e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 1019–1026
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Effects of lead nitrate and sodium selenite on DNA damage and oxidative stress in diabetic and non-diabetic rat erythrocytes and leucocytes Hatice Bas¸ a,∗ , Yusuf Kalender b , Dilek Pandir a , Suna Kalender c a b c
Bozok University, Faculty of Arts and Science, Department of Biology, 66100 Yozgat, Turkey Gazi University, Faculty of Science, Department of Biology, 06500 Ankara, Turkey Gazi University, Gazi Education Faculty, Department of Science Education, 06500, Ankara, Turkey
a r t i c l e
i n f o
a b s t r a c t
Article history:
The adverse effects of lead nitrate (LN) and the preventive role of sodium selenite were inves-
Received 23 December 2014
tigated in diabetic and non-diabetic rat blood by measuring trolox equivalent antioxidant
Received in revised form
capacity (TEAC), ferric reducing antioxidant power (FRAP), malondialdehyde (MDA) levels
17 March 2015
and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)
Accepted 19 March 2015
and glutathione-S-transferase (GST) also by evaluating DNA damage with comet assay. LN
Available online 27 March 2015
increased the levels of MDA, tail DNA%, mean tail length and tail moment, decreased the enzymes activities, FRAP and TEAC values. In sodium selenite + LN group, we observed the
Keywords:
protective effect of sodium selenite on examining parameters. Diabetes caused alterations
Lead
on these parameters, too. We found that sodium selenite did not protect against diabetes
Selenium
caused damages. As a result, LN caused toxic effects on blood cells and sodium selenite alle-
Diabetes
viated this toxicity but it did not show preventive effect against diabetes. Also, LN caused
Oxidative stress
more harmfull effects in diabetic groups than non-diabetic groups. © 2015 Elsevier B.V. All rights reserved.
Antioxidant enzyme DNA damage
1.
Introduction
Metals have important roles in the functioning of enzymes and cell signaling processes. Increasing concern has been expressed about the rapidly rising level of metals in the environment, specially lead, which has well-known harmfull effects (Dewanjee et al., 2013). Lead can hinder biological functions by changing the molecular interactions, cell signaling processes and cellular function. LN is leads to a broad range of biochemical, physiological and behavioral dysfunctions (Lakshmi et al., 2013).
∗
Selenium has substantial roles in many biological processes and it prevents against so many diseases such as cancers and cardiovascular disease. It is suggested that selenium can support antioxidant capacity by increasing activities of antioxidant enzymes and by enhancing levels of the antioxidants (Kalender et al., 2013). Tissues of selenium deficient animals are more susceptible to oxidative stress when compared to control animals. Selenium addition increased thioredoxin reductase and GPx activities and resulted in improved recovery of functions of organs after damages (Venardos et al., 2004).
Corresponding author. Tel.: +90 354 242 10 21/255; fax: +90 354 242 10 22. E-mail address: [email protected] (H. Bas¸).
http://dx.doi.org/10.1016/j.etap.2015.03.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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Several studies have shown that diabetes is associated with enhanced generation of free oxygen radicals and decrease in antioxidant ability due to these events. There are so many alterations in diabetic people that are associated with oxidative stress like products of glycation (Diabetes Care, 2010) and hyperglycemia which makes an imbalance in cellular oxidation and reduction (Farokhi et al., 2012). Against oxidative damage, human body has enhanced various defence mechanisms, involving enzymatic and nonenzymatic systems. SOD, CAT, GPx and GST are important antioxidant enzymes (Bas¸ et al., 2014). To evaluate the genotoxic effects there are several test systems. Comet assay is used to determine the potential genotoxicity. This assay is an identifier of effects of carcinogen exposure. It has been proved to be good marker of DNA damage (Zengin et al., 2011). The FRAP and TEAC assays are simple and cheap procedures which measure the total antioxidant levels (Nawirska-Olszanska et al., 2013). Due to the widespread use of lead, it is important to determine the harmful effects of lead on living things. In recent years, in terms of antioxidant properties selenium is one of the widely studied materials. Also, there are no studies for propound the role of sodium selenite against LN induced oxidative stress in diabetic and non-diabetic rat blood. Therefore, this study was done to investigate the effects of LN and sodium selenite on values of FRAP and TEAC, MDA levels and the activities of SOD, CAT, GPx and GST, tail DNA%, mean tail length and tail moment in blood cells of rats.
2.
Materials and methods
FRAP and TEAC values were quantified in plasma, SOD, CAT, GPx, GST, MDA were studied in erythrocytes and leucocytes, DNA damage was measured in leucocytes.
2.1.
Animals and chemicals
The protocol was approved by the Gazi Univ. Animal Experiments Local Ethics Committee (G.Ü.ET–11.028). Wistar rats (200–250 g), were obtained from the Gazi Univ. Laboratory Animals Growing and Experimental Research Center, Ankara, Turkey. They were supplied with standard laboratory chow and water ad libitum at 22 ± 2 ◦ C. Procedures were performed in accordance with international guidelines for care and use of laboratory animals. Streptozotocin (STZ), LN, sodium selenite and other chemicals were procured from Sigma Aldrich. LN and sodium selenite were dissolved in distilled water (Sharma et al., 2010; Sakr et al., 2011).
2.2.
Animal grouping and treatment
Eight groups (6 animals in each group) were formed for this study. These groups were control, sodium selenite, LN, sodium selenite + LN, diabetic control, diabetic sodium selenite, diabetic LN, diabetic sodium selenite + LN. During the experimental period (28 days) 1 ml/kg b.w (body weight) distilled water for control groups, 1 mg/kg b.w sodium selenite for sodium selenite treatment groups and 22,5 mg/kg b.w (1/100 LD50 ) LN (Sharma et al., 2010) for LN treatment groups were
given to rats daily via gavage. Animals were rendered diabetic by a single i.p. injection of STZ (55 mg/kg). STZ was prepared in 1 ml of freshly prepared sodium citrate buffer at 4.5 pH. After 2 days, the blood glucose levels were measured with a glucometer and whose levels were 300 mg/dl or higher, they selected for diabetic groups (Schmatz et al., 2009).
2.3.
