Experimental Eye Research 80 (2005) 535–543

www.elsevier.com/locate/yexer

The protective role of squalene in alcohol damage

in the chick embryo retina
Yolanda Aguileraa,*, Manuel E. Doradoa, Francisco A. Pradaa, Juan J. Martı́neza,
Adela Quesadaa, Valentina Ruiz-Gutiérrezb
a
Instituto de Biologı́a del Desarrollo, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez Pizjuán s/n, 41009 Seville, Spain
b
Instituto de la Grasa, CSIC, 41012 Seville, Spain
Received 27 July 2004; accepted in revised form 12 November 2004
Available online 19 January 2005

Abstract
The developing CNS, and in particular the visual system, is very sensitive to the effects of alcohol. Alcohol causes lipid peroxidation.
Squalene, the major olive oil hydrocarbon, is a quencher of singlet oxygen and prevents the corresponding lipid peroxidation. We presumed
that squalene can protect against the alcohol-induced damage already observed during the development of the chick retina. AlcoholC
squalene was administered directly into the yolk sac of the egg of White Leghorn chicks at day 6 of incubation. The lipid composition of the
retina was analyzed in embryos at E7, E11, E15 and E18. The proportions of phospholipids, free and esterified cholesterol, diacylglycerides
and free fatty acids were estimated using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to
determine the fatty acid composition. The morphological study was carried out at E11 using semithin sections, and by means of
immunohistochemical techniques at E19. Comparing the results obtained in control embryos, the administration of alcoholCsqualene
reduces the effects of alcohol on the total lipid composition of the retina during development. The effects were, in fact, of less magnitude than
in embryos treated only with alcohol. The major phospholipid species of alcoholCsqualene-treated embryos exhibited total recuperation at
E15. As far as fatty acids are concerned, no significant changes were observed with regard to control embryos during development. From a
morphological point of view, the retinas of alcoholCsqualene-treated embryos show at E11 fewer cellular alterations than the retinas of
alcohol-treated embryos. In this respect, the retinas of alcoholCsqualene-treated embryos exhibited: a columnar cell arrangement similar to
that observed in control retinas; few pycnotic cells and very few alterations in ganglion cell layers and in the optic nerve fibers layer. At E19
the recuperation of the expression of myelin oligodendrocyte specific protein (MOSP) in alcoholCsqualene-treated embryos was recorded.
Since squalene reduces the deleterious effects caused by alcohol on the lipid composition and the structure of the retina, squalene could act as
a naturally occurring agent for the prevention of damage caused by abusive alcohol ingestion during pregnancy.
q 2004 Elsevier Ltd. All rights reserved.

Keywords: squalene; fetal alcohol syndrome; retinal development; lipids; fatty acids

