Transcript Figure 1. - İstanbul Bilim Üniversitesi

The Antioxidant Effect of Boric Acid in
Chronic Alcohol Abuse
Ibrahim Sogut 1, Melike Ersoz 2, Sıla Ozlem Paltun 3, Melis Savasan Sogut 4, Canan Hurdag 3
1 Istanbul
Bilim University, Vocational School of Health Services, Department of Medical Services and Techniques, Istanbul/ Turkey
2 Istanbul Bilim University, Science and Arts Faculty, Department of Molecular Biology and Genetics, Istanbul/ Turkey
3 Istanbul Bilim University, Faculty of Medicine, Department of Histology and Embryology, Istanbul/ Turkey
4 Yeditepe University, Department of Genetics and Bioengineering, Istanbul/ Turkey
Abstract
Alcohol is a toxin that causes serious damage on many organs depending on the dose and duration of use. Chronic alcohol consumption is the most important factor that leads to cirrhosis and liver failure.
In this study, oxidative stress that was generated due to chronic alcohol intake and the protective effect of boric acid was evaluated.
Experimental animals were divided into four groups: control, alcohol, alcohol+boric acid and boric acid. The levels of alcohol-induced oxidative stress indicators malondialdehyde ((MDA), total sialic acid
(TSA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) were measured in liver tissues. While the MDA and TSA levels increased significantly in the alcohol group compared
to the control group (p<0.05, p<0.01), that of the alcohol+boric acid group decreased significantly compared to the alcohol group (p<0.01, p<0.001). The TSA level was significantly low in the boric acid group
as compared to the alcohol group (p<0.001). In the alcohol group, SOD and GPx activities were significantly lowered (p<0.01, p<0.001), while there was an increase in that of the alcohol + boric acid group
compared to the alcohol group (p<0.01, p<0.05). SOD and GPx activities increased significantly in the boric acid group compared to the alcohol group (p<0.01, p<0.001). There was no significant difference
between the groups in CAT activity. Consequently, these results show that alcohol triggers membrane damage on liver and boric acid can act to increase the antioxidant mechanisms against alcohol-induced
oxidative stress.
Methods
Male Sprague–Dawley rats of 250–300 g weights were used in our study. Four groups of 8 rats were prepared as control, alcohol, alcohol+boric acid and
boric acid groups. They were placed in a secluded, temperature- and humidity-controlled room (22±3˚C and 55±5% respectively) in which a 12:12 h
light-dark cycle was maintained. All experiments were carried out in accordance with institutional guidelines for animal welfare and were approved by the
local ethics committe of Bezmialem University.
 Rats in the control group received isocaloric dexrose solution by gavage.
 Rats in the alcohol and alcohol+boric acid groups were given 1.5 g/kg/day ethanol for the initial first week and 3 g/kg/day for second week and 6 g/kg/day
for another two weeks through gavage (1). The gradient increasing of ethanol concentration used in our study was to ensure the successful model and avoid
animal death.

The dose of boric acid was selected to be 100 mg/kg/day according to our previous study (2).
 Boric acid group received isocaloric dexrose solution + boric acid by gavage. 1 h after ethanol administration, rats of all groups were anesthetized by ketamine–xylazine mixture. Liver issues were
excised, frozen in liquid nitrogen and kept at -80 C for biochemical analysis.
 Blood alcohol concentration (BAC) were measured by Roche-HITACHI Cobas C 501 autoanalyzer. ALT and AST activities in the serum were measured by Roche-HITACHI Cobas c 311
autoanalyzer Lipid peroxidation was quantified at 532 nm by the measurement of malondialdehyde (MDA) reacted with thiobarbituric acid (TBA) according to the method of Ohkawa et al (3).
 TSA levels in liver homogenates were determined according to the method of Katopodis et al (4), which is based on the principle of measuring the color that resorcinol forms with SA at 580 nm.
 SA values were expressed in milligrams of SA per gram of wet weight. The results were expressed in nmol/mg protein.
 SOD activity was determined according to the method of Winterbourn et al (5). One unit of SOD expressed in U/mg protein was designated as the amount of enzyme that inhibits the reduction of
nitroblue tetrazolium reduction by 50% .
 CAT activities were calculated using the method of Beutler (6). The reduction in optical density per minute was determined and the enzyme activity was expressed in U/mg protein.
 GPx activity in U/g protein was spectrophotometrically determined at 340 nm using the methods of Paglia and Valentine (7).
 The protein concentration of homogenates gathered from liver tissues were determined using the Bradford assay (8).
 SPSS software, version 22 for Windows (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis of biochemical data. In order to assess differences between groups, one-way analysis of
variance (ANOVA) and Tukey’s multiple comparison test were used. Results are presented as mean ± standard deviation and P<0.05 was considered to indicate a statistically significant result.