Preparation of erythrocytes
Blood samples were collected into heparinized tubes after treatment. Erythrocytes were separated from plasma by centrifugation (1600 rpm for 5 min) and then washed with a cold isotonic saline solution. After separation, erythrocytes were suspended in phosphate buffer at pH 7.4 to obtain a 50% cellular suspension. Erythrocytes were destroyed by osmotic pressure and the resulting mixtures were subjected to centrifugation. Supernatants were isolated, and MDA levels and enzyme activities were measured by Shimadzu UV-1800, Japan. The concentration of hemoglobin was determined by the method of Drabkin (1946).
2.4.
Preparation of leucocytes
30 ml of blood was collected in sodium citrate and mixed with 6% dextran in isotonic saline and allowed to stand for 30 min. The upper layer was transferred to other tube which is containing 2.25 ml EDTA and centrifuged at 1000 rpm. Then the leucocytes were washed with Tris buffer at 4 ◦ C (Chen et al., 1997). After this step MDA levels and enzyme activities were measured by Shimadzu UV-1800, Japan.
2.5.
Measurement of MDA levels
MDA is the most abundant aldehyde resulting from LPO in biological process. For determination of MDA levels cells were incubated with thiobarbituric acid (TBA) at 95 ◦ C (pH 3.4). MDA reacts with TBA to form a colored complex. MDA levels were specified at 532 nm (Ohkawa et al., 1979).
2.6.
Antioxidant enzyme assays
The activity of SOD was measured assaying the autooxidation and illumination of pyrogallol by the method of Marklund and Marklund (1974). This reaction was monitored at 440 nm. For CAT activity samples were diluted with Triton X-100. The activity was measured as the changing rate of H2 O2 decomposition at 240 nm according to study of Aebi (1984). GPx activity was determined with H2 O2 as substrate. Reaction mixtures contained reduced glutathione, glutathione reductase, NADPH and Tris–HCl. GPx activity was measured as the change in absorbance at 340 nm (Paglia and Valentine,1967). Activity of GST was analysed by measuring the formation of the glutathione and 1-chloro-2,4-dinitrobenzene conjugate. The increase in absorbance was recorded at 340 nm (Habig et al., 1974).
2.7.
FRAP assay
The antioxidant capacity was evaluated by FRAP assay according to the method of Benzie and Strain (1996). This assay
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 1019–1026
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Fig. 1 – Effects of LN and sodium selenite on FRAP values (mol/L). Each bar represents mean ± SD in each group. Columns superscripts with different letters are significantly different. Significance at P < 0.05.
measures the changes in absorbance at 593 nm due to the generation of FeII -tripyridyltriazine compound from oxidised FeIII form. The FRAP reagent was prepared by mixing 300 mmol/L acetate buffer with 10 mmol/L 2,4,6- tripyridyl-s-triazine in 40 mmol/L HCl and with 20 mmol/L ferric chloride.
2.8.
TEAC assay
The TEAC assay is based on the inhibition of the absorbance of the ABTS.+ by tested antioxidant (Re et al., 1999). Solution of ABTS.+ was diluted with phosphate buffer saline (PBS) to an absorbance of 0.70 (± 0.02) at 734 nm. When we add the diluted ABTS.+ to biological samples the mixtures was incubated for 6 min. Then at 734 nm, the decrease in absorbance was measured.
2.9.
Determination of DNA damage (comet assay)
The cells were suspended in low melting point agarose (0.65%) and 75 l of suspension was layered over microscope slides which were precoated with normal melting point agarose
(0.65%) then they covered with a coverglass and the slides were placed at +4 ◦ C. After rigidification, the coverglass was removed and immersed in lysing solution. After this step, the slides were removed placed on a gel electrophoresis platform covered with electrophoresis buffer and they were left in the solution for 20 min to allow the unwinding of the DNA (Ozkan et al., 2009). The slides were then placed in a neutralizing tank and washed with neutralizing buffer. Ethidium bromide was dispensed directly onto slides and covered with a coverglass. Data were analyzed using BS 200 ProP with software (BAB Bs Comet Assay software) image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey).
2.10.
Statistical analyses
Statistical analyses were performed using SPSS, version 11.0 for Windows. To evaluation the differences, one way analysis of variance (ANOVA) followed by Tukey multiple comparison were used. P < 0.05 was considered statistically significant. All data was described as the means ± standard deviation (S.D).
Fig. 2 – Effects of LN and sodium selenite on TEAC values (mol/L). Each bar represents. mean ± SD in each group. Columns superscripts with different letters are significantly different. Significance at P < 0.05.
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Fig. 3 – Comet appearances of the LN and/or Sodium selenite treated blood in rats (A) control, (B) Sodium selenite, (C) LN, (D) Sodium selenite + LN, (E) diabetic control, (F) diabetic Sodium selenite, (G) diabetic LN, (H) diabetic Sodium selenite + LN.