1. Introduction However, it is widely distributed in nature, with high

Squalene, a polyunsaturated triterpene that contains six
isoprene units, is a key intermediate in cholesterol
biosynthesis. It received its name because of its occurrence
in shark liver oil (Squaluss spp.) which is considered to be
the richest source of squalene (Gershbein and Singh, 1969).
* Corresponding author. Dra Yolanda Aguilera, Instituto de Biologı́a del
Desarrollo, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez
Pizjuán s/n, 41009 Seville, Spain.
E-mail address: yaguilera@us.es (Y. Aguilera).
0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2004.11.003
proportions in olive oil compared to other vegetable oils. It
is the major olive oil hydrocarbon. It makes up more than
90% of the hydrocarbon fraction, ranging from 200 to
7500 mg kgK1 oil (Perrin, 1992).
Distributed ubiquitously in human tissue, it has its
greatest concentrations in the skin (Liu et al., 1976) and the
adipose tissue (Liu et al., 1976; Tilvis and Miettinen, 1983).
In general, levels of squalene in the vertebrate retina are
very low. However, the squalene appears to play an
important role in the frog retina. In addition, it is present
in bovine retinal homogenates. Both in vitro and in vivo
536 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543
models animals, the majority of newly synthesized squalene
in the retina is transported to the rod out segment
membranes where it turns over in parallel with the disk
membranes (Fliesler and Keller, 1997).
In humans, a substantial amount of dietary squalene is
absorbed and converted to cholesterol. Presumably due to a
concomitant increase in fecal elimination (Strandberg et al.,
1990), this increase in synthesis is not, however, associated
with a corresponding increase of serum cholesterol levels.
The exogenous squalene stimulates the activity of ACAT
(acyl coenzyme A: cholesterol acyltransferase) but strongly
inhibits the activity of 3-hydroxy-3-methylglutaryl coen-
zyme A (HMG-CoA) reductase (Strandberg et al., 1989).
In vitro, experimental evidence indicates that squalene is
a quencher of singlet oxygen, prevents the corresponding
lipid peroxidation in human skin surface (Kohno et al.,
1995) and confers some cellular and systemic radioprotec-
tion to mice receiving lethal whole-body radiation doses
(Storm et al., 1993). Several studies have revealed that
squalene is an important dietary cancer chemo-preventive
agent (reviewed in Kelly, 1999; Newmark, 1997). Kamikura
et al. (1992) have also suggested that, by enhancing drug
elimination from the body, squalene might be an antidote to
reduce the toxicity of accidentally ingested drugs.
Researchers and clinicians are worried about women
who consume alcohol in large quantities before they realize
they are pregnant as well as during early pregnancy. Heavy
alcohol consumption during pregnancy leads to a significant
risk of brain injury in the developing fetus which is
especially vulnerable to alcohol-induced brain injuries
during specific stages of brain development such as, e.g.
early pregnancy (Ikonomidou et al., 2000). Women who
drink in excess during pregnancy may deliver offspring who
could be diagnosed with fetal alcohol syndrome (FAS),
which is associated with growth retardation, disorders of the
central nervous system (CNS), a characteristic pattern of
facial anomalies (Jones and Smith, 1973) and more or less
severe deficits involving cognitive and behavioural dis-
orders. Fortunately, most women reduce or stop their
alcohol consumption as soon as they realize they are
pregnant; in some cases, however, some injury to the fetal
brain may have already occurred (Maier and West, 2001).
Strabismus, blepharoptosis, epicanthus, telecanthus and
short palpebral fisures are typical ocular features in children
with FAS. The most frequent finding in the eyes of children
with FAS are anomalies of the retina and optic nerve
hypoplasia (Strömland and Pinazo-Duran, 1994). Alcohol
alters the development of the retina provoking retarded and
defective cell migration, loss of neurones in the inner layers
of the retina, especially so in the layer of ganglion cells.
Moreover, the reduction in the number of myelin-axons of
the optic nerve fiber layer is very noticeable. (Aguilera et al.,
2004; Chmielewski et al., 1997). On the other hand, chicken
embryos alcohol-treated show alterations on the retina lipid
composition with a remarkable increase in free cholesterol
and diacylglicerides and a decrease in esterified cholesterol.
In addition, C14:0 increase and C18:1(nK9) and PUFAs,
such as C20:4(nK6) and C22:6(nK3) decrease, at E7
(Aguilera et al., 2004).
Given that the harmful effects of alcohol on health are
thought to be caused mostly by reactive oxygen species,
especially by some oxygen and other free radicals (Mantle
and Preedy, 1999; Zloch, 1994) and that the retina and optic
nerve are often involved in FAS (Strömland, 1987), the aim
of this work has been to study the protective role of squalene,
a natural antioxidant, in alcohol-induced damage during the
chick retinal development. The alcohol-induced lipid and
morphological changes were previously analyzed in our
laboratory (Aguilera et al., 2004; Chmielewski et al., 1997).
2. Materials and methods
2.1. Materials and experimental procedures
Retinas were obtained from White Leghorn embryos.
Chick embryos were incubated at 378C in a humidified
forced draft incubator. Embryos received treatment by
direct injection into the yolk sac at day 6 of incubation (E6)
(Aguilera et al., 2004; Chmielewski et al., 1997; Quesada
et al., 1990). Embryos were injected with 10 ml of absolute
ethanol (Merck, Ref. 983) and 10 ml of squalene (Sigma,
Ref. 3626) (for the study of lipids) and 20 ml of absolute
ethanol and 20 ml of squalene (for the morphological study),
according to the system of Means et al. (1988). The data
from the embryos of the so-called ACS group were
compared with the results of two groups: a control group,
whose individuals were injected with a sterile physiological
saline solution of 10 ml (for the study of lipids) and 20 ml
(for the morphological study) and the alcohol group, whose
individuals were likewise injected with 10 and 20 ml of
absolute ethanol, respectively. Simultaneously, a squalene
group was injected with the same of squalene volume for
