Abstract
Abstract
Methods
Methods
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.
Acknowledgements
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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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.
Results
Results
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.
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 alcohol-induced oxidative stress.
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 1. Blood Alcohol Concentration Figure 1. Blood Alcohol Concentration
( Data shown are mean ± standard error.**
p<0,01)
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 Control Alcohol
Alcohol+
Boric acid Boric acid
ALT (U/L) Control 53,83±1,1 *** ** ns
Alcohol 66,5±0,96 *** * ***
Alcohol+Boric acid 60,9±1,75 ** * ***
Boric acid 52,06±1,71 ns *** ***
Table 3. AST activity (One way ANOVA test, Post Hoc Tukey
parametric multiple comparisons, ns: not significant)
Variables
Groups F(3:29)=0,98
P <0,4154 Mean±SE Control Alcohol
Alcohol+
Boric acid Boric acid
AST (U/L) Control 162,9±13,03 ns ns ns
Alcohol 191,5±11,77 ns ns ns
Alcohol+Boric acid 174,0±10,53 ns ns ns
Boric acid 165,7±15,79 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.
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 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 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.
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.
