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Advances in the Development of Novel Antioxidant Therapies
as an Approach for Fetal Alcohol Syndrome Prevention
Xavier Joya
1,2
, Oscar Garcia-Algar*
1,2,3
, Judith Salat-Batlle
1,2
, Cristina Pujades
4
,
and Oriol Vall
1,2,3
Ethanol is the most common human teratogen, and its consumption during
pregnancy can produce a wide range of abnormalities in infants known as
fetal alcohol spectrum disorder (FASD). The major characteristics of FASD
can be divided into: (i) growth retardation, (ii) craniofacial abnormalities, and
(iii) central nervous system (CNS) dysfunction. FASD is the most common
cause of nongenetic mental retardation in Western countries. Although the
underlying molecular mechanisms of ethanol neurotoxicity are not completely
determined, the induction of oxidative stress is believed to be one central
process linked to the development of the disease. Currently, there is no
known effective strategy for prevention (other than alcohol avoidance) or
treatment. In the present review we will provide the state of art in the
evidence for the use of antioxidants as a potential therapeutic strategy for the
treatment using whole-embryo and culture cells models of FASD. We
conclude that the imbalance of the intracellular redox state contributes to the
pathogenesis observed in FASD models, and we suggest that antioxidant
therapy can be considered a new efficient strategy to mitigate the effects of
prenatal ethanol exposure.
Birth Defects Research (Part A) 00:000–000, 2014.
V
C2014 Wiley Periodicals, Inc.
Key words: fetal alcohol syndrome; neuroprotective; treatment; oxidative stress;
teratogenesis
Introduction
Alcohol is known to be a teratogen and its consumption
during pregnancy can produce a wide range of adverse
effects in the developing fetus. The severity of fetal dam-
age due to ethanol exposure depends on several factors
which include the timing, pattern, and dose of consump-
tion (Abel and Hannigan, 1995). Maternal ethanol con-
sumption can develop a spectrum of physical, cognitive,
and behavioral disabilities in newborns known as fetal
alcohol spectrum disorder (FASD). The most severe form,
that includes morphological abnormalities is defined as
fetal alcohol syndrome (FAS) (de Sanctis et al., 2011; Joya
et al., 2012; Memo et al., 2013). The classical dysmorphic
facial features of FAS include microcephaly, a rather flat
midface with short palpebral fissures, low nasal bridge
with short nose and long smooth or flat phylum with a
narrow vermilion of the upper lip (de Sanctis et al., 2011).
This disease is also characterized by failure to thrive, that
starts in the prenatal age and persists postnatally, and by
neurocognitive defects (Memo et al., 2013). Using these
criteria, the diagnosis of FAS missed many individuals
without phenotipical diagnostic clues. The term FASD was
not intended to be used as a clinical diagnosis, but an
umbrella containing diagnoses as FAS, partial FAS and
alcohol related neurodevelopmental disorders (ARND).
FASD includes the range of individuals who have from the
full syndrome to only a few issues about learning and
behavior, and no facial or growth signs (May et al., 2010).
Currently, in Europe there are no systematic data on FAS
and FASD prevalence, nor on prenatal exposure to ethanol.
In Canada, the prevalence of FAS and FASD has been
reported to be 1 to 3 and 9 per 1000 live births, respec-
tively, higher than the FAS prevalence observed in the
United States (0.5–2.0 per 1000 live births (Goh et al.,
2008).
It is well known that in adults, ethanol-induced dam-
age is mediated by induction of oxidative stress and its
plays a major role in different mechanisms such in the
case of liver injury (Dey and Cederbaum, 2006). Similarly,
prenatal ethanol exposure has been shown to cause an
increase in oxidative stress in developing organs, including
the brain (Reyes et al., 1993; Heaton et al., 2003). Even a
brief exposure to ethanol, the fetal brain alters its redox
balance (Dong et al., 2010). On the other hand, it is gener-
ally admitted that antioxidants treatment cause the oppo-
site effect (Busby et al., 2002; Neese et al., 2004).
The brain is the principal target tissue of prenatal
ethanol exposure and it possesses the highest oxygen
1
Unitat de Recerca Inf
ancia i Entorn (URIE), Institut Hospital del Mar d’Inves-
tigacions Me
`diques (IMIM), Barcelona, Spain
2
Red de Salud Materno-Infantil y del Desarrollo (SAMID), Programa RETICS,
Instituto Carlos III, Madrid, Spain
3
Departament de Pediatria, Ginecologia i Obstetricia i de Medicina Preventiva,
Universitat Aut
onoma de Barcelona (UAB), Bellaterra, Spain
4
Department of Experimental and Health Sciences, Universitat Pompeu Fabra
(UPF), Parc de Recerca Biomedica de Barcelona, Barcelona, Spain.
This study was supported by Grants from Fondo de Investigaciones Sanitarias
(FIS) (PI10/02593; PI13/01135), from the Instituto Carlos III (Madrid, Spain),
RecerCaixa (OG085818) and Red de Salud Materno-Infantil y del Desarrollo
(SAMID) (RD12/0026/0003) from the Instituto Carlos III (Spain), intramural
funding of the Neuroscience Program at IMIM (Institut Hospital del Mar
d’Investigacions Me
`diques) and partially supported by Generalitat de Catalu-
nya (Spain) AGAUR (2009SGR1388)
*Correspondence to: Oscar Garcia Algar, Unitat de Recerca Inf
ancia i Entorn
(URIE), Institut Hospital del Mar d’Investigacions Me
`diques (IMIM), Parc de
Recerca Biome
`dica de Barcelona (PRBB), C/ Dr. Aiguader 88, 08003, Barce-
lona, Spain. E-mail: 90458@parcdesalutmar.cat
Published online 0 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
Doi: 10.1002/bdra.23290
V
C2014 Wiley Periodicals, Inc.