morphological study.
Later the embryos were extracted and the retinas
dissected at E7, E11, E15 and E18 for the analysis of the
lipid composition of the retina; E11 for the optical analysis
of semithins; and E19 for immunohistochemical techniques.
The development at the moments of injection and extraction
was determined systematically by means of the tables of
Hamburger and Hamilton (1951).
The animal care protocols used in our laboratory and
university vivaria comply with the relevant national
legislation (Decreto 223/1988, B.O.E. no. 67) and the
Directive 86/609/EEC of the Council of the European
Communities.
2.2. Lipid determination
2.2.1. Lipid analysis
Dissected retinas were placed in a saline solution in an
Eppendorf tube and centrifuged at 6000 rpm at 48C for
 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543
10 min. The saline supernatant was discarded and the retinas
were weighed and frozen in liquid nitrogen until determi-
nation. Each group were performed on retinas pooled at
each age; E7 pooled 32 retinas for each group; E11 had 30
retinas; and E15 and E18 pooled 24 for each group.
The total lipid analysis of the retinas was carried out
following the method of Folch et al. (1957) using
chloroform:methanol (2:1, v/v)) as the solvent. The lipid
extract was kept in a sealed vessel under a nitrogen
atmosphere at K308C until the assay. Lipid and phospho-
lipid compositions were determined by the Iatroscan
TLC/FID method (Ruiz-Gutierrez et al., 1995). An
Iatroscan MK-5 (Technical Marketing Associated, Missi-
sanga, Ont.) was used in combination with Chromarods S
(Technical Marketing Associated, Missisanga, Ont.) pre-
coated with a thin active silica layer. To separate total lipids,
chromorods were developed in hexane/diethyl ether/formic
acid (90:10:2, v/v/v). The phospholipids were resolved in
two steps: (a) initial development of the chromorods in
chloroform:methanol:acetic acid:water (67:28:2:3, v/v/v/v)
and drying at 708C for 10 min, followed by (b) a second
development in hexane:diethyl ether:formic acid (90:10:2,
v/v/v). The chromorods were scanned under the following
conditions: hydrogen flow, 175 ml minK1; air flow
1850 ml minK1; scanning speed, 47 mm sK1; chart speed,
42 mm minK1. An Iatrocorder TC-11 integrator was used to
record and integrate peak areas.
2.2.2. Fatty acid analysis
The fatty acid analysis was performed using gas
chromatography (Ruiz-Gutierrez et al., 1996). Samples
were saponified by heating for 20 min with 5 ml of 0$2 M
sodium methylate and then reheating at 808C for 20 min
with 6% (w/v) H2SO4 in anhydrous methanol. The fatty acid
methyl esters (FAME) thus formed were analyzed using a
5890 series II gas chromatographer (Hewlett-Packard Co.,
Avondale, PA) equipped with a flame ionization detector
and an Supelcowax (Supelco, Bellefonte, USA) fused silica
capillary column (30 m!0$32 mm ID, 0$25-mm film).
Individual FAMEs were identified by comparing the
retention times with standard records (Ruiz-Gutierrez et
al., 1992). FAME for which no standard were available were
identified by gas chromatography-mass spectrometry on a
Konik KNK-2000 chromatograph (Konik Co., Barcelona,
Spain) interfaced directly to an AEJ MS30/790 VG mass
spectrometer (VG Analytical, Manchester, UK) using
electron impact ionization mode. The ion source tempera-
ture was maintained at 2008C, the accelerating voltage was
4$0 kV, the emission current was 100 mA and the electron-
impact ionization was 70 eV. The data were processed with
a VG 11/250 data system.
2.2.3. Statistical analysis
Mean and S.D. were calculated. Analysis of variance
(ANOVA) with a least significant difference test (LSD)
were used, and p!0$05 was the criterion for significance.
537
2.3. Optical microscopy study
2.3.1. Semithin sections
The retinas were processed according to the technique of
Palay and Chan-Palay (1974), cut to 1 mm with a glass blade
and stained with 1% toluidine blue in 1% sodium borate
(Richardson et al., 1960). The observations were carried out
with a Zeiss microscope fitted with an incorporated
AxioCam HR camera scanner.
2.3.2. Immunohistochemical methods
Eye globes of embryos at E19 were fixed in 4%
paraformaldehyde in a 0$1M phosphate buffer at pH 7$6.
After fixation, tissues were cryoprotected, embedded in
Tissue-Tek medium (Miles Laboratories, Elkhart, IN), and
frozen by dipping in liquid nitrogen. Cryostat sections were
cut at 10–20 mm and mounted on chromium potassium
sulphate-gelatin-coated slides, dried at room temperature
and stored at K608C.
For single immunohistochemical labelling, we used a
monoclonal antibody against myelin oligodendrocyte
specific protein (MOSP) from Chemicon International
(MAB328). To block nonspecific staining, sections were
first incubated in 10% chick serum in phosphate-buffered
saline (PBS) containing 0$25% Triton X-100 for 30 min.
Sections were then incubated for 24 hr at room temperature
with the primary antibody diluted 1/100 in PBS with 0$1%
Triton X-100 and 1% chick serum. After washing for 10 min
three times in PBS with 0$1% Triton X-100, sections were
incubated with secondary antibody conjugated to floures-
cein isothio-cyanate, anti-mouse Ig M (Sigma, St Louis,
MO) for MOSP, diluted 1/50 in PBS for 2 hr at room
temperature in the dark. The slides were then washed three
times for 10 min in 0$1% Triton X-100 in PBS, rinsed in
PBS and coverslipped. Control sections were processed in
each set of preparations. The solution used to block
nonspecific staining replaced either the primary or the
secondary antibody. Sections were studied with an epi-
fluorescence microscope Zeiss Axioplan.
3. Results
3.1. Effects on the lipid composition of the retina
Table 1 shows the effects of age on the total lipid content
of chick embryo retinas of embryos treated with 10 ml of
alcohol and 10 ml of squalene (ACS) as compared to
control embryos (treated with 10 ml of a sterile saline
solution) and embryos treated with 10 ml of alcohol. The
data express the meanGS.D.
During development alcohol induces changes in the lipid
composition of the retina which mostly revert at the end of
development. The joint administration of alcohol and squalene
speeds up the reversion of the lipid changes provoked by
the administration of alcohol only. When studying the lipid
538 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543