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Table 1 – Evaluated mean values of MDA levels and antioxidant enzyme activities of LN and/or sodium selenite treatment diabetic and non-diabetic rat erythrocytes. Groups
MDA (nmol/mgHb)
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
6.04 6.25 11.31 8.37 8.07 7.92 15.91 10.98
± ± ± ± ± ± ± ±
a
0.22 0.41a 0.54b 0.42c 0.11c 0.63c 0.79d 0.66 b
SOD (U/mgHb)
CAT (U/mgHb)
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
612.04 624.50 510.45 589.52 594.04 598 424.7 516.5
a
5.03 7.47a 9.12b 8.06c 4.03c 5.83c 9.85d 12.12b
323.5 330.25 241.46 297.17 301.9 295.29 201.6 232.19
GPx (U/mgHb)
a
3.82 5.11a 5.33b 4.34c 4.03c 6.45c 6.30d 4.65b
33.8 35.51 23.68 28.21 27.23 27.85 19.96 24.27
± ± ± ± ± ± ± ±
a
0.31 1.13a 1.37b 0.67c 1.01a 0.91a 1.1b 1.44c
GST (U/mgHb) 19.8 18.5 11.13 15.1 14.8 15.41 8.18 10.24
± ± ± ± ± ± ± ±
1.01a 0.83a 0.7b 0.71c 0.32a 1.2a 0.51b 1.16c
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
Table 2 – Evaluated mean values of MDA levels and antioxidant enzyme activities of LN and/or sodium selenite treatment diabetic and non-diabetic rat leucocytes. Groups
MDA(nmol/mgprotein) SOD (U/mgprotein) CAT (U/mgprotein) GPx (U/mgprotein) GST (U/mgprotein)
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
1.27 ± 0.11a 1.16 ± 0.14a 2.9 ± 0.16b 2.01 ± 0.17c 2.11 ± 0.1c 2.24 ± 0.13c
348.12 ± 6.23a 343.71 ± 8.14a 256.67 ± 6.92b 296.77 ± 9.61c 321.21 ± 7.27c 314.6 ± 8.53c
223.75 ± 4.62a 214.69 ± 5.87a 161.22 ± 6.03b 187.37 ± 4.91c 184.21 ± 7.83c 175.06 ± 5.28c
41.33 ± 2.11a 37.18 ± 3.05a 27.02 ± 1.63b 30.23 ± 1.12c 32.6 ± 1.2a 30.52 ± 1.87a
8.17 ± 0.37a 8.62 ± 0.52a 6.46 ± 0.3b 7.28 ± 0.41c 7.31 ± 0.32a 7.35 ± 0.28a
3.5 ± 0.12d 3.08 ± 0.16 b
219.68 ± 11.57d 254.43 ± 6.04b
141.34 ± 6.01d 166.9 ± 4.11b
20.65 ± 0.83b 26.71 ± 1.66c
5.02 ± 0.55b 6.39 ± 0.43c
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
Table 3 – Estimated mean values of tail DNA%, tail length and tail moment of comets by image analysis on treatment groups in rats blood. Groups
Tail DNA%
Tail length
Tail moment
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
57.94 ± 5.09 a 64.25 ± 2.51a 91.51 ± 4.04b 80.79 ± 1.54c 77.97 ± 5.10c 79.00 ± 2.61c 99.81 ± 3.19d 89.17 ± 1.64 b
17.04 ± 1.03a 20.50 ± 2.47a 68.50 ± 2.12b 45.50 ± 1.06c 41.04 ± 4.03c 43.00 ± 2.83c 118.50 ± 19.85d 65.50 ± 2.12b
13.75 ± 0.91a 13.29 ± 2.10a 55.66 ± 3.51b 40.37 ± 1.24 c 37.90 ± 2.93c 35.22 ± 5.45c 121.66 ± 24.30d 52.58 ± 2.12b
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
3.
Results
There were no statistically significant changes in values of FRAP and TEAC, levels of MDA, in enzyme activities and in tail DNA%, mean tail length and tail moment between the sodium selenite group compared with the control also between diabetic sodium selenite group compared with the diabetic control for erythrocytes and leucocytes (P > 0.05, Figs. 1–3 and Tables 1–3).
3.1.
MDA levels of erythrocytes and leucocytes
Treatment with LN increased the level of MDA compared with control significantly. The MDA levels were decreased in the sodium selenite + LN treated group compared to LN
treated group (P < 0.05). Also, similar values were evaluated in diabetic control, diabetic sodium selenite, diabetic LN and diabetic sodium selenite + LN treated groups. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group, a significant increasing in levels of MDA were determined in diabetic groups (Tables 1 and 2).
3.2. Antioxidant enzyme activities of erythrocytes and leucocytes SOD, CAT, GPx and GST activities were measured in all groups of rats. Decreasing in SOD, CAT, GPx and GST activities were detected in erythrocytes and leucocytes in LN treated rats.
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The activity of enzymes was increased in sodium selenite + LN treated group compared to LN group, significantly (P < 0.05). Similar results were searched also in diabetic control, diabetic sodium selenite, diabetic LN and diabetic sodium selenite + LN treated groups. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group significant reduction in enzyme activities were observed in diabetic groups (Tables 1 and 2).
3.3.
Values of FRAP and TEAC
The changes observed in FRAP and TEAC levels are shown in Figs. 1 and 2, respectively. The values of FRAP and TEAC were found to decrease significantly in LN treated rats (P < 0.05) when compared to the control. There was a significant increase in FRAP and TEAC levels of sodium selenite + LN rats when compared to the LN group. Similar results were observed between diabetic groups, too. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group decreasing in values of FRAP and TEAC were observed significantly in diabetic animals.
3.4.
Commet assay results
According to comet assay results the tail moment, mean tail length and tail DNA% significantly increased with LN treatment when compared to control. It was observed decreasing of these values in sodium selenite + LN treated group. Similar values were observed between diabetic groups, too. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group increasing in values of the tail moment, mean tail length and tail DNA% were observed significantly in diabetic animals (Table 3). The figures of the comet assay were observed in Figure 3 for non-diabetic and diabetic groups.
4.
Discussion
In our work LN caused reduction in activities of SOD, CAT, GPx and GST in erythrocytes and leucocytes. There are studies proving that lead cause decreasing in enzyme activities (Liu et al., 2010; Haleagrahara et al., 2010). There is a study indicating that the lead binds to proteins and causes changes in several enzyme activities (Mehana et al., 2012). The antioxidant enzyme activities have been used to determine the oxidative stress in other studies (Liu et al., 2010). Changes reported in this paper about reducing in these enzyme activities following LN treatment may have resulted from increasing generation of free radicals. In present study our MDA level results support this probability of the LN treated groups. Recent in vivo studies in lead treated animals showed the generation of ROS, stimulation of LPO and decreased antioxidant defense system supporting the function of oxidative stress in lead toxicity (Haleagrahara et al., 2010).