metabolic rate in the body because its cells use 20% of
the total oxygen consumed by the organism (Sokoloff,
1999). For this reason, it presents the highest quantity of
reactive oxygen species (ROS) production during oxidative
metabolism. Moreover, the production of ROS can be
increased by the presence of high content of unsaturated
fatty acids that can be substrates for ROS production. Fur-
thermore, the antioxidant defense system of the brain is
limited with respect to other organs. Particularly, the activ-
ities of antioxidant enzymes such superoxide dismutase
(SOD), catalase, and/or glutathione peroxidase (GPx) are
lower (Floyd and Carney, 1992). For all of these reasons
mentioned above, the neural fetal cells are more vulnera-
ble to neurotoxic effects of oxidative stress than the adult
brain cells, because the levels of antioxidant enzymes and
nonenzymatic endogenous antioxidants in the developing
fetus are lower compared with adults (Bergamini et al.,
2004).
Oxidative stress is a general term used to describe an
imbalance between the systemic manifestation of ROS, and
a biological system’s ability to readily detoxify these reac-
tive intermediates or to repair the resulting damage. The
main intracellular source of ROS is the oxidative phospho-
rylation generated by the mitochondria. Moreover, other
enzymes such xanthine oxidase and NADPH oxidases
(NOX/XOX) can produce ROS in the cytoplasm. NOX is a
multi-subunit enzyme complex that is activated and
induced by inflammatory signals (Infanger et al., 2006).
This inflammatory signals include several external sources
such UV light, chemical reagents, cigarette smoke, drugs
and/or ethanol consumption (Zadak et al., 2009). Activa-
tion of glial cells, especially microglia, that release of pro-
inflammatory factors (TNFa,IL-1b, IL-6, etc.) and ROS
have been implicated in several models of neurodegenera-
tion (Lucas et al., 2006; Block and Hong, 2007). ROS react
with cellular molecules including proteins, lipids and DNA
causing genetic alterations (Finkel and Holbrook, 2000)
and finally culminate in cell death (activation of apoptosis
cascades). In humans, oxidative stress is a pathogenic
mechanism involved in the development of cancer (Halli-
well, 2007) Parkinson’s disease (Valko et al., 2007) fragile
X syndrome (de Diego-Otero et al., 2009) or autism (James
et al., 2004).
The organism presents a variety of defense mecha-
nisms that can be referred to as the endogenous antioxi-
dant system (Halliwell and Gutteridge, 1995). Endogenous
antioxidants can inhibit the ROS formation or promote the
free radicals scavenging. These endogenous antioxidants
can be broadly divided into: nonenzymatic and enzymatic
origin. Endogenous nonenzymatic antioxidants include thi-
ols and glutathione (GSH) (Halliwell, 2006). On the other
hand, enzymatic antioxidants include: (1) SOD, (2) cata-
lase, (3) the glutathione system, which encompasses the
enzymes: (3.1) glutathione reductase (GR) and uses GSH
and NADPH as co-factors. (3.2.) glutathione peroxidase
(GPx) reduces hydrogen peroxide and other organic perox-
ides at the expense of GSH, which is in turn oxidized to
form glutathione disulfide (GSSG) and (3.3.) glutathione
S-transferases (GSTs) that catalyze the conjugation of the
reduced form of GSH to xenobiotic substrates for the pur-
pose of detoxification. In the brain, most GST is located in
glial cells (which are also rich in GSH), helping protect
neuronal populations that have a low content of this co-
factor (Astor et al., 1988; Hayes and Strange, 1995; Salinas
and Wong, 1999). Given its extensive functions list, GSH is
probably the most important endogenous nonenzymatic
antioxidant.
It is well known that alcohol produces high levels of
ROS production through its metabolism (Fig. 1). Ethanol is
metabolized to acetaldehyde by the alcohol dehydrogenase
(ADH) in the liver. Alternatively, ethanol can also be
metabolized by cytochrome P450 2E1 present in the liver
and brain. Of interest, the reaction catalyzed by cyto-
chrome P450 2E1 leads to an increase in the generation of
acetaldehyde and hydroxyl radicals in both tissues. Acetal-
dehyde can then be further oxidized into acetate by the
enzyme acetaldehyde dehydrogenase, these reaction
results in an increase in the activity of respiratory chain
and consequently produces ROS.
An increasing body of evidence has postulated the role
of oxidative stress in FASD evidenced by ROS production
in animals and in vitro models (Heaton et al., 2002, 2003;
Smith et al., 2005; Kane et al., 2008; Dong et al., 2010).
These evidences have been related to the damage on: (1)
lipid peroxidation (Henderson et al., 1995; Chen et al.,
1997; Perez et al., 2006), (2) protein peroxidation (Marino
et al., 2004; Shirpoor et al., 2009), and (3) DNA (Chu
et al., 2007; Dong et al., 2010).
The principal difficulty in this scenario is the heteroge-
neous experimental designs available in the literature.
Parameters such: methodology, ethanol exposure period,
peak of blood alcohol concentration (BAC) reached and
the time of analysis, as well as tissues analyzed and
FIGURE 1. Ethanol metabolism and induction of oxidative stress. Main molecu-
lar mechanisms which ethanol causes oxidative stress. ADH, alcohol dehydro-
genase; ALDH, aldehyde dehydrogenase; NAD, nicotinamide adenine
dinucleotide; NADH, reduced form of NAD; ROS, reactive oxygen species.
