Prenatal alcohol-induced neuroapoptosis in rat brain: Protection by folic acid and betaine

(1)

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:120 34394 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 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.

Methods

Background

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 (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).

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 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).

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 (0.203±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). Figure 3. Figure 4. Figure 5. Table 1. Experimental Groups

 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

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and Bilim University for their financial support to attend the congress. This work was supported by TUBITAK-1002 109S510 project.