Table 1
Lipid composition of chick embryo neural retina

Fraction Animal group Stages

E7 E11 E15 E18
a a b
Phospholipids Control 77.63G0.10 78.52G0.89 81.20G1.71 80.47G0.28a
Alcohol 82.89G0.32b 72.35G5.41a 73.85G0.49a 79.41G0.54a
ACS 82.57G0.23b 81.15G0.83a 79.38G1.10b 81.92G2.70a
Free cholesterol Control 9.45G0.26a 8.58G0.61a 6.69G0.06a 8.75G0.13a
Alcohol 7.00G0.96a 23.67G3.84b 18.85G0.50c 15.73G0.43b
ACS 7.42G0.13a 10.08G0.44a 14.10G0.30b 10.74G1.69a
Esterified cholesterol Control 11.92G0.20b 11.87G0.28c 10.94G1.90b 9.75G0.13c
Alcohol 8.15G1.30a 0.27G0.07a 0.97G0.03a 2.25G0.07a
ACS 7.82G0.16a 6.21G0.09b 4.07G2.81a 6.30G0.41b
Diacylglycerides Control 0.99G0.06b 1.03G0.01a 1.16G0.24b 1.04G0.01a
Alcohol 1.97G0.01a 3.70G0.21b 6.34G0.01a 2.60G0.04b
ACS 2.18G0.06c 2.56G0.30a 2.46G1.43b 1.04G0.58a

Data (%w/w) are the meanGS.D. of three individual determinations. The letters (a–c) indicate significant differences with p!0$05 according to the least
significant difference test (LSD).

composition of the retina 24 hr after treatment, we observe that
alcohol and ACS-treated embryos show statistically similar
values. Differences between these two groups only appear as
development proceeds, the total lipid values in ACS-treated
embryos becoming then similar to those of control embryos.
With the exception of sterified cholesterol, at E18 the total
lipids of ACS-treated embryos had values similar to those of
control animals.
As compared to control data, phospholipids and
diacylglycerides of ACS-treated embryos showed a
significant increase at E7, a record that was similar or
higher than the increase observed in alcohol-treated
embryos. However, in the other developmental stages
under consideration the results were statistically similar
to those of control embryos and ACS-treated embryos.
Free cholesterol shows statistically similar values both in
control embryos and in ACS-treated embryos, with
Table 2
Composition of major phospholipid species in chick embryo neural retina
the exception of E15, where an increase of 110$8% as
regards to the control group may be observed. This
increase, however, is not as marked as the one observed
in the alcohol group (181$8%). At E7 the esterified
cholesterol dropped about 33% in embryos experimen-
tally treated with alcohol or ACS. In alcohol-treated
embryos this difference was much more noticeable in
E11 and E15 (97$7 and 91$2%, respectively), a value
that decreased to 76$9% in E18. As compared with the
control group, the ACS-treated embryos showed a
significant decrease that is considerably smaller (47$7%
at E11, 62$8% at E15 and 35$3% at E18) than alcohol-
treated embryos.
At E18 the percentages of major phospholipids species
in ACS-treated embryos, alcohol-treated embryos and
control embryos are much the same (Table 2). The largest
number of differences between control embryos and

Fraction Animal group Stages

E7 E11 E15 E18
b c a
PE Control 40.91G2.12 34.32G0.56 33.83G0.50 37.60G0.01a
Alcohol 31.85G1.38a 26.36G0.01a 38.56G0.25b 39.57G0.84a
ACS 46.07G1.92b 31.84G0.53b 33.66G0.10a 35.68G2.29a
PC Control 46.41G4.88b 58.02G0.15c 58.02G0.76b 55.06G0.01a
Alcohol 29.59G1.03a 24.93G0.01a 24.27G1.12a 52.79G1.02a
ACS 31.05G0.23a 39.07G0.90b 58.73G0.12b 56.19G2.77a
PI Control 1.78G0.78a 1.26G0.13a 1.20G0.01a 0.96G0.01a
Alcohol 3.55G3.52a 3.30G0.01b 2.64G0.06c 1.00G0.01a
ACS 2.86G2.39a 1.64G0.21a 2.28G0.06b 1.60G0.25b
PS Control 5.30G4.31a 3.56G0.56a 3.27G0.05a 3.25G0.01a
Alcohol 18.06G1.05a 17.71G0.01b 15.96G0.09b 3.45G0.19a
ACS 3.43G3.07a 11.76G2.08b 2.55G0.25a 3.46G0.25a
SPH Control 5.59G3.48a 2.83G0.01a 3.66G0.69a 3.12G0.01a
Alcohol 16.94G0.07b 27.66G0.06c 18.56G0.85b 3.17G0.01a
ACS 17.08G0.31b 15.70G0.45b 2.79G0.09a 3.07G0.37a

