIV-1 patients experiencing a clinically manifest
peripheral neuropathy while staying on AZT and other nucleoside
analogue treatment , and in patients with AZT-induced
mtDNA depletion . Hence, LAC deficiency could play a role
in neurotoxic effects of these drugs. AZT treatment reduces
carnitine levels in blood and tissues [5,6] and LAC treatment
could mediate an improvement of mitochondrial dysfunction in a
straightforward manner, namely, by replenishing carnitine levels.
Other clinical studies have shown that treatment with LAC
improved NRTI-induced polyneuropathy symptoms in HIV-1-
positive patients, including electrophysiological variables relating
to motor conduction velocity . In a morphological study in
HIV-1 patients with distal symmetrical polyneuropathy, regener-
ation of all fiber types and in particular of small sensory fibers in
the dermis, epidermis and sweat glands was observed after oral
LAC treatment . All this considered, LAC can be viewed as a
pleiotropic molecule metabolically active, with neuromodulatory,
neurotrophic, cytoprotective, and antioxidant activity in brain.
The use of ARV drugs to prevent mother-to-child transmission
of HIV-1 infection is one of the most successful achievements in
HIV-1 prevention and, based on several clinical and epidemio-
logical studies, benefits of this therapy appear to outweigh its
adverse side effects in exposed infant/children [2,95]. However,
there is still considerable uncertainty on the prevalence and
severity of mitochondrial toxicity associated to ARV treatment in
pregnancy . In this framework, research on therapeutic
intervention effective in reducing side effect risks of this life-saving
regimen should be implemented. The present preclinical findings,
besides providing a mechanistic hypothesis for the neurobehav-
ioral effects of AZT, strongly suggest that preventive administra-
tion of LAC already shown to improve NRTI-induced polyneu-
ropathy in HIV-1-positive patients [45–48] might also be effective
in reducing the neurological side-effects of ARV therapy in fetus/
It is known that an excessive production of pro-inflammatory
cytokines and free radicals might occur during HIV infection.
Because pro-inflammatory cytokines and free radicals can travel
through placenta to the fetus, they could contribute to increase the
fetal oxidative stress induced by NRTI drugs. Nonetheless, our
results highlight the importance of counteracting the neurotoxic
effects of NRTIs in terms of rescue of neuronal function based on
the maintenance of mitochondrial activity. Since LAC treatment
has generally shown a good profile of safety and tolerability, our
study strongly supports the rationale for the development of
clinical trials aimed at investigating the efficacy of the adminis-
tration of LAC to HIV-1 infected pregnant women treated with
NRTIs to counteract potential adverse consequences on their
The authors wish to express their gratitude to Dr. Assia Catalani for kindly
advice relating to this research and to Dr. Flavia Chiarotti for her statistical
Conceived and designed the experiments: GC PC AVP. Performed the
experiments: ARZ CG AVP AT MAAC CC AG GP. Analyzed the data:
ARZ CG AVP GSA LM RN GC PC. Wrote the paper: GC PC LM RN.
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