Results
Table 1. Blood Alcohol Concentration
Groups
Mean ± SE
t TEST, Mann-Whitney U p<0,0016
Alcohol
191,1 ± 13,53
p<0,01
Alcohol+Boric acid
127,4 ± 6,67
Table 2. ALT activity (One way ANOVA test, Post Hoc Tukey
parametric multiple comparisons, * P<0,05; ** P<0,01; ***P<0,001;
ns: not significant)
Variables
Groups F(3:29)=20,76
P <0,0001
Mean±SE
ALT (U/L)
Control
53,83±1,1
Alcohol
66,5±0,96
***
Alcohol+Boric acid
60,9±1,75
**
*
Boric acid
52,06±1,71
ns
***
Control
Alcohol
Alcohol+
Boric acid
Boric acid
***
**
ns
*
***
Variables
AST (U/L)
F(3:29)=0,98
P <0,4154
Control
Alcohol
Alcohol+Boric acid
Boric acid
Mean±SE
162,9±13,03
191,5±11,77
174,0±10,53
165,7±15,79
Control
Figure 5. Effect of boric acid on
the activity of catalase (CAT) in
rats exposed to alcohol. There
were statistically insignificant
(P>0.05) differences among the
groups Data shown are mean ±
error. Control 138±21,7; Alcohol
88,8±10,0; Alcohol+Boric acid
135,7±12,8; Boric acid 132,5±12,1.
***
Alcohol
ns
ns
ns
ns
Figure 2. Effect of boric acid on
malondialdehyde (MDA) levels in rats
exposed
to
chronic
alcohol. *P<0.05; **P<0.01. Data shown are
mean ± error. Control 4,95±0,21; Alcohol
6,08±0,29; Alcohol+Boric acid 4,87±0,17;
Boric acid 5,30±0,25.
***
Table 3. AST activity (One way ANOVA test, Post Hoc Tukey
parametric multiple comparisons, ns: not significant)
Groups
Figure
4.
Comparison
of
superoxide dismutase (SOD)
activity among the groups. **
P<0,01. Data shown are mean ±
error. Control 7,05±0,36; Alcohol
4,33±0,17; Alcohol+Boric acid
6,76±0,69; Boric acid 6,92±0,59.
Figure 1. Blood Alcohol Concentration
( Data shown are mean ± standard error.**
p<0,01)
ns
ns
Alcohol+
Boric acid
ns
ns
Figure 3. Comparing SA (nmol/dl) levels
among the groups. ** P<0,01; ***P<0,001.
Data shown are mean ± error. Control
99,37±2,35;
Alcohol
125,50±5,11;
Alcohol+Boric acid 90,25±4,28; Boric acid
96,00±4,46.
Figure
6.
Comparison
of
glutathione peroxidase (GPx)
among the groups. * P<0,05; **
P<0,01; ***P<0,001. Data shown
are mean ± error. Control
42,6±1,4;
Alcohol
25,6±1,6;
Alcohol+Boric acid 33±1,3; Boric
acid 40,1±1,6.
Boric acid
ns
ns
ns
ns
Discussion & Conclusion
 To the best of our knowledge, this is the first study concerning the effect of BA administration on rats with chronic alcohol abuse and the possible antioxidant mechanisms.
 The constitutive formation of oxidants can be balanced by the production of antioxidants at a similar rate. The imbalance between oxidants and antioxidant species causes oxidative stress,
resulting from the peroxidation of lipids.
 Enzymatic (SOD, CAT and GPx) and non-enzymatic (thiol and GSH) antioxidants have important roles in preventing the damage resulting from alcohol-induced ROS (9). At present, the
complete antioxidant mechanism of BA is not fully understood however, BA is a well-known component of cell membrane functions and enzymatic reactions (10).
 According to this information, in the current study, it is hypothesized that a decline in lipid peroxidation was associated with an increase in the SAM/SAH ratio and GSH, resulting in balancing
of the cell membrane functions. Consequently, these results show that alcohol triggers membrane damage on liver and boric acid can act to increase the antioxidant mechanisms against alcoholinduced oxidative stress.
Acknowledgements
The authors would like to express their deepest gratitude to the FEBS 2015 Committee
and Bilim University for their financial support to attend the congress. This work was supported by
TUBITAK-113S546 project.
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