2 ANTIOXIDANT THERAPIES FOR FAS PREVENTION
markers evaluated, are not standardized due to make com-
parisons between experiments is impossible. Given this
scenario, the present review is focused on the most recent
findings in novel antioxidant therapeutic approach for the
mitigation of prenatal alcohol exposure effects.
Antioxidant Supplementation as a Therapeutic
Intervention for FASD Prevention
Despite the role of oxidative stress in FASD is also con-
firmed by numerous studies showing the beneficial
impact of antioxidant therapy upon prenatal ethanol
exposure effects, there are a great number of experimen-
tal variables (including the mode and period of ethanol
exposure, the antioxidant selected, the time of adminis-
tration and the age of animals at the time of analysis as
well as the neuropathological parameters evaluated). This
fact complicates direct comparison among the studies
published. Thus, to facilitate the following discussion, the
experimental parameters and the major findings in a
wide range of literature studies are summarized in
Tab l e 1 .
EFFECT OF ANTIOXIDANT SUPPLEMENTATION ON THE AMELIORATION
OF BIRTH DEFECTS
The defects associated with FASD are variable and lie
along a continuum spectrum going from the most severe
form, represented by deficiencies in brain growth (reduced
head circumference and/or structural brain anomaly) to
distinct facial features (microcephaly, short palpebral fis-
sures, thin upper lip and/or smooth philtrum) (Jones and
Smith, 1973). Related with this, some in vivo studies have
indicated that antioxidant treatments can prevent or
reduce growth retardation and/or the occurrence of mal-
formations as a consequence of ethanol exposure during
development. Using Xenopus laevis co-treated with vitamin
C, Peng et al. showed a decrease in microencephaly inci-
dence and growth retardation (Peng et al., 2005). Further-
more, Chen et al. administered ethanol to pregnant mice
dams in combination with EUK-134, a synthetic
manganese-porphyrin complexe similar to SOD and cata-
lase. The co-treatment with EUK-134 reduced the inci-
dence of forelimb malformations in the offspring pups
(Chen et al., 2004). The treatment with vitamin E in preg-
nant mice dams treated with ethanol normalized fetal
development (Wentzel et al., 2006). Similarly, black gin-
seng (Panax ginseng) improved most of the morphological
scores in mice embryos (Lee et al., 2009). Another birth
outcome commonly seen in children exposed prenatally to
ethanol are congenital heart defects (Karunamuni et al.,
2014). Using zebrafish embryos as animal model Reimers
et al. evaluated the effect of lipoic acid, vitamin E and Tro-
lox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid, a powerful free radical scavenger). These antioxi-
dants partially attenuated the pericardial edema incidence
(Reimers et al., 2006).
EFFECT OF ANTIOXIDANT SUPPLEMENTATION ON THE
NEUROANATOMICAL ARCHITECTURE
In several cases, antioxidant treatment was also shown to
have a positive impact at the neuroanatomical level. In
this sense, embryos prenatally exposed to ethanol and
treated with vitamin E attenuated the reduction in the
number of Purkinje cells in the lobule I of the cerebellum
(Heaton et al., 2000a). Similar results were obtained by
Lee et al. using black ginseng as a therapeutic approach.
Embryos co-treated with ethanol and black ginseng
showed similar head length compared with the control
group (including fore-, mid-, and hindbrain) (Lee et al.,
2009). Using a Guinean pig model treated with a combina-
tion of high doses of vitamin C and E, protects against the
loss of hippocampal weight (Nash et al., 2007). In accord-
ance with these results, Marino et al. observed an amino-
ration of hippocampal neuronal loss (Marino et al., 2004).
Finally, co-treatment of ethanol-exposed pregnant dams
with silymarin showed to be useful to prevent the
ethanol-induced impairment in corpus callosum develop-
ment (Moreland et al., 2002). On the other hand, not all
the studies reported beneficial results. U83836E, a new
vitamin E derivate generated by Upjohn Company (Kala-
mazoo, MI), did not attenuate neonatal alcohol-induced
microencephaly or Purkinje cell loss in lobule I (Grisel and
Chen, 2005). In fact, it has been reported that the protec-
tive efficacy of U83836E may be dose related, and high
doses of the drug can be cytotoxic (Mertsch et al., 1998).
Anthocyanins, a large subgroup of flavonoids present in
many vegetables and fruits, are safe and potent antioxi-
dants that can cross the blood–brain barrier and be dis-
tributed in the CNS (Passamonti et al., 2005). An example
of anthocyanins is cyanidin-3-glucoside (C3G) obtained
from blackberries. C3G has been demonstrated that
presents a potent antioxidant and anti-tumor capacity
(Ding et al., 2006). Promising results provided by Chen
et al. showed that C3G can ameliorate ethanol-induced
neuronal death blocking GSK3bactivation (Ke et al.,
2011).
The role of antioxidants treatment in relation to behav-
ioral deficits is inconclusive and not all experimental or
clinical studies find beneficial effects. In some cases, the
neuroprotection conferred by antioxidant therapy was
translated into an improvement of the behavioral deficits
and learning abnormalities associated with perinatal etha-
nol exposure (Busby et al., 2002; Vink et al., 2005; Miller
et al., 2013). For example, Busby et al. co-administered
silymarin and ethanol throughout gestation and detected
that silymarin improve several behavioral deficits in the
rat adult offspring (Busby et al., 2002). The co-treatment
with vitamin C and E in pregnant Guinean pigs mitigated
the ethanol-induced deficit in the task-retention compo-
nent of the water-maze activity. However, other study,
using the same vitamin regimen did not mitigate the
ethanol-induced impairment in hippocampal long-term
BIRTH DEFECTS RESEARCH (PART A) 00:00–00 (2014) 3
TABLE 1. Summary of the principal characteristics of novel targets with antioxidant activity for the prevention of FASD
Natural antioxidants
Drug
Experimental design /
Animals used
Range of drug concen-
trations
used / Administration /
Time
Range of EtOH concen-
trations / Administra-
tion / Time Observations Reference
In vivo experiment.