We measured MDA level for evaluation of oxidative stress caused by LPO in erythrocytes and leucocytes. Increased MDA levels were determined in LN treated rats in this study. There are lots of studies showing that heavy metals cause increasing in MDA levels (Mehana et al., 2012; Dewanjee et al., 2013). Previous studies indicated that MDA may be increase due to adverse effects of LN on fatty acids of cell membranes (Haleagrahara et al., 2010). Comparison of different analytical methods is helpful for better understanding and interpretation of the results about the antioxidant capacity of a sample (Katalinic et al., 2005). Therefore, FRAP and TEAC assays were studied in this work in addition to antioxidant enzyme activity assays. In this study as a result of appling LN, it was observed significant decrease of FRAP and TEAC values. These results reinforce the findings about antioxidant enzyme activities. During the last years, determination on damage of DNA caused by toxins has been performed by comet assay (Zengin et al., 2011). So in this study, the comet assay was used to examine the DNA damage of rat leucocytes when they are exposed to LN. In our study we observed significant increasing in tail DNA%, mean tail length and tail moment, indicating DNA damage, in LN treatment rat leucocytes when compared to control. Oxidative stress and depletion of antioxidant defence system have important roles in the pathogenesis of diabetes mellitus. There are some investigations about diabetes caused tissue injury that it is based on breaking oxidant/antioxidant balance (Suresh et al., 2013). In this study, compared with diabetic groups with non-diabetic groups, we were observed decreasing in values of FRAP, TEAC and activities of SOD, CAT, GST, GPx and increasing in MDA levels and in tail DNA%, mean tail length and tail moment in diabetic rats. Previous experimental studies have been proved that in diabetic rats increased LPO and altered antioxidant enzymes such as SOD, CAT, GPx and GST (Shanmugam et al., 2011; Suresh et al., 2013). Kuppusamy et al. (2005) reported a significantly lower FRAP level in type 2 diabetic patients and Colak et al. (2005) documented a similar decrease in type 2 diabetic patients, too. Also when we compared LN group with diabetic LN group, we were determined that there were more alterations in examining parameters in diabetic LN group. Thus, we can say diabetic rats are more susceptible to LN induced oxidative stress than non-diabetic rats. In the present study we observed sodium selenite has protective effects against LN induced adverse effects on examining parameters of blood cells of rats. It is known that selenium has important roles about detoxification of toxic heavy metals (Kalender et al., 2013). Sodium selenite becomes protective because of its antioxidant properties, like other studies (Orun et al., 2008; Kalender et al., 2013). It may be indirectly scavenger of ROS or increase antioxidant enzyme activities; therefore it may prevent the toxicity produce by LN. Accumulating evidences have shown that lead induces oxidative stress by inducing the producing of ROS (Dai et al., 2010). Extreme generation of ROS can cause the damages of DNA, proteins and lipids and this situation led to adverse effects (Liu et al., 2010). In this study, we determined that exposure to LN, induced over-production of ROS and led to oxidative damage
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in rats. Whereas, selenium significantly reverse LN caused toxicity by regulating the ROS level in the blood cells of rats.
5.
Conclusion
In this study, even though LN was given orally at low dose to diabetic and non-diabetic rats, MDA, FRAP, TEAC levels, DNA damage and enzymatic changes were observed in the rat blood cells, but none of the rats died during the experimental period. From the data of this study, it was evident that LN caused toxicity, including LPO and disturbances in antioxidant enzyme activities, DNA damages both diabetic and non-diabetic rats. These results were probably due to generation of ROS, causing damage to cell membranes. With the present study we determined the protective effects of sodium selenite on LN caused oxidative stress in human erythrocytes and leucocytes. Results showed important toxic changes in the diabetic groups, which could be due to oxidative damage in diabetes because oxidative stress has an important role play in the pathogenesis of diabetes. Data in this study showed that sodium selenite did not alleviate diabetes induced unwanted effects but it has beneficial effects on LN mediated toxicity, but not protect completely.
Conflict of Interest The authors have nothing to disclose.
references
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Bas¸, H., Kalender, S., Pandır, D., 2014. In vitro effects of quercetin on oxidative stress mediated in human erythrocytes by benzoic acid and citric acid. Folia Biol. (Krakow) 62 (1), 59–66. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Chen, L., Haught, W.H., Yang, B., Saldeen, T.G.P., Parathasarathy, S., Mehta, J.L., 1997. Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine. J. Am. Coll. Cardiol. 30 (2), 569–575. Colak, E., Majkic-Singh, N., Stankovic, S., Sreckovic Dimitrijevic, V., Djordjevic, P.B., Lalic, K., Lalic, N., 2005. Parameters of antioxidative defense in type 2 diabetic patients with cardiovascular complications. Ann. Med. 37, 613–620. Dai, W., Fu, L., Du, H., Liu, H., Xu, Z., 2010. Effects of montmorillonite on Pb accumulation oxidative stress, and DNA damage in Tilapia (Oreochromis niloticus) exposed to dietary Pb. Biol. Trace Elem. Res. 136, 71–78. Dewanjee, S., Sahu, R., Karmakar, S., Gangopadhyay, M., 2013. Toxic effects of lead exposure in Wistar rats: Involvement of oxidative stress and the beneficial role of edible jute (Corchorus olitorius) leaves. Food Chem. Toxicol. 55, 78–91. Diabetes Care, 2010. Excecutive summary, standards of medical care in diabetes. 33, 4–10. Drabkin, D.I., 1946. Spectrophotometric studies. XIV, the crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. J. Biol. Chem. 164, 703–723. Farokhi, F., Farkhad, N.K., Togmechi, A., Band, K.S., 2012. Preventive effects of Prangos ferulacea (L.) lindle on liver
1025
damage of diabetic rats induced by alloxan. Avicenna J. Phytomed. 2 (2), 63–71. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Haleagrahara, N., Jackie, T., Chakravarthi, S., Rao, M., Pasupathi, T., 2010. Protective effects of Etlingera elatior extract on lead acetate-induced changes in oxidative biomarkers in bone marrow of rats. Food Chem. Toxicol. 48, 2688–2694. Kalender, S., Uzun, F.G., Demir, F., Uzunhisarcıklı, M., Aslanturk, A., 2013. Mercuric chloride-induced testicular toxicity in rats and the protective role of sodium selenite and vitamin E. Food Chem. Toxicol. 55, 456–462. Katalinic, V., Modun, D., Music, I., Boban, M., 2005. Gender differences in antioxidant capacity of rat tissues determined by 2,2V-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comp. Biochem. Physiol. 140, 47–52. Kuppusamy, U.R., Indran, M., Rokiah, P., 2005. Glycaemic control in relation to xanthine oxidase and antioxidant indices in Malaysian Type 2 diabetes patients. Diabet. Med. 22, 1343–1346. Lakshmi, B.V.S., Sudhakar, M., Aparna, M., 2013. Protective potential of Black grapes against lead induced oxidative stress in rats. Environ. Toxicol. Pharmacol. 35, 361–368. Liu, C., Ma, J., Sun, Y., 2010. Quercetin protects the rat kidney against oxidative stress mediated DNA damage and apoptosis induced by lead. Environ. Toxicol. Pharmacol. 30, 264–271. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Mehana, E.E., Meki, A.R., Fazili, K.M., 2012. Ameliorated effects of green tea extract on lead induced liver toxicity in rats. Exp. Toxicol. Pathol. 64, 291–295. Nawirska-Olszanska, A., Kita, A., Biesiada, A., Sokol-Letowska, A., Kucharska, A.Z., 2013. Characteristics of antioxidant activity and composition of pumpkin seed oils in 12 cultivars. Food Chem. 139, 155–161. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Orun, I., Selamoglu Talas, Z., Ozdemir, I., Alkan, A., Erdogan, K., 2008. Antioxidative role of selenium on some tissues of (Cd2+ , Cr3+ )-induced rainbow trout. Ecotox. Environ. Safe. 71, 71–75. Ozkan, D., Yuzbasıoglu, D., Unal, F., Yılmaz, S., Aksoy, H., 2009. Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate. Cytotechnology 59, 73–80. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantative and qualitative characterization of glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radical Biol. Med. 26, 1231–1237. Sakr, S.A.R., Mahran, H.A., Nofal, A.E., 2011. Effect of selenium on carbimazole induced testicular damage and oxidative stress in albino rats. J. Trace Elem. Med. Biol. 25, 59–66. Schmatz, R., Mazzanti, C.M., Spanevello, R., Stefanello, N., Gutierres, J., Corrêa, M., da Rosa, M.M., Rubin, M.A., Schetinger, M.R.C., Morsch, V.M., 2009. Resveratrol prevents memory deficits and the increase in acetylcholinesterase activity in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 610, 42–48. Shanmugam, K.R., Mallikarjuna, K., Nishanth, K., Kuo, C.H., Reddy, K.S., 2011. Protective effect of dietary ginger on antioxidant enzymes and oxidative damage in experimental diabetic rat tissues. Food Chem. 124, 1436–1442.
