Download Figure 4 - İstanbul Bilim Üniversitesi

Melis Savasan Sogut 1 , Ibrahim Sogut 2, Aysegul Oglakci 3, Kazim Kartkaya 3, Kevser Kusat Ol 3, Gungor Kanbak 3, Mine Erden Inal 3
1
Yeditepe University, Department of Genetics and Bioengineering, 34755, Kayisdagi, Istanbul / TURKEY
2Istanbul Bilim University, Vocational School of Health Services, Department of Medical Services and Techniques, 34394, Istanbul / TURKEY
3 Eskisehir Osmangazi University, Faculty of Medicine, Department of Biochemistry, 26480, Eskisehir / TURKEY
Background
Depending on its acute or chronic usage and the dosage, alcohol has toxic effects on both mother and fetus when it is administered during pregnancy 1. Up till now, fetal alcohol spectrum disorders
(FASD) has been shown to include alcohol-related birth defects (ARBD), alcohol-related neurological disorders (ARND) and fetal alcohol syndrome (FAS). Children born with FAS have some difficulties
in learning, memory, attention span, communication, hearing and vision. In order to overcome this problem, new strategies should be developed to prevent the teratogenic effects of alcohol on fetus.
Oxidative stress has been shown to be related to many diseases including FAS 2. Oxidative stress leads to increasing lipid peroxidation and reactive oxygen species (ROS) formation that results in
malfunction of the enzymes and membrane disruption 3. Antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) have role in ROS detoxification.
Boric acid (BA) is an essential trace element for human beings, plants and animals that supports the metabolic events. BA forms complexes with glycolipids, glycoproteins and phosphoinositides that have
hydroxyl group on them. By this way, it affects membrane integrity, calcium chealator function and redox metabolism. Recent studies have demonstrated that BA has also antioxidant properties 4.
In this study, the aim was to investigate the prenatal alcohol-induced oxidative stress on rat cerebral cortex of newborn pups and assess the protective and beneficial effects of Boric acid (BA)
supplementation on rats with Fetal alcohol syndrome (FAS). As markers of alcohol-induced oxidative stress in cerebral cortex of newborn pups, MDA, SOD, CAT and GPx levels were measured.
Methods
Results
 10 adult male and 30 adult female Spraque-Dawley rats between 200-250 grams were used for
breeding in the present study. For mating, a male rat was randomly picked up and placed into a
female's cage. The females were checked each morning for the presence of a vaginal plug which was
evaluated as an evidence of fertilization. The day the plug was seen was regarded as E0. Only at 15
females vaginal plug was seen, so they were used to set up the three experimental groups.
 MDA levels (nmol/mg protein) in alcohol-group
(7.53 ± 1.41) was significantly increased (p<0.05)
compared to control level (6.11 ± 1.11). MDA level in
the alcohol+boric acid group (5.74 ± 1.22) showed a
significant decrease (p<0.01) compared to only-alcohol
group (Figure 2).
Figure 2. The effect of boric acid on MDA level
(nmol/mg protein) in rats exposed alcohol.
 There was no statistical difference among groups
(control group 4.73 ± 1.22, alcohol group 3.50 ± 0.92,
alcohol+boric acid group 4.31 ± 1.96) for SOD activities
(U/mg protein) (Figure 3).
 CAT activity (U/mg protein) of alcohol+boric acid
group (15.55 ± 3.65) was found higher compared
(p<0.05) to the alcohol group (10.80 ± 2.79). There was
no statistical diffference (p>0.05) among the control
group (13,90 ± 4,34) and alcohol group (10,80 ± 2,79)
(Figure 4).
Figure 1. Experimental Procedure
(E = Embryonic day and P = postnatal days; E0: First embryonic day, P0 = the first 24 h after birth)
 The animal model of prenatal alcohol consumption was modified from Uzbay and Kayaalp
methods 5. Control group was given an isocaloric pair-fed modified liquid diet containing sucrose as
a caloric substitude for alcohol (96 gr sucrose and 75 ml cow milk was replaced with 60.75 g or 75 ml
ethanol). Modified liquid diet (MLD) was given to the alcohol or alcohol + boric acid groups.
Figure 3. Comparing SOD (U/mg protein)
activity in groups.
 On days E15, E18 and E20, every morning 6 and 22 hours after administration of fresh diets, 20 µl
blood sample was taken from tails of the rats and blood alcohol concentration (BAC) was
determined (NAD-ADH Reagent Multiple Test Vial, Sigma).
 GPx activity (U/mg protein) in the alcohol
administered group (67.49±18.88) was decreased
(p<0.05) as compared to the control group (95.70 ±
25.85). Alcohol+boric acid group (85.67 ± 29.71) was not
significantly different (p>0.05) than other groups
(Figure 5).
 Lipid peroxidation was quantified at 532 nm by the measurement of MDA reacted with
thiobarbituric acid (TBA) according to the method of Ohkawa et al.6. The results were expressed as
nmol/mg protein. SOD activity was determined according to Winterborn et al. method 7. One unit of
SOD expressed as U/mg protein was designed as the amount of enzyme that inhibits the NBT
reduction by 50%. CAT activities were calculated by using the method of Beutler 8. The decrease in
optical density per minute was found and the enzyme activity was expressed as U/mg protein. GPx
activity as U/g protein was spectrophotometrically determined at 340 nm by using Paglia and
Valentine methods 9.
Figure 5. Comparing GPx (Glutathione
Peroxidase, U/mg protein) activity in groups.
Figure 4. The effect of boric acid on CAT activity
(U/mg protein) in rats exposed alcohol.
 SPSS 15.0 Windows program was used for statistical analysis of biochemical data.
Discussion & Conclusion
 According to our knowledge, this was the first study about the effect of BA administration on rats with FAS and the possible antioxidant mechanisms.
 A possible mechanism for the decline of alcohol-induced lipid peroxidation (LPO) might be the increasing levels of S-adenosyl methionine (SAM) with BA administration. Being a Lewis acid, BA may
form complexes with many biological compounds through its hydroxyl groups. It has high affinity for SAM that is known as a methyl donor in biochemical processes, a metabolite in transsulfuration
pathway and a precursor in sythesis of polyamines 10. In a previous study, chronic alcohol model decreased SAM levels while increasing S-adenosylhomocysteine (SAH) together with decreasing the
methylation capacity indicator ratio, SAM/SAH 11. In another study on boron deprivation in the liver, SAM level was reported to decrease while homocystein level increased 10. According to those
information, in the current study, our assumption is that a decline in LPO was related to an increase in SAM/SAH ratio, resulting in balancing the cell membrane functions.
 In this study, the increasing CAT activity after BA administration may stem from the hydroxyl ion acceptive property of BA (a Lewis acid). BA may have two crucial functions in this mechanism. Firstly,
it detoxifies hydrogen peroxides resulting from alcohol by converting it into peroxyboric acid. Secondly, BA converts CAT enzyme to its native state and maintains that enzyme.
 Taken together, alcohol-related oxidative stress on cell membrane could be decreased by BA supplementation. According to this study, BA had no impact on blood alcohol levels but it may be a great
candidate to protect and regulate the membrane in the rat brain. However, future studies will be needed to evaluate this recovery effect of BA on FAS.
Acknowledgements
The authors would like to express their deepest gratitude to Bilim University and Yeditepe
University for their financial support to attend the congress. To my beloved son, Deha…
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