Guinea pigs.
250 mg/kg / PO / GD2
– GD67
9 g/kg/day / PO / GD2
– GD67
- Reduces lipid peroxidation in the liver.
-Enhances the activities of
glutathione peroxidase and reductase.
-Reduces the activity of GGT.
Suresh et al., 1999
In vivo experiment.
Xenopus laevis
0.1 mM / 2 h before
the EtOH treatment
0 – 2% (v/v) / Stage 13
to 22
- Inhibits ROS production.
- Activates NF-kB pathway.
-Prevents microencephaly.
Peng et al., 2005
Vitamin C In vivo experiment.
Guinea pigs
250 mg/day / PO / GD2
– GD67
4 g/kg/day / PO / GD2
– GD67
- Protects hippocampal weight versus brain
weight.
-Does not mitigate impairment of hippocampal
long-term potentiation.
Nash et al., 2007
In vitro experiment.Primary hippocam-
pal neuronal cells from Sprague–
Dawley rats at GD17.5
1 mM / 24 h 100 mM / 24 h - Decreases expression of Bax, caspase-9, cas-
pase-3, cytochrome-c
- Increases expression of Bcl-2
Naseer et al., 2011
EGCG In vitro experiment.Primary fetal rhom-
boencephalic neurons from Sprague-
Dawley rats at GD14.
0.001 mM / 24 h 75 mM / 24 h - Decreases the number of apoptotic cells Antonio and Druse,
2008
In vivo experiment.C57BL/6J mice 200 - 400 mg/kg/day /
PO / GD7 - 8
0.005 20.02 ml/g /
IP / GD8
- Normalizes head sizes.
-Normalizes Otx1 and Sox2 expression levels.
-Decreases H2O2 and MDA.
Long et al., 2010
Ginkgolide B In vitro experiment.PC12 cells 0.001 – 0.050 mM / 4h
before EtOH
treatment
100 mM / 24 h - Decreases caspase-3.
- Decreases ROS production.
-Does not affect ethanol-induced ADH and
CYP2E1 activities.
Zhang et al., 2011
In vitro experiment. Primary fetal rhom-
boencephalic neurons from Sprague-
Dawley rats at GD14.
0.01 mM / 24 h 75 mM / 24 h - Decreases the number of apoptotic cells Antonio and Druse,
2008
4 ANTIOXIDANT THERAPIES FOR FAS PREVENTION
TABLE 1. Continued
Natural antioxidants
Drug
Experimental design /
Animals used
Range of drug concen-
trations
used / Administration /
Time
Range of EtOH concen-
trations / Administra-
tion / Time Observations Reference
Resveratrol In vitro experiment. Primary CGNs from
Long-Evans rats at PD7
2 – 100 mg/kg / 1
– 24 h before EtOH
treatment
BAC: 80 mM / 5 h - Decreases ROS levels.
-Restores the expression levels of Nrf2 in the
nucleus.
-Retains the expression and activity of NADPH
quinine oxidoreductase 1 and SOD.
Kumar et al., 2011
In vitro experiment. Primary DRG
neurons from Wistar rats at GD15.
0.0001 – 0.030 mM /
24 h
325.6 mM / 24 h - Increases the number of extended nerve fibers
and neurons that migrated from the DRG
explants.
-Inhibits EtOH-induced apoptosis.
-Recovers SOD and GSH expression.
Yuan et al., 2013b
In vitro experiment.Primary SCs from
Wistar rats at PD3.
30 mM / 96 h 1500 mg/dl / 96 h - Recovers cell viability.
-Increases the BDNF and GDNF expression.
Yuan et al., 2013a
Curcumin In vitro experiment.Primary fetal
rhomboencephalic neurons from
Sprague–Dawley rats at GD14.
0.001 mM / 24 h 75 mM / 24 h - Decreases the number of apoptotic cells. Antonio and Druse,
2008
In vivo experiment.Wistar rats PD5 30 – 60 mg/kg / PO /
PD6 – 28
5 g/kg / PO / PD7 – 9 - Ameliorates neuroinflamation (oxidative nitro-
sative stress, TNF-a, IL-1b, and TGF-b1).
-Decreases neuronal apoptosis (NF-jb and cas-
pase 3) in both cerebral cortex and
hippocampus.
Tiwari and Chopra,
2012
Melatonin In vivo experiment.Sprague-Dawley rats 20 mg/kg/day / PD4 – 9 6 g/kg/day / PD4 – 9 - Does not decrease the apoptotic Purkinje cell
number.
Grisel and Chen,
2005
In vitro experiment.Primary
rhomboencephalic neurons from
Sprague–Dawley rats at GD14.
0.001 mM / 24 h 75 mM / 24 h - Decreases the number of apoptotic cells. Antonio and Druse,
2008
Thymoquinone In vitro experiment.Primary cortical neu-
rons from Sprague-Dawley rats at
GD17.5
0.01 – 0.035 mM /
12 h
100 mM / 12 h - Inhibits apoptotic events (increasing Bcl-2
expression)- Reduces the cleavage of PARP-1.