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Sharma, V., Sharma, A., Kansal, L., 2010. The effect of oral administration of Allium sativum extracts on lead nitrate induced toxicity in male mice. Food Chem. Toxicol. 48, 928–936. Suresh, S., Prithiviraj, E., Lakshmi, N.V., Ganesh, M.K., Ganesh, L., Prakash, S., 2013. Effect of Mucuna pruriens (Linn.) on mitochondrial dysfunction and DNA damage in epididymal sperm of streptozotocin induced diabetic rat. J. Ethnopharmacol. 145, 32–41.
Venardos, K., Harrison, G., Headrick, J., Perkins, A., 2004. Effects of dietary selenium on glutathione peroxidase and thioredoxin reductase activity and recovery from cardiac ischemia–reperfusion. J. Trace Elem. Med. Biol. 18, 81–88. Zengin, N., Yuzbasıoglu, D., Unal, F., Yılmaz, S., Aksoy, H., 2011. The evaluation of the genotoxicity of two food preservatives: sodium benzoate and potassium benzoate. Food Chem. Toxicol. 49, 763–769.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Effects of lead nitrate and sodium selenite on DNA damage and oxidative stress in diabetic and non-diabetic rat erythrocytes and leucocytes Hatice Bas¸ a,∗ , Yusuf Kalender b , Dilek Pandir a , Suna Kalender c a b c
Bozok University, Faculty of Arts and Science, Department of Biology, 66100 Yozgat, Turkey Gazi University, Faculty of Science, Department of Biology, 06500 Ankara, Turkey Gazi University, Gazi Education Faculty, Department of Science Education, 06500, Ankara, Turkey
a r t i c l e
i n f o
a b s t r a c t
Article history:
The adverse effects of lead nitrate (LN) and the preventive role of sodium selenite were inves-
Received 23 December 2014
tigated in diabetic and non-diabetic rat blood by measuring trolox equivalent antioxidant
Received in revised form
capacity (TEAC), ferric reducing antioxidant power (FRAP), malondialdehyde (MDA) levels
17 March 2015
and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)
Accepted 19 March 2015
and glutathione-S-transferase (GST) also by evaluating DNA damage with comet assay. LN
Available online 27 March 2015
increased the levels of MDA, tail DNA%, mean tail length and tail moment, decreased the enzymes activities, FRAP and TEAC values. In sodium selenite + LN group, we observed the
Keywords:
protective effect of sodium selenite on examining parameters. Diabetes caused alterations
Lead
on these parameters, too. We found that sodium selenite did not protect against diabetes
Selenium
caused damages. As a result, LN caused toxic effects on blood cells and sodium selenite alle-
Diabetes
viated this toxicity but it did not show preventive effect against diabetes. Also, LN caused
Oxidative stress
more harmfull effects in diabetic groups than non-diabetic groups. © 2015 Elsevier B.V. All rights reserved.
Antioxidant enzyme DNA damage
1.
Introduction
Metals have important roles in the functioning of enzymes and cell signaling processes. Increasing concern has been expressed about the rapidly rising level of metals in the environment, specially lead, which has well-known harmfull effects (Dewanjee et al., 2013). Lead can hinder biological functions by changing the molecular interactions, cell signaling processes and cellular function. LN is leads to a broad range of biochemical, physiological and behavioral dysfunctions (Lakshmi et al., 2013).
∗
Selenium has substantial roles in many biological processes and it prevents against so many diseases such as cancers and cardiovascular disease. It is suggested that selenium can support antioxidant capacity by increasing activities of antioxidant enzymes and by enhancing levels of the antioxidants (Kalender et al., 2013). Tissues of selenium deficient animals are more susceptible to oxidative stress when compared to control animals. Selenium addition increased thioredoxin reductase and GPx activities and resulted in improved recovery of functions of organs after damages (Venardos et al., 2004).
Corresponding author. Tel.: +90 354 242 10 21/255; fax: +90 354 242 10 22. E-mail address: [email protected] (H. Bas¸).
http://dx.doi.org/10.1016/j.etap.2015.03.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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Several studies have shown that diabetes is associated with enhanced generation of free oxygen radicals and decrease in antioxidant ability due to these events. There are so many alterations in diabetic people that are associated with oxidative stress like products of glycation (Diabetes Care, 2010) and hyperglycemia which makes an imbalance in cellular oxidation and reduction (Farokhi et al., 2012). Against oxidative damage, human body has enhanced various defence mechanisms, involving enzymatic and nonenzymatic systems. SOD, CAT, GPx and GST are important antioxidant enzymes (Bas¸ et al., 2014). To evaluate the genotoxic effects there are several test systems. Comet assay is used to determine the potential genotoxicity. This assay is an identifier of effects of carcinogen exposure. It has been proved to be good marker of DNA damage (Zengin et al., 2011). The FRAP and TEAC assays are simple and cheap procedures which measure the total antioxidant levels (Nawirska-Olszanska et al., 2013). Due to the widespread use of lead, it is important to determine the harmful effects of lead on living things. In recent years, in terms of antioxidant properties selenium is one of the widely studied materials. Also, there are no studies for propound the role of sodium selenite against LN induced oxidative stress in diabetic and non-diabetic rat blood. Therefore, this study was done to investigate the effects of LN and sodium selenite on values of FRAP and TEAC, MDA levels and the activities of SOD, CAT, GPx and GST, tail DNA%, mean tail length and tail moment in blood cells of rats.