Data (%w/w) are the meanGS.D. of three individual determinations. The letters (a–c) indicate significant differences with p!0$05 according to the least
significant difference test (LSD). PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; SPH,
sphingomyelin.
 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543 539

Table 3
Fatty acid composition of total lipids in chick embryo neural retina

Fatty acid Animal group Stages

E7 E11 E15 E18
a a a
C14:0 Control 2.64G0.10 2.58G0.45 2.43G0.13 2.18G0.04a
Alcohol 7.08G0.72c 2.42G0.03a 3.22G0.18a 1.78G0.07a
ACS 4.25G0.14b 3.04G0.07a 2.98G0.27a 2.80G0.17b
C16:0 Control 36.51G0.16a 36.51G0.94a 35.99G0.52b 35.47G2.68a
Alcohol 44.64G0.95b 36.66G0.21a 36.69G0.35b 33.88G0.74a
ACS 36.54G0.45a 36.38G2.16a 32.48G0.95a 32.27G2.07a
C16:1(nK7) Control 2.67G0.16a 2.08G0.26a,b 1.67G0.20a 1.72G0.08a
Alcohol 4.72G0.06c 1.88G0.02a 2.52G0.27a,b 1.47G0.11a
ACS 3.89G0.31b 2.57G0.19b 2.86G0.23b 2.41G0.02b
C18:0 Control 14.14G0.18a 15.53G0.81a 16.82G0.35a 17.59G0.55a
Alcohol 14.95G0.41a 14.97G0.24a 16.76G1.08a 18.05G0.87a
ACS 14.21G0.79a 15.13G0.26a 15.75G0.03a 17.42G0.52a
C18:1(nK9)C(nK7) Control 22.75G0.94b 17.94G0.38a 16.90G0.32a 16.71G0.38a
Alcohol 14.15G0.41a 17.92G1.23a 18.52G0.90a 17.06G0.27a
ACS 21.23G0.31b 17.91G0.09a 18.62G0.36a 18.86G0.30b
C18:2(nK6) Control 2.86G1.66a 1.35G0.23a 1.15G0.02a 1.31G0.01a,b
Alcohol 1.20G0.02a 1.50G0.70a 1.66G0.32a 1.17G0.10a
ACS 1.51G0.25a 1.40G0.24a 1.66G0.27a 1.58G0.15b
C18:3(nK3) Control 0.43G0.03a 0.27G0.14a 0.26G0.22a 0.29G0.25a
Alcohol 1.17G0.18b 0.24G0.09a 0.74G0.05a 0.15G0.02a
ACS 0.37G0.15a 0.50G0.55a 0.63G0.74a 0.27G0.14a
C20:0 Control 0.44G0.11a 0.23G0.09a 0.18G0.03a 0.29G0.09a
Alcohol 0.89G0.25a 0.20G0.08a 0.36G0.09b 0.26G0.10a
ACS 0.55G0.07a 0.31G0.04a 0.33G0.09b 0.75G0.06b
C20:1(nK9) Control 0.53G0.13a 0.39G0.01a 0.48G0.01a 0.44G0.03a
Alcohol 1.01G0.06b 0.47G0.11a 0.56G0.28a 0.25G0.20a
ACS 1.11G0.01b 0.72G0.06b 0.54G0.01a 0.51G0.28a
C20:4(nK6) Control 6.99G0.87b 9.29G1.60a 8.94G0.19b 7.71G0.13a
Alcohol 2.51G0.29a 9.69G1.14a 6.92G0.04a 7.40G0.01a
ACS 6.38G0.60b 8.29G0.40a 8.33G0.34b 7.96G0.18a
C22:4(nK6) Control 2.62G1.71a 5.74G0.12b 3.28G0.14a 2.63G0.14a
Alcohol 3.61G0.30a 3.74G0.31a 1.83G0.48a 3.34G0.09b
ACS 3.37G0.30a 3.60G0.45a 2.94G0.73a 4.00G0.17b
C22:5(nK3) Control 0.52G0.16a 0.36G0.10a 0.62G0.03b 0.51G0.04a
Alcohol 0.36G0.28a 0.50G0.02a 0.39G0.03a 0.51G0.01a
ACS 0.31G0.10a 0.43G0.11a 0.48G0.04a,b 0.46G0.16a
C22:6(nK3) Control 6.89G0.60b 7.73G0.18a 11.27G0.18a 13.15G0.23b
Alcohol 3.70G0.67a 9.82G0.17b 9.82G1.65a 14.68G0.19c
ACS 6.29G0.41a,b 9.72G0.74b 12.41G0.12a 10.73G0.93a
Total sat Control 53.72G1.99a 54.85G2.28a 55.42G0.76a 55.53G2.00a
Alcohol 67.57G1.51b 54.25G0.33a 57.03G1.70a 53.98G0.17a
ACE 55.54G0.41a 54.86G2.45a 51.54G0.62a 53.23G1.66a
Total mono Control 25.96G0.65b 20.41G0.63a 19.05G0.52a 18.87G0.50a
Alcohol 19.88G0.42a 20.27G1.14a 21.60G0.34a 18.77G0.59a
ACE 26.23G0.61b 21.20G0.04a 22.01G0.60a 21.78G0.59a
Total poly Control 20.32G2.64b 24.74G2.91a 25.53G0.24b 25.60G1.50a
Alcohol 12.55G1.09a 25.48G0.81a 21.36G1.36a 27.24G0.42a
ACE 18.22G1.02b 23.94G2.50a 26.44G0.01b 25.00G1.07a

