Prenatal Alcohol-Induced Neuroapoptosis in Rat Brain: Protection
by Folic Acid and Betaine Ibrahim Sogut 1, Onur Uysal 2, Aysegul
Oglakci 2, Ferruh Yucel 2, Kazim Kartkaya 2, Gungor Kanbak 2 1
Istanbul Bilim University, Buyukdere Cad. No: Esentepe Sisli,
Istanbul/TURKEY 2 Eskisehir Osmangazi University, Meselik Kampusu,
Ataturk Blv, Osmangazi, Eskisehir/TURKEY F etal alcohol syndrome
(FAS) was first defined as mental retardation and growth deficits
together with facial anomalies in newborns from chronic alcohol
consumer mothers. All over the world, 1-2 out of every 1000 births
are born with FAS. This ratio increases in populations with higher
welfare. It is stated in many studies that prenatal alcohol
consumption trigger apoptotic neurodegeneration. Betaine, being a
molecular chaperone, protects cells against oxidative stress,
mitochondrial damage and lowers SAH levels. Betaine is known to
diminish ethanol-dependent damage by increasing reduced glutathione
with trans-sulfuration. The calcium levels, oxidative stress and
neurotoxic homocysteine levels in the cell are affected by the
deficiency of folic acid. Depending on the change in homocysteine
level, DNA and mitochondrial damage occurs, caspase-3 levels change
leading to apoptotic cell death. In this study, alcohol-induced
neuroapoptosis in cerebral cortex of pups from rats that were given
alcohol during pregnancy, the protective effect of betaine in these
regions and the benefits of folic acid supplementation that was
required for normal brain development were investigated.Conclusions
Adult male and female Spraque-Dawley rats of the same age were used
for breeding. The presence of a vaginal plug was evidence of a
successful fertilization and the day that a positive plug was
present was regarded as E0. The animal model of prenatal ethanol
consumption was prepared by modifying Uzbay and Kayaalp method. The
rats were given a modified liquid diet (MLD) with or without
ethanol. Extra chow or water was not supplied. This mixture
contained kcal/l. Pregnant rats were given MLD without ethanol
between days P0-P7. Pups were killed by decapitation on day P7 and
a pool was prepared for each group (Table 1). Their cerebral cortex
was removed surgically. Tissues used for biochemical studies were
frozen in liquid nitrogen and kept at -80C until they were tested.
Blood Alcohol Concentration (BAC) is measured in grams in 100 ml of
blood. Cytochrome C levels were detected both in cytosolic and
mitochondrial fractions with a commercial kit. The ratio of
cytosolic fraction to mitochondrial fraction was used as an
indicator of mitochondrial damage. Caspase 3 activity was
determined according to the method of Zovein et al. Calpain
activity was determined according to the method of McDonald et al.
as the difference between the calcium-dependent fluorescence and
the non-calcium-dependent fluorescence. The ratio of the cathepsin
activities measured separately from cytosolic and lysosomal
fractions showed the amount of lysosomal integrity. The protein
concentration of homogenates were determined using the Bradford
assay. Histological examinations (Hematoxylin-Eosin staining, in
situ apoptosis detection, TUNEL, morphometric examinations) were
performed. All slides were scored by a histopathologist blinded to
the experimental group. SPSS 15.0 Windows program was used for
statistical analysis of data. MethodsBackground Results Overall
histological features and the tissue damage were examined on the
cerebral cortical region, where is stained with hemotoxylin and
eosin (H&E). Normal tissue appearance was observed in the
control group (Figure 3a). In the ethanol group, there was mild
congestion, moderate edema and severe necrosis and chromatolysis
(Figure 3b) together with some microglia/macrophage/MNL
infiltration (Figure 3c). Our findings demonstrated that folic acid
alone may exert anti-edematous effects and the combine use of folic
acid and betaine may induce a significant improvement for necrosis.
There was not a significant difference between the groups in terms
of PMNL and microglia/macrophage/MNL infiltrations (p>0.05)
(Figure 3d, 3e, 3f). For evaluating apoptotic cells at cerebral
cortex layer, Brown stained nuclei were defined as TUNEL (+). When
the apoptotic cell count was considered, a statistically
significant difference was found between ethanol and
ethanol+betaine groups and ethanol and ethanol+folic acid+betaine
groups (p0.05). The cathepsin B level of ethanol group (0.82 0.29)
was moderately higher than the control (0.63 0.11), ethanol+betaine
(0.66 0.17), ethanol+folic acid (0.61 0.14) and
ethanol+betaine+folic acid groups (0.66 0.17) but that was not
statistically significant (p>0.05). Figure 2b shows the change
of cathepsin L levels (cytosolic / lysosomal cathepsin L activity).
There was not a significant difference between the control (0.66
0.11), ethanol (0.74 0.14), ethanol+betaine (0.77 0.13),
ethanol+folic acid (0.71 0.14) and ethanol+betaine+folic acid
groups (0.75 0.20) (p>0.05). Figure 1. Figure 2. Figure 1a shows
the change of cytochrome c release (cytosolic/mitochondrial
cytochrome c). On pups from ethanol- administered rats, the
cytochrome c release at the cerebral cortex (0.2030.04) was
significantly higher than the control (0.1420.03) and the
ethanol+folic acid groups (0.1450.03) (p