2.
Materials and methods
FRAP and TEAC values were quantified in plasma, SOD, CAT, GPx, GST, MDA were studied in erythrocytes and leucocytes, DNA damage was measured in leucocytes.
2.1.
Animals and chemicals
The protocol was approved by the Gazi Univ. Animal Experiments Local Ethics Committee (G.Ü.ET–11.028). Wistar rats (200–250 g), were obtained from the Gazi Univ. Laboratory Animals Growing and Experimental Research Center, Ankara, Turkey. They were supplied with standard laboratory chow and water ad libitum at 22 ± 2 ◦ C. Procedures were performed in accordance with international guidelines for care and use of laboratory animals. Streptozotocin (STZ), LN, sodium selenite and other chemicals were procured from Sigma Aldrich. LN and sodium selenite were dissolved in distilled water (Sharma et al., 2010; Sakr et al., 2011).
2.2.
Animal grouping and treatment
Eight groups (6 animals in each group) were formed for this study. These groups were control, sodium selenite, LN, sodium selenite + LN, diabetic control, diabetic sodium selenite, diabetic LN, diabetic sodium selenite + LN. During the experimental period (28 days) 1 ml/kg b.w (body weight) distilled water for control groups, 1 mg/kg b.w sodium selenite for sodium selenite treatment groups and 22,5 mg/kg b.w (1/100 LD50 ) LN (Sharma et al., 2010) for LN treatment groups were
given to rats daily via gavage. Animals were rendered diabetic by a single i.p. injection of STZ (55 mg/kg). STZ was prepared in 1 ml of freshly prepared sodium citrate buffer at 4.5 pH. After 2 days, the blood glucose levels were measured with a glucometer and whose levels were 300 mg/dl or higher, they selected for diabetic groups (Schmatz et al., 2009).
2.3.
Preparation of erythrocytes
Blood samples were collected into heparinized tubes after treatment. Erythrocytes were separated from plasma by centrifugation (1600 rpm for 5 min) and then washed with a cold isotonic saline solution. After separation, erythrocytes were suspended in phosphate buffer at pH 7.4 to obtain a 50% cellular suspension. Erythrocytes were destroyed by osmotic pressure and the resulting mixtures were subjected to centrifugation. Supernatants were isolated, and MDA levels and enzyme activities were measured by Shimadzu UV-1800, Japan. The concentration of hemoglobin was determined by the method of Drabkin (1946).
2.4.
Preparation of leucocytes
30 ml of blood was collected in sodium citrate and mixed with 6% dextran in isotonic saline and allowed to stand for 30 min. The upper layer was transferred to other tube which is containing 2.25 ml EDTA and centrifuged at 1000 rpm. Then the leucocytes were washed with Tris buffer at 4 ◦ C (Chen et al., 1997). After this step MDA levels and enzyme activities were measured by Shimadzu UV-1800, Japan.
2.5.
Measurement of MDA levels
MDA is the most abundant aldehyde resulting from LPO in biological process. For determination of MDA levels cells were incubated with thiobarbituric acid (TBA) at 95 ◦ C (pH 3.4). MDA reacts with TBA to form a colored complex. MDA levels were specified at 532 nm (Ohkawa et al., 1979).
2.6.
Antioxidant enzyme assays
The activity of SOD was measured assaying the autooxidation and illumination of pyrogallol by the method of Marklund and Marklund (1974). This reaction was monitored at 440 nm. For CAT activity samples were diluted with Triton X-100. The activity was measured as the changing rate of H2 O2 decomposition at 240 nm according to study of Aebi (1984). GPx activity was determined with H2 O2 as substrate. Reaction mixtures contained reduced glutathione, glutathione reductase, NADPH and Tris–HCl. GPx activity was measured as the change in absorbance at 340 nm (Paglia and Valentine,1967). Activity of GST was analysed by measuring the formation of the glutathione and 1-chloro-2,4-dinitrobenzene conjugate. The increase in absorbance was recorded at 340 nm (Habig et al., 1974).
2.7.
FRAP assay
The antioxidant capacity was evaluated by FRAP assay according to the method of Benzie and Strain (1996). This assay
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Fig. 1 – Effects of LN and sodium selenite on FRAP values (mol/L). Each bar represents mean ± SD in each group. Columns superscripts with different letters are significantly different. Significance at P < 0.05.
measures the changes in absorbance at 593 nm due to the generation of FeII -tripyridyltriazine compound from oxidised FeIII form. The FRAP reagent was prepared by mixing 300 mmol/L acetate buffer with 10 mmol/L 2,4,6- tripyridyl-s-triazine in 40 mmol/L HCl and with 20 mmol/L ferric chloride.
2.8.
TEAC assay
The TEAC assay is based on the inhibition of the absorbance of the ABTS.+ by tested antioxidant (Re et al., 1999). Solution of ABTS.+ was diluted with phosphate buffer saline (PBS) to an absorbance of 0.70 (± 0.02) at 734 nm. When we add the diluted ABTS.+ to biological samples the mixtures was incubated for 6 min. Then at 734 nm, the decrease in absorbance was measured.
2.9.
Determination of DNA damage (comet assay)
The cells were suspended in low melting point agarose (0.65%) and 75 l of suspension was layered over microscope slides which were precoated with normal melting point agarose
(0.65%) then they covered with a coverglass and the slides were placed at +4 ◦ C. After rigidification, the coverglass was removed and immersed in lysing solution. After this step, the slides were removed placed on a gel electrophoresis platform covered with electrophoresis buffer and they were left in the solution for 20 min to allow the unwinding of the DNA (Ozkan et al., 2009). The slides were then placed in a neutralizing tank and washed with neutralizing buffer. Ethidium bromide was dispensed directly onto slides and covered with a coverglass. Data were analyzed using BS 200 ProP with software (BAB Bs Comet Assay software) image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey).