Data (%w/w) are the meanGS.D. of three individual determinations. The letters (a–c) indicate significant differences with p!0$05 according to the least
significant difference test (LSD). Sat, saturated fatty acids; Mono, monounsaturated fatty acids; Poly, polyunsaturated fatty acids.

ACS-treated embryos are observed at E11, when
phosphatidylethanolamine (PE) and phosphatidylcholine
(PC) show a significative decrease as compared to the
control group. This decrease is not so remarkable as
the one observed in alcohol-treated embryos. At E11 the
levels of phosphatidylserine (PS) and sphingomyelin
(SPH) in ACS-treated embryos increase as compared to
the control, but, once more, not as much as in the alcohol
group. At E15 phosphatidylinositol (PI) is the only
phospholipid which exhibits variations with regard to
the control group, but these are not as pronounced as in
the alcohol group.
540 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543

The fatty acid profile of control, alcohol and ACS-

treated embryos is given in Table 3. At first, control
embryos and ACS-treated embryos shared almost the same
pattern of fatty acid, while alcohol-treated embryos showed
changes in the content of myristic (C14:0), palmitic (C16:0),
palmitoleic (C16:1, nK7), oleic (C18:1), linolenic (C18:3,
nK3), arachidonic (C20:4, nK6) and docosahexaenoic
(C22:6, nK3) acids. At the last studied developmental
stage (E18), we recorded an increase of C18:1 (16$71 in
the control group and 18$86 in ACS) and a decrease
of C:22:6(nK3) (13$15 in the control group and 10$73 in
ACS), two of the main fatty acids in the retina. In spite of
that, control and ACS-treated embryos showed similar
statistically values in the percentages of total monounsatu- Fig. 1. Semithin sections of chick embryo retina at E11: control (a), alcohol-
rated and polyunsaturated fatty acids (PUFAs). treated (b), and alcoholCsqualene-treated (ACS) (c). Arrows in (b) show
At E7, the contents of saturated, monounsaturated and pycnotic cells in the inner nuclear layer (INL) and ganglion cell layer
polyunsaturated fatty acids in alcohol-treated embryos were (GCL). In (a–c), the horizontal bars indicate the thickness of GCL. IPL,
different as those of control embryos, whereas ACS-treated inner plexiform layer. Scale bars: 10 mm.
embryos were much the same as those of control embryos.
In accordance with the results of this study and others
Significant changes were absent during the remaining stages
above realized (Aguilera et al., 2004; Chmielewski et al.,
(Table 3).
1997; Strömland and Pinazo-Duran, 1994), alcohol pro-
vokes a marked decrease in number of myelinic axons on
3.2. Effects on retinal structure
the optic nerve fiber layer. This fact justifies the use of an
immunological marker which recognizes one myelin
Although, at a chemical level, changes are striking, at a
morphological level these are more difficult to detect. For oligodendrocyte specific protein (MOSP). Alcohol causes
this reason we doubled the alcohol dose. MOSP expression reduced. The retinas of embryos treated
The morphological effects on the retina development of with alcohol at E6 and sacrificed at E19, indicate that
alcohol injected at E6 have already been described in alcohol dramatically reduces the expression of the above-
previous works (Aguilera et al., 2004; Chimelewski et al., mentioned protein (Fig. 2(b)) (Aguilera et al., 2004). The
1997). Alcohol causes a basic alteration of the migration and expression of MOSP is very pronounced at E19 in the
development of the inner retina layers. Thus, alcohol retinas of control embryos (Fig. 2(a)), especially in the inner
induces an increase in the intercellular spaces and numerous and outer processes of Müller cells (arrows) and in the axon
pyknosis in amacrine cells, a loss of the vertical cell terminations of horizontal cells in the outer plexiform layer
arrangement peculiar to the process of neuron migration, a (OPL, arrow heads). Alcohol-treated embryos show an
decrease in the thickness of the inner plexiform layer and,
most importantly, very pronounced alterations of the
ganglion cell layer and the optic nerve fiber layer.
Numerous pyknosis can be observed in the ganglion cell
layer, which leads to a considerable loss of ganglion
neurons. The optic nerve fiber layer undergoes a decrease in
the number of axons, especially myelinating axons of
medium and large size.
Fig. 1(b), a vertical section at E11 of the retina of an
embryo, injected at E6 with 20 ml of alcohol, shows some of
the alcohol-induced effects already mentioned. When
compared to retinal sections of control embryos (Fig. 1(a))
and ACS-treated embryos (Fig. 1(c)), general cell disor-