Ullah et al., 2012
BIRTH DEFECTS RESEARCH (PART A) 00:00–00 (2014) 5
TABLE 1. Continued
Natural antioxidants
Drug
Experimental design /
Animals used
Range of drug concen-
trations
used / Administration /
Time
Range of EtOH concen-
trations / Administra-
tion / Time Observations Reference
Sulforaphane In vitro experiment.NCC (JoMa1.3 cells) 2.5310
24
–4310
23
mM/24h–72h
50 – 200 mM /
24 – 72 h
- Increases Nrf2 activation and activates the
downstream expression of endogenous
antioxidants.
Chen et al., 2013
Capsaicin In vitro experiment.Whole embryo cul-
ture of Sprague–Dawley rats at
GD8.5
10
28
–10
27
lg/ml / 17 h 1 lg/ml / 17 h - Recovers SOD and GSH activity. Kim et al., 2008
Black ginseng In vitro experiment.Culture ICR rat
embryo from GD8.5
0 - 100 mg/ml / 48 h 0.017 mM / 48 h - Normalizes morphological scores (including
head length, fore-, mid- and hindbrain).
Lee et al., 2009
In vitro experiment.Primary embryonic
hippocampus from Long-Evans rats
at GD18
0.05 mM / 2 h or 16 h 0 – 346.6 mM /
2 h or 16 h
- Protects neuronal viability of embryonic hippo-
campal cultures against ethanol.
Mitchell et al.,
1999a,b
In vivo experiment.Long-Evans rats. 30 - 60 IU/100 ml /
IG / PD4 – 5
12% / IG / PD4 – 5 - Prevents the loss of Purkinje cells. Heaton et al.,
2000a,b
In vitro experiment.Primary CGN from
Long-Evans rats at PD8.
0.050 mM / 24 h 86.7 – 346.6 mM / 24 h - Restores the expression of NTFs (BDNF and
neurotrophin-3).
-Diminishes the cellular disturbances in oxida-
tive processes.
Heaton et al., 2004
In vivo experiment.Long-Evans rats 2000 g/kg / PD6 5.25 g/kg / PD7 – 9 - Alleviates the increase in protein carbonyls.
-Does not improve spatial learning in the
ethanol-exposed animals.
Marino et al., 2004
In vivo experiment.Long-Evans rats. 12.26 mg/kg/day /
PD4 – 9
2.625 g/kg/day /
PD4 – 9
- Fails to protect against reduction of cerebellar
Purkinje cells.
Tran et al., 2005
In vitro experiment.Primary CGN from
Long-Evans rats at PD9.
0.050 mM / 24 h 86.7 – 346.6 mM /
24 h
- Amplifies the g-GCS and total GSH protein
expression levels.
Siler-Marsiglio et al.,
2005
Vitamin E In vivo experiment. Zebrafish embryos. Data not shown /
3 – 24hpf
200 mM / 3 – 24 hpf - Attenuates the incidence of pericardial edema.
-Does not provide protection against cell death.
Reimers et al., 2006
6 ANTIOXIDANT THERAPIES FOR FAS PREVENTION
TABLE 1. Continued
Natural antioxidants
Drug
Experimental design /
Animals used
Range of drug concen-
trations
used / Administration /
Time
Range of EtOH concen-
trations / Administra-
tion / Time Observations Reference
In vivo experiment. Sprague–Dawley
rats
5% / GD1 – 20 20% / GD1 – 20 - Does not affect BAC.
-Normalizes fetal development.
-Normalizes fetal hepatic levels of 8-iso-
PGF2a.
Wentzel et al., 2006
In vivo experiment. Guinea pigs. 100 – 250 mg/day / PO
/ GD2 – 67
4 g/kg/day / PO / GD2
–67
- Protects hippocampal weight relative to brain
weight.
-Does not mitigate the EtOH-induced impair-
ment of hippocampal long-term potentiation.
Nash et al., 2007
In vivo experiment. Wistar rats. 300 mg/day / SC / GD7
– PD21
4.5 g/kg/day / SC / GD7
– PD21
- Decreases DNA damage.
-Restores the elevated level of Hcy to control
levels.
Shirpoor et al., 2009
In vitro experiment. Primary CGNs from
rats at PD8.
0.050 mM / 24 h 86.6 mM / 24 h - Reduces Bax translocation.
-Decreases ROS production.
Heaton et al., 2011
In vitro experiment. Chicken embryos 50 mM 15 – 50% - Diminished mortality and growth retardation. Satiroglu-Tufan and
Tufan, 2004
Silymarin In vivo experiment. Fisher/344 rats 400 mg/kg / OA BAC: 6.7% / OA - Amelioration of the effects upon the develop-
ing fetal rat brain.
Moreland et al., 2002
C3G In vitro experiment. Neuro2a cells 5mM 87 mM - Restores the neurite outgrowt h Chen et al., 2009
In vivo experiment. C57BL/6 mice 10 – 30 mg/kg / IP 2.5 g/kg / SCPD7 - Reduces EtOH-meditated caspase-3 activation
in the cerebral cortex blocking GSK3b
Ke et al., 2011
Synthetic antioxidants
U83836E In vivo experiment. Sprague-Dawley rats 20 mg/kg/day / PD4 – 9 6 g/kg/day / PD4 – 9 - Melatonin does not decrease the Purkinje cell
number.
-Does not change BAC measured on PD 6.
Grisel and Chen,
2005
Trolox In vivo experiment. Zebrafish embryos. Data not shown / 3 –
24hpf
200 mM / 3 – 24hpf - Attenuated the incidence of pericardial
edema.
-Does not provided protection against cell
death.
