Transcript Protection by Folic Acid and Betaine Ibrahim Sogut 1

Prenatal Alcohol-Induced Neuroapoptosis in Rat Brain:
Protection by Folic Acid and Betaine
Ibrahim Sogut1, Onur Uysal2, Aysegul Oglakci2, Ferruh Yucel2, Kazim Kartkaya2, Gungor Kanbak2
1 Istanbul
Bilim University, Buyukdere Cad. No:120 34394 Esentepe Sisli, Istanbul/TURKEY
2 Eskisehir Osmangazi University, Meselik Kampusu, Ataturk Blv, Osmangazi, Eskisehir/TURKEY
Background
Methods
Fetal 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.
Table 1.
Experimental Groups
 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 1000.7 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 -80°C 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.
Results
Figure 1a shows the change of cytochrome c release
(cytosolic/mitochondrial cytochrome c). On pups from ethanoladministered rats, the cytochrome c release at the cerebral cortex
(0.203±0.04) was significantly higher than the control (0.142±0.03)
and the ethanol+folic acid groups (0.145±0.03) (p<0.01, p<0.05,
respectively). Cytochrome c release level for ethanol+betaine group
(0.213±0.01) was significantly higher than the control (0.142±0.03),
ethanol+folic acid (0.145±0.03) and ethanol+betaine+folic acid groups
(0.156±0.01) (p<0.01, p<0.01 and p<0.05, respectively).
When the caspase-3 activities (umol pNA/minute/mg protein) of
the groups shown in Figure 1b were assessed statistically, it was
seen that the caspase-3 activity of the ethanol group (1.34±0.46) were
statistically significant (p<0.01) with respect to the control group
(0.85±0.16) and ethanol+betaine+folic acid group (0.84±0.09).
Figure 1c shows the change of calpain activity (pmol/min/mg
protein). The calpain level of ethanol group (5.45±1.36) was
significantly higher than control (3.49±1.03), ethanol+folic acid
(3.68±0.51) and ethanol+betaine+folic acid groups (3.45±0.65)
(p<0.01).
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).
Figure 3.
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 (p<0.05). Based on our findings, a statistically
significant reduction was found in the apoptotic cell count when
betaine is used alone and combined with folic acid (Figure 4 and
Figure 5).
Figure 1.
Figure 2a shows the change of cathepsin B levels (cytosolic /
lysosomal cathepsin B activity). There was not a significant
difference between groups (p>0.05). 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).
A statistically significant difference was not found between the
control group and ethanol group with respect to the mean diameters
of apoptotic cells. This finding suggests that the increased number of
apoptotic cells per unit volume does not derive from neuronal
morphology. On the contrary, mean cell diameters significantly
decreased in protective groups between ethanol group and
ethanol+betaine group and between ethanol group and ethanol+folic
acid+betaine groups. According to these data, it is observed that the
diameters of apoptotic neurons in the ethanol group significantly
decreased due to the protective effect.
Figure 2.
Figure 4.
Figure 5.
Conclusions
 It can be concluded that betaine and folic acid both alone or together may decrease neuroapoptosis on pups with FAS from the rats that were administered alcohol during pregnancy. This may be explained by the
methyl donating property of both folic acid and betaine and their functioning in parallel pathways.
 In addition to this, while folic acid effects on intracellular apoptotic pathways, betaine works on extracellular apoptotic pathways, thereby protecting the membranes.
 This study on brain tissue of newborn rat pups is important for decreasing the damage on children with FAS with betaine and folic acid supplementation, future studies will be necessary.
Acknowledgements
The authors would like to express their deepest gratitude to the FEBS-EMBO 2014 Committee
and Bilim University for their financial support to attend the congress. This work was supported by
TUBITAK-1002 109S510 project.
References
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