ganization can be observed. There are numerous pyknosis in Fig. 2. MOSP immunoreactivity in sections of E19 retina of control chick
the inner half of the inner nuclear layer and in the ganglion embryo (a), alcohol-treated embryo (b), and ACS treated embryo (c).
cell layer (GCL, arrows). In addition, the loss of neurones in Control embryos show a strong immunoreactivity, especially in the inner
this latter layer has been so remarkable that it has been and outer processes of Müller cells (arrows) and in the axon terminations of
reduced to a mere cell row (see horizontal bars), something horizontal cells in the outer plexiform layer (OPL, arrow heads). The retinas
of alcohol-treated embryos show a loss of immunoreactivity for MOSP,
which is not found in the retinas of control embryos which, in the case of axon terminations, is absent. The horizontal bars show
(Fig. 1(a)) or ACS-treated embryos (Fig. 1(c)) or squalene- the thickness of the inner plexiform layer (IPL). GCL, ganglion cell layer;
treated embryos (data not shown). NFL, nerve fiber layer. Scale bars: 15 mm.
 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543
almost complete absence of MOSP expression (Fig. 2(b)),
whereas in the retinae of ACS-treated embryos the MOSP
expression is quite good in the outer plexiform layer and in
both the ganglion cell layer and the optic nerve fiber layer
(Fig. 2(c), arrow and arrow head). In addition, when
compared with the retinas of control embryos and ACS
embryos (see Fig. 2(a–c), horizontal bars), a slight reduction
of the thickness of the inner plexiform layer is observed in
embryos treated only with alcohol. Embryos injected with
squalene showed a similar pattern to that in the control and
ACS embryos. Although, particular sections of the retina
do not clearly reveal this fact, it is significant if the whole
retina is taken into account.
4. Discussion
The results of our research suggest that squalene can help
diminish the alterations induced by alcohol in retinal lipid
composition and structure.
After treating embryos with alcoholCsqualene we
recorded an increase in free cholesterol levels as compared
to control embryos, but these differences were not as marked
as they were in embryos treated only with alcohol. This
leads us to think that squalene dramatically reduces the
damage induced by alcohol. The protective role of squalene
against the effects of alcohol has not been studied up to now.
However, some authors have recorded that squalene-rich
diets increase cholesterol levels in the hepatic tissue
(Strandberg et al., 1989), but not in serum (Strandberg
et al., 1990). As Strandberg et al. (1989) have already
suggested, this effect may be caused by a squalene-induced
reduction of the HMG-CoA reductase activity while, at the
same time, squalene triggers ACAT in rat liver and
provokes an increase in the level of esterified cholesterol.
A similar effect can be observed in our research in the ACS
group as compared to the alcohol group.
Previous studies carried out in our laboratory prove that
the percentage of diacylglycerides increases in alcohol-
treated embryos with an absolute peak at E15 (Aguilera
et al., 2004), whereas, with the exception of E7, the ACS-
treated embryos show values statistically similar to those of
control embryos. As already said, the role of diacylglycer-
ides is to activate protein kinase C, a central stimulating
factor of a large number of biological processes involving
cell division, proliferation and differentiation. Thus, our
results suggest that in cases of alcohol consumption an
additional treatment with squalene could probably stabilize
the metabolic functions involving diacylglycerides.
As further supportive evidence of the protective role played
by squalene, in ACS-treated embryos phospholipids
behave much in the same way as diacylglycerides.
As already observed in previous studies on the main
types of phospholipids (Aguilera et al., 2004), the changes
induced on the lipid composition of the retina by an
intensive treatment with alcohol tend to disappear at E18.
541
However, the combined treatment with squalene speeds up
recovery with results similar to those recorded in control
embryos at earlier stages. It is true that there are still
differences with regard to the control group, but since these
are smaller, the data contribute to support the theory of the
protective role of squalene.
A detailed examination of the composition of fatty acids
from alcohol-treated embryos at day 7 shows that the
increase in saturated fatty acids, and the loss of oleic acid and
long-chain PUFAs such as C20:4(nK6) and C22:6(nK3)
have resulted in a more rigid membrane, how it was