2.10.
Statistical analyses
Statistical analyses were performed using SPSS, version 11.0 for Windows. To evaluation the differences, one way analysis of variance (ANOVA) followed by Tukey multiple comparison were used. P < 0.05 was considered statistically significant. All data was described as the means ± standard deviation (S.D).
Fig. 2 – Effects of LN and sodium selenite on TEAC values (mol/L). Each bar represents. mean ± SD in each group. Columns superscripts with different letters are significantly different. Significance at P < 0.05.
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Fig. 3 – Comet appearances of the LN and/or Sodium selenite treated blood in rats (A) control, (B) Sodium selenite, (C) LN, (D) Sodium selenite + LN, (E) diabetic control, (F) diabetic Sodium selenite, (G) diabetic LN, (H) diabetic Sodium selenite + LN.
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Table 1 – Evaluated mean values of MDA levels and antioxidant enzyme activities of LN and/or sodium selenite treatment diabetic and non-diabetic rat erythrocytes. Groups
MDA (nmol/mgHb)
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
6.04 6.25 11.31 8.37 8.07 7.92 15.91 10.98
± ± ± ± ± ± ± ±
a
0.22 0.41a 0.54b 0.42c 0.11c 0.63c 0.79d 0.66 b
SOD (U/mgHb)
CAT (U/mgHb)
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
612.04 624.50 510.45 589.52 594.04 598 424.7 516.5
a
5.03 7.47a 9.12b 8.06c 4.03c 5.83c 9.85d 12.12b
323.5 330.25 241.46 297.17 301.9 295.29 201.6 232.19
GPx (U/mgHb)
a
3.82 5.11a 5.33b 4.34c 4.03c 6.45c 6.30d 4.65b
33.8 35.51 23.68 28.21 27.23 27.85 19.96 24.27
± ± ± ± ± ± ± ±
a
0.31 1.13a 1.37b 0.67c 1.01a 0.91a 1.1b 1.44c
GST (U/mgHb) 19.8 18.5 11.13 15.1 14.8 15.41 8.18 10.24
± ± ± ± ± ± ± ±
1.01a 0.83a 0.7b 0.71c 0.32a 1.2a 0.51b 1.16c
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
Table 2 – Evaluated mean values of MDA levels and antioxidant enzyme activities of LN and/or sodium selenite treatment diabetic and non-diabetic rat leucocytes. Groups
MDA(nmol/mgprotein) SOD (U/mgprotein) CAT (U/mgprotein) GPx (U/mgprotein) GST (U/mgprotein)
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
1.27 ± 0.11a 1.16 ± 0.14a 2.9 ± 0.16b 2.01 ± 0.17c 2.11 ± 0.1c 2.24 ± 0.13c
348.12 ± 6.23a 343.71 ± 8.14a 256.67 ± 6.92b 296.77 ± 9.61c 321.21 ± 7.27c 314.6 ± 8.53c
223.75 ± 4.62a 214.69 ± 5.87a 161.22 ± 6.03b 187.37 ± 4.91c 184.21 ± 7.83c 175.06 ± 5.28c
41.33 ± 2.11a 37.18 ± 3.05a 27.02 ± 1.63b 30.23 ± 1.12c 32.6 ± 1.2a 30.52 ± 1.87a
8.17 ± 0.37a 8.62 ± 0.52a 6.46 ± 0.3b 7.28 ± 0.41c 7.31 ± 0.32a 7.35 ± 0.28a
3.5 ± 0.12d 3.08 ± 0.16 b
219.68 ± 11.57d 254.43 ± 6.04b
141.34 ± 6.01d 166.9 ± 4.11b
20.65 ± 0.83b 26.71 ± 1.66c
5.02 ± 0.55b 6.39 ± 0.43c
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
Table 3 – Estimated mean values of tail DNA%, tail length and tail moment of comets by image analysis on treatment groups in rats blood. Groups
Tail DNA%
Tail length
Tail moment
Control Sodium selenite LN Sodium selenite + LN Diabetic Control Diabetic Sodium selenite Diabetic LN Diabetic Sodium selenite + LN
57.94 ± 5.09 a 64.25 ± 2.51a 91.51 ± 4.04b 80.79 ± 1.54c 77.97 ± 5.10c 79.00 ± 2.61c 99.81 ± 3.19d 89.17 ± 1.64 b
17.04 ± 1.03a 20.50 ± 2.47a 68.50 ± 2.12b 45.50 ± 1.06c 41.04 ± 4.03c 43.00 ± 2.83c 118.50 ± 19.85d 65.50 ± 2.12b
13.75 ± 0.91a 13.29 ± 2.10a 55.66 ± 3.51b 40.37 ± 1.24 c 37.90 ± 2.93c 35.22 ± 5.45c 121.66 ± 24.30d 52.58 ± 2.12b
Values are mean ± SD of six rats in each group. Significance at P < 0.05. Within each column, means superscript with different letters are significantly different.
3.
Results
There were no statistically significant changes in values of FRAP and TEAC, levels of MDA, in enzyme activities and in tail DNA%, mean tail length and tail moment between the sodium selenite group compared with the control also between diabetic sodium selenite group compared with the diabetic control for erythrocytes and leucocytes (P > 0.05, Figs. 1–3 and Tables 1–3).
3.1.
MDA levels of erythrocytes and leucocytes
Treatment with LN increased the level of MDA compared with control significantly. The MDA levels were decreased in the sodium selenite + LN treated group compared to LN
treated group (P < 0.05). Also, similar values were evaluated in diabetic control, diabetic sodium selenite, diabetic LN and diabetic sodium selenite + LN treated groups. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group, a significant increasing in levels of MDA were determined in diabetic groups (Tables 1 and 2).
3.2. Antioxidant enzyme activities of erythrocytes and leucocytes SOD, CAT, GPx and GST activities were measured in all groups of rats. Decreasing in SOD, CAT, GPx and GST activities were detected in erythrocytes and leucocytes in LN treated rats.
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The activity of enzymes was increased in sodium selenite + LN treated group compared to LN group, significantly (P < 0.05). Similar results were searched also in diabetic control, diabetic sodium selenite, diabetic LN and diabetic sodium selenite + LN treated groups. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group significant reduction in enzyme activities were observed in diabetic groups (Tables 1 and 2).
3.3.