Reimers et al., 2006
BIRTH DEFECTS RESEARCH (PART A) 00:00–00 (2014) 7
TABLE 1. Continued
Natural antioxidants
Drug
Experimental design /
Animals used
Range of drug concen-
trations
used / Administration /
Time
Range of EtOH concen-
trations / Administra-
tion / Time Observations Reference
In vivo experiment. Zebrafish embryos. 0.1 mM / 3 – 24hpf 200 mM / 3 – 24 hpf - Attenuates the incidence of pericardial edema.
-Does not provided protection against cell
death.
Reimers et al., 2006
Alpha lipoic acid In vitro experiment. Primary rhomboen-
cephalic neurons from Sprague–
Dawley rats at GD14
0.01 mM / 24 h 75 mM / 24 h - Decreases the number of apoptotic cells. Antonio and Druse,
2008
In vivo experiment. Wistar rats. 100 mg/kg / IP / GD7 –
PD21
4.5 g/kg / SC / GD7 –
PD21
- Decreases DNA damage.
-Restores the elevated protein carbonyl and
lipid hydroperoxide levels.
Shirpoor et al., 2008
Pycnogenol In vitro experiment. Primary CGNs from
Long-Evans rats at PD9
25 - 100 mg/ml / 5s –
24 h
86.7 – 346.6 mM / 5s
–24h
- Decreases cell death and reduces the activa-
tion of caspase-3.
Siler-Marsiglio et al.,
2004
tBHQ In vitro experiment. Primary NCC from
C57BL/6J mice from GD10.5
0.010 mM / 16h before
EtOH treatment.
100 mM / 24 h - tBHQ alone increases the protein expression
of Nrf2 and its downstream antioxidants.
-tBHQ-mediated antioxidant response pre-
vents oxidative stress andapoptosis.
Yan et al., 2010
Diphenylene iodo-
nium (DPI)
In vivo experiment. C57BL/6J mice 4 mg/kg / IP / GD9 2.9 g/kg / IPA / GD9 - DPI prevented ethanol-induced increases NOX
enzyme activity, ROS generation and oxida-
tive DNA damage.
-DPI reduces caspase-3 activation and
diminished prevalence of apoptosis.
Dong et al., 2010
D3T In vivo experiment. C57BL/6J mice 5 mg/kg / IP / GD8 2.9 g/kg / IP / GD8 - D3T increases Nrf2 protein levels and Nrf2-
ARE binding, and strongly induces the
mRNA expression of Nrf2 downstream target
genes.
-D3T decreases the levels of ROS.
Dong et al., 2008
In vitro experiment. PC12 cells. 0.05 mM / 16h before
EtOH
200 mM / 24 h - D3T treatment reduces ethanol-induced apo-
ptosis stabilizing Nrf2.
Dong et al., 2011
8 ANTIOXIDANT THERAPIES FOR FAS PREVENTION
potentiation (Nash et al., 2007). Overall, the results of
these studies indicate that maternal administration of
high-dose vitamins C plus E throughout gestation has lim-
ited efficacy and potential adverse effects (such low birth
weight) as a therapeutic intervention (Poston et al., 2006).
Furthermore, a recent prospective observational study
conducted by a Canadian work team in pregnant women
supplementing with mega-doses of vitamin E, detected an
apparent decrease in mean birth weight that could not be
explained by other variables including maternal age, gesta-
tional (Boskovic et al., 2005). In light of the results, the
EViCE (Effectiveness of Vitamin C and E in alcohol exposed
pregnancies) study was suspended (Goh et al., 2007).
EFFECT OF ANTIOXIDANT THERAPY ON THE ENDOGENOUS OXIDATIVE
STRESS LEVELS
The use of compounds with antioxidant properties has
also been consistently shown to reduce oxidative stress
levels and/or to increase the endogenous antioxidant
capacity in the rodent brains of different models of FASD.
The antioxidant vitamin C inhibited ROS production in
Xenopus laevis embryos exposed to ethanol (Peng et al.,
2005). Vitamin E is the natural antioxidant most com-
monly used and several studies have also shown its bene-
ficial effects in decreasing oxidative stress in different
models of FASD. Recently, in offspring rat pups exposed
prenatally to ethanol, vitamin E reversed the levels of pro-
tein and lipid oxidation in both hippocampus and cerebel-
lum (Shirpoor et al., 2009). In the same tissue, using rat
pups vitamin E, alleviated oxidative stress (Marino et al.,
2004). Similarly, maternal vitamin E treatment restores
the fetal hepatic isoprostanes (Wentzel et al., 2006). (–)-
Epigallocatechin-3-gallate (EGCG) is another powerful anti-
oxidant and is believed to be responsible for most of the
health benefits attributed to green tea consumption (Nagle
et al., 2006). Long et al., using a FASD murine model found
that EGCG provided significant protection against ethanol-
associated embryonic developmental retardation. This pro-
tection seems to be mediated by its antioxidative proper-
ties (Long et al., 2010). Resveratrol (3,5,40-trihydroxy-
trans-stilbene) has been shown to be a promising natural
compound with antiapoptotic, free radical-scavenging, and
antilipoprotein peroxidation properties (Shakibaei et al.,
2009). Using a mice model of FASD, the treatment with
resveratrol, before ethanol exposure, restores nuclear fac-
tor (erythroid-derived 2)-like 2 (Nrf2) transcription factor
levels in cerebellum granule neurons (CGNs) and in the
same tissue, and this fact promotes the survival of these
cells (Kumar et al., 2011). Nrf2 has been demonstrated to
be a critical transcription factor that regulates the induc-
tion of phase 2 antioxidant enzymes detoxifying and anti-
oxidant genes (Zhang, 2006; Nguyen et al., 2009).
Thymoqinone (TQ), the active component of Nigella sativa
seeds, has broad and versatile pharmacological effects.