suggested in previous studies (Aguilera et al., 2004). The
ACS-treatment of embryos provides results similar to those
recorded in control embryos, which leads us to conclude that
squalene contributes to maintaining membrane fluid. In
addition, both alcohol and ACS treated embryos show a
redistribution of free and esterified cholesterol. However,
total (freeCesterified) cholesterol content, which increases
in ethanol-treated embryos along development, is not
increased in ACS embryos. This fact, plus the reduced
increase in SPH in ACS embryos, compared to those treated
with ethanol, might help to explain squalene effects on
preserving membrane fluidity.
At E7 there is a considerable decrease in C22:6(nK3) in
alcohol-treated embryos, a PUFA which is essential for the
nervous system development and is related to functional
disorders, CNS developmental abnormalities and deterio-
rated visual acuity (Scott et al., 1988; Neuringer et al.,
1986). The results obtained in ACS-treated embryos, which
at E7 showed values close to those of control embryos, lead
us to conclude that a squalene-rich diet could be an effective
means to avoid the alcohol-induced loss of PUFAs.
Alcohol toxicity is mainly caused by the production of
free radicals in a number of tissues. Lipid membrane
peroxidation induced by free radicals causes irreversible
damage leading to cell death (Mantle and Preedy, 1999). Of
all tissues the retina has the highest oxygen consumption per
weight and has a very high pressure of oxygen. However,
oxygen can be very toxic for the retina. Although, the retina
possesses certain antioxidant components (e.g. vitamin E,
superoxide dismutase, selenium), the levels of glutathione
peroxidase and catalase (enzymes which destroy hydrogen
peroxide) are relatively low in this tissue (Fliesler and
Anderson, 1983, review). In this case and given its
antioxidant properties, diet squalene could be a protective
agent as an efficient captor of free oxygen and other reactive
oxygen species in the lipid peroxidation (Dessi et al., 2002;
Kohno et al., 1995). Heaton et al. (2000) have already
suggested that treatment with antioxidants could be an
effective therapy to prevent or diminish the damage of CNS
observed in FAS.
At the end of embryo development, some fatty acids
showed differences between control embryos and ACS-
treated embryos. However, these differences did not affect
to the percentages of total saturated, monounsaturated
and polyunsaturated fatty acids. Generally speaking,
542 Y. Aguilera et al. / Experimental Eye Research 80 (2005) 535–543
as development proceeds, the content of fatty acid tends to
be similar in the three groups (control, alcohol and ACS).
This suggests that either alcohol or ACS does not
significantly condition the final availability of fatty acids
in embryo tissues.
Although, there are numerous experiments proving that
alcohol provokes morphological alterations in the structure
of the retina (Aguilera et al., 2004; Ashwell and Zhang,
1994; Chmielewski et al., 1997; Parson et al., 1995; Parson
and Sojitra, 1995; Strömland and Pinazo-Duran, 1994), this
is the first survey which examines the effect of alcohol when
administered in combination with squalene.
From a morphological viewpoint the retinal structure of
embryos treated with ACS at E6 shows little changes from
E11 onwards as compared to the retinas of control embryos.
As a result, we think that squalene reduces the deleterious
effects induced by alcohol during retinal development. The
morphological recovery provoked by treatment with
squalene coincides with the results obtained in the lipid
analysis. The absence of immediate beneficial effects, i.e.
directly after the injection of ACS, is probably due to a
slower absorption of squalene as compared to alcohol or to a
subsequent metabolization by the embryo.
Given that the olive oil contains high proportions of
squalene in compared to other vegetable oils, the results of
this experiment also hint at the fundamental role which olive
oil could play as the main source of squalene in the diet to
prevent the neuronal and glial damages induced by an
excessive consumption of alcohol in the early stages of
pregnancy.
Acknowledgements
This work was supported by funds from the Comisión
Interministerial de Ciencia y Tecnologı́a (CICYT) (AGL2002-
00495) and (BFI2002-04481-C02-01); Consejerı́a de
Agricultura y Pesca (CAO01-002) and Red FIS.S de
investigatión cooperativa G03-140 (PREDIMED). This work
has received the prize ‘II Premio Nacional de la Asociación
Española de Municipios del Olivo a la Investigación sobre
Aceite y Salud 2003’.
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