Values of FRAP and TEAC
The changes observed in FRAP and TEAC levels are shown in Figs. 1 and 2, respectively. The values of FRAP and TEAC were found to decrease significantly in LN treated rats (P < 0.05) when compared to the control. There was a significant increase in FRAP and TEAC levels of sodium selenite + LN rats when compared to the LN group. Similar results were observed between diabetic groups, too. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group decreasing in values of FRAP and TEAC were observed significantly in diabetic animals.
3.4.
Commet assay results
According to comet assay results the tail moment, mean tail length and tail DNA% significantly increased with LN treatment when compared to control. It was observed decreasing of these values in sodium selenite + LN treated group. Similar values were observed between diabetic groups, too. When compared control with diabetic control group, sodium selenite with diabetic sodium selenite group, LN with diabetic LN group, sodium selenite + LN with diabetic sodium selenite + LN group increasing in values of the tail moment, mean tail length and tail DNA% were observed significantly in diabetic animals (Table 3). The figures of the comet assay were observed in Figure 3 for non-diabetic and diabetic groups.
4.
Discussion
In our work LN caused reduction in activities of SOD, CAT, GPx and GST in erythrocytes and leucocytes. There are studies proving that lead cause decreasing in enzyme activities (Liu et al., 2010; Haleagrahara et al., 2010). There is a study indicating that the lead binds to proteins and causes changes in several enzyme activities (Mehana et al., 2012). The antioxidant enzyme activities have been used to determine the oxidative stress in other studies (Liu et al., 2010). Changes reported in this paper about reducing in these enzyme activities following LN treatment may have resulted from increasing generation of free radicals. In present study our MDA level results support this probability of the LN treated groups. Recent in vivo studies in lead treated animals showed the generation of ROS, stimulation of LPO and decreased antioxidant defense system supporting the function of oxidative stress in lead toxicity (Haleagrahara et al., 2010).
We measured MDA level for evaluation of oxidative stress caused by LPO in erythrocytes and leucocytes. Increased MDA levels were determined in LN treated rats in this study. There are lots of studies showing that heavy metals cause increasing in MDA levels (Mehana et al., 2012; Dewanjee et al., 2013). Previous studies indicated that MDA may be increase due to adverse effects of LN on fatty acids of cell membranes (Haleagrahara et al., 2010). Comparison of different analytical methods is helpful for better understanding and interpretation of the results about the antioxidant capacity of a sample (Katalinic et al., 2005). Therefore, FRAP and TEAC assays were studied in this work in addition to antioxidant enzyme activity assays. In this study as a result of appling LN, it was observed significant decrease of FRAP and TEAC values. These results reinforce the findings about antioxidant enzyme activities. During the last years, determination on damage of DNA caused by toxins has been performed by comet assay (Zengin et al., 2011). So in this study, the comet assay was used to examine the DNA damage of rat leucocytes when they are exposed to LN. In our study we observed significant increasing in tail DNA%, mean tail length and tail moment, indicating DNA damage, in LN treatment rat leucocytes when compared to control. Oxidative stress and depletion of antioxidant defence system have important roles in the pathogenesis of diabetes mellitus. There are some investigations about diabetes caused tissue injury that it is based on breaking oxidant/antioxidant balance (Suresh et al., 2013). In this study, compared with diabetic groups with non-diabetic groups, we were observed decreasing in values of FRAP, TEAC and activities of SOD, CAT, GST, GPx and increasing in MDA levels and in tail DNA%, mean tail length and tail moment in diabetic rats. Previous experimental studies have been proved that in diabetic rats increased LPO and altered antioxidant enzymes such as SOD, CAT, GPx and GST (Shanmugam et al., 2011; Suresh et al., 2013). Kuppusamy et al. (2005) reported a significantly lower FRAP level in type 2 diabetic patients and Colak et al. (2005) documented a similar decrease in type 2 diabetic patients, too. Also when we compared LN group with diabetic LN group, we were determined that there were more alterations in examining parameters in diabetic LN group. Thus, we can say diabetic rats are more susceptible to LN induced oxidative stress than non-diabetic rats. In the present study we observed sodium selenite has protective effects against LN induced adverse effects on examining parameters of blood cells of rats. It is known that selenium has important roles about detoxification of toxic heavy metals (Kalender et al., 2013). Sodium selenite becomes protective because of its antioxidant properties, like other studies (Orun et al., 2008; Kalender et al., 2013). It may be indirectly scavenger of ROS or increase antioxidant enzyme activities; therefore it may prevent the toxicity produce by LN. Accumulating evidences have shown that lead induces oxidative stress by inducing the producing of ROS (Dai et al., 2010). Extreme generation of ROS can cause the damages of DNA, proteins and lipids and this situation led to adverse effects (Liu et al., 2010). In this study, we determined that exposure to LN, induced over-production of ROS and led to oxidative damage
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in rats. Whereas, selenium significantly reverse LN caused toxicity by regulating the ROS level in the blood cells of rats.
5.
Conclusion
In this study, even though LN was given orally at low dose to diabetic and non-diabetic rats, MDA, FRAP, TEAC levels, DNA damage and enzymatic changes were observed in the rat blood cells, but none of the rats died during the experimental period. From the data of this study, it was evident that LN caused toxicity, including LPO and disturbances in antioxidant enzyme activities, DNA damages both diabetic and non-diabetic rats. These results were probably due to generation of ROS, causing damage to cell membranes. With the present study we determined the protective effects of sodium selenite on LN caused oxidative stress in human erythrocytes and leucocytes. Results showed important toxic changes in the diabetic groups, which could be due to oxidative damage in diabetes because oxidative stress has an important role play in the pathogenesis of diabetes. Data in this study showed that sodium selenite did not alleviate diabetes induced unwanted effects but it has beneficial effects on LN mediated toxicity, but not protect completely.
Conflict of Interest The authors have nothing to disclose.
references
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Bas¸, H., Kalender, S., Pandır, D., 2014. In vitro effects of quercetin on oxidative stress mediated in human erythrocytes by benzoic acid and citric acid. Folia Biol. (Krakow) 62 (1), 59–66. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Chen, L., Haught, W.H., Yang, B., Saldeen, T.G.P., Parathasarathy, S., Mehta, J.L., 1997. Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine. J. Am. Coll. Cardiol. 30 (2), 569–575. Colak, E., Majkic-Singh, N., Stankovic