These effects include strong antioxidant activity against
free radical-generating agents (Houghton et al., 1995). TQ
stimulates resistance to oxidative stress decreasing the
elevated levels of malondialdehyde (MDA), and stimulating
catalase and SOD expression (Al-Majed et al., 2006). Sul-
foraphane (SFN) is a natural isothiocyanate, found abun-
dantly in broccoli sprouts. Compelling evidence indicates
that SFN-rich broccoli sprouts and other SFN food sources
trigger the induction of phase 2 detoxifying genes and
antioxidant enzymes, through activation of Nrf2 signaling,
and can aid in preventing cancer and other diseases (Din-
kova-Kostova, 2002). Chen et al. (2013) showed the Nrf2-
mediated antioxidant response on neural crest cells
(NCCs) exposed to ethanol. Capsaicin (8-methyl-N-vanillyl-
6-nonemide) is the major pungent principle of hot peppers
of the plant genus Capsicum. Kim et al. treated with capsa-
icin embryos exposed prenatally to ethanol. These animals
recovered their SOD activity and GPx and GPx mRNAs
expression (Kim et al., 2008). Lipoic acid and its reduced
form, dihydrolipoic acid (DHLA) eliminate hydroxyl radi-
cals and hypochlorous acid with a potency comparable to
GSH (Biewenga and Bast, 1995). Tert-butylhydroquinone
(tBHQ) increases Nrf2 protein stability through inhibition
of the Keap1-mediated ubiquitination. An in vitro model
that used NCCs co-exposed to ethanol and tBHQ showed
less oxidative stress and apoptosis (Yan et al., 2010).
Diphenylene Iodonium (DPI) is a NOX inhibitor. NOX
enzymes can catalyze NADPH-dependent reduction of oxy-
gen to generate superoxide anion (Banfi et al., 2003) and
interestingly, ethanol activates NOX and the subsequent
ROS generation (Wang et al., 2012). Dong et al. (2010)
examined the effect of co-administration of DPI with etha-
nol on pregnant mouse. The results support the hypothe-
sis that DPI is a promising molecular target for blocking
NOX, a critical source of ROS in ethanol-exposed embryos.
3H-1,2 dithiole-3-thione (D3T) is a potent cancer
chemopreventive agent that prevents mutation and pro-
vides protection against neoplasia initiation (Otieno et al.,
2000). In addition, activation of the Nrf2 pathway, by oral
administration of D3T, has recently been reported to con-
fer partial protection against 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP)-induced neurotoxicity (Burton
et al., 2006). The protective effects of D3T in animals have
been associated with induction of the detoxifying and anti-
oxidant enzymes SOD, catalase and c-glutamylcysteine syn-
thetase (c-GCS) (Otieno et al., 2000; Munday and Munday,
2004; Cao et al., 2006). Dong et al. (2008) exposed mice
embryos to D3T decreasing ROS generation.
PROTECTIVE EFFECT OF ANTIOXIDANTS USING IN VITRO MODELS
The beneficial role of antioxidants have been corroborated
by several in vitro studies (Mitchell et al., 1999a ; Heaton
et al., 2000a; Lee et al., 2009). A prospective apoptotic
effect has been described for vitamin C. Using primary-
cultured neuronal cells co-treated with Vitamin C and
ethanol the expression of Bax, caspase-9, caspase-3 and
BIRTH DEFECTS RESEARCH (PART A) 00:00–00 (2014) 9
cytochrome-c decreased while the expression of antiapop-
totic Bcl-2 protein increased significantly (Naseer et al.,
2011). Furthermore, vitamin E has been extensively dem-
onstrated that prevents alcohol-induced cell loss using in
vitro models (Mitchell et al., 1999a; Heaton et al., 2000a;
Siler-Marsiglio et al., 2004, 2005). For example, neuronal
viability was significantly higher in cell cultures previously
treated with ethanol and maintained on vitamin E or
b-carotene (Mitchell et al., 1999b). A recent study has
demonstrated that EGCG and resveratrol could protect
fetal rhomboencephalic neurons from ethanol-induced
apoptosis (Antonio and Druse, 2008). In agreement with
that, resveratrol prevents ethanol-induced apoptosis dur-
ing mouse blastocyst development (Huang et al., 2007).
Ginkgolide B (GB), originally extracted from Ginkgo biloba
leaves, is one of the major components of traditional Chi-
nese medicine (Maclennan et al., 2002). It has been shown
that GB can scavenge free radicals and inhibit seryl and
aspartyl proteases (Brunetti et al., 2006), protecting
against neural damage. Preliminary in vitro results have
demonstrated the powerful antioxidant characteristics of
GB inhibiting ethanol-induced cell apoptosis (Zhang et al.,
2011). Another antioxidant with natural origin is curcu-
min, the principal curcuminoid found in turmeric. Curcu-
min has potent antiamyloid (Wang et al., 2010) anti-
ischemic (Shukla et al., 2008) and anti-inflammatory prop-
erties (Basnet and Skalko-Basnet, 2011). All of these char-
acteristics seem to be mediated by its pharmacological
actions with respect to its antioxidant effect. As a conse-
quence, curcumin presents protective effects against
ethanol-induced apoptosis. This was initially observed
using primary fetal rhomboencephalic neurons (Antonio
and Druse, 2008) and postnatal pup rats (Tiwari and Cho-
pra, 2013). Recently, it has been reported that TQ, by
means of direct reduction of intracellular ROS, protects
against cell death induced by serum/glucose deprivation
in PC12 cells by means of a direct reduction in intracellu-
lar ROS (Mousavi et al., 2010). Moreover, TQ maintained
normal physiological mitochondrial transmembrane poten-
tial. These findings suggest that TQ is a potential protec-
tive agent against ethanol-induced neuronal apoptosis. All
of these results strongly support the idea that an increase
in oxidative stress is one of the mechanisms by which
ethanol induces apoptotic cell death in fetal neurons.
Finally, with respect to the action of anthocyanins, Chen
et al. demonstrated that C3G can recover the reduction of
neurite outgrowth caused by ethanol treatment. Moreover,
this process is mediated by glycogen synthase kinase 3b
(GSK3b) (Chen et al., 2009).
The alterations observed in these systems occur as a
consequence of oxidative stress but the intermediate
mechanisms are already unknown and depend on the
magnitude, pattern and timing of the exposure (as certain
as the genetic susceptibilities to ethanol also exert an
influence on the dose and period of exposure). Thus, it is
possible that oxidative stress only represents a single
molecular process involved in ethanol-induced damage.
Consequently, treatment with antioxidants might not be
enough to counter act the effects of perinatal ethanol
exposure.
Nevertheless, antioxidants (alone or in combination
with other therapeutic agents) might still be good candi-
dates for the mitigation of some of the deficits observed in
individuals with FASD. Further studies in animal models
are warranted to identify the optimal cocktail of antioxi-
dant compounds in addition to test therapeutic strategies
that use antioxidants in combination with other pharmaco-
logical drugs.
CONCLUSIONS
FASD is a major public health problem, being the leading
cause of preventable mental retardation and birth defects
in the Western countries (May et al., 2009). The simplest
method for the prevention of FASD is avoiding any alcohol
intake during pregnancy. However, a widespread and appa-
rently increasing incidence of FASD has been observed
recently (Fig. 2) (Abel, 2006; Riley et al., 2011). Whereas a
great effort should be made to avoid ethanol consumption,
several pharmacological approaches for the prevention of
FASD are currently under active research and some of
them have already generated patents (Martinez and Egea,
2007). Identification of effective interventions and treat-
ments for FASD is, therefore, critical. Ideally, one would
intervene at the time of alcohol exposure, thereby directly
preventing or reducing the amount of alcohol-related dam-
age. Based on the mechanisms involved in the ethanol-
induced damage include neurotrophic agents (Heaton
et al., 2000b), neuroactive peptides (Vink et al., 2005) and
antioxidants. Nutritional supplementation may also miti-
gate alcohol’s teratogenic effects. Nutritional supplements
may compensate for changes in the bioavailability of
FIGURE 2. Current strategies for the prevention and treatment of FASD.
Extracted from Martinez and Egea (2007).
10 ANTIOXIDANT THERAPIES FOR FAS PREVENTION
nutrients due to alcohol metabolism (Lieber, 2000). Chol-
ine supplementation during early postnatal development
reduces the severity of some ethanol-induced neurobeha-
vioral alterations (Thomas et al., 2000, 2009, 2010). Simi-
larly, folic acid (FA) supplementation in young women can
prevent intrauterine growth restriction, neural tube
defects and other congenital anomalies (Eskes, 1997;
Scholl and Johnson, 2000). FA can also ameliorate toxicity
induced by ethanol (Gutierrez et al., 2007; Yanaguita et al.,
2008).
Nevertheless, it is well-known that oxidative stress
plays a pivotal role in the development of the disease.
This increase in the levels of ROS production has direct
consequences on the ethanol metabolism due to its actions
on mitochondrial bioenergetics and in the antioxidant sys-
tem. Future research is warranted to test these
hypotheses.
Different animal models have been used for the study
of FASD but the knowledge about the cellular and molecu-
lar processes are not completely understood. Given the
use of different modes of ethanol administration and dif-
ferent exposure periods, to make comparisons among
studies is a challenging issue and drawing clear conclu-
sions may be difficult to understand though the compari-
son between studies. For this reason, it will be necessary
uniform methodologies (including the same model and
controlling external confusing variables such as dose and
administration procedure, BAC peak achieved, time and/or
duration of exposure) to analyze the effect of prenatal
ethanol exposure on different indicators of oxidative stress
in a systematic manner.
Furthermore, it would be interesting to explore
whether the use of antioxidants later on in life would also
have beneficial effects in FASD models. To date, there is
only one clinical study, showing no significant differences
in the urine levels of lipid peroxidation products in
women who drunk during pregnancy compared with non-
drinkers pregnant women (Signore et al., 2008). It is
important to mention that this study did not evaluate the
oxidative stress levels in the newborns. This is particularly
important because, to date, most studies have only ana-
lyzed the effects of antioxidant compounds in models of
FASD when these are administered concurrently with etha-
nol. The majority of these publications evidence that anti-
oxidant treatment can be beneficial in the amelioration of
some characteristics of FASD (Table 1). However, this strat-
egy has not been explored on humans. Nevertheless,
remains to be clear if the antioxidant therapy can be bene-
ficial in the amelioration of some biochemical and behav-
ioral characteristics in children with FASD. However, for
other neurodevelopmental disorders such as autism (Akins
et al., 2010), attention deficit/hyperactivity disorder
(ADHD) (Chovanova et al., 2006) or fragile-X syndrome
(de Diego-Otero et al., 2009), have been explored the
administration of antioxidants showing beneficial effects in
the mitigation of these disease effects. Within this sce-
nario, antioxidants (either alone or in combination with
other therapies) are strong candidates for clinical trials
design in FASD-affected children to prevent or to revert
deleterious effects of ethanol during neurodevelopment.
Acknowledgment
The authors thank J. Klein for excellent language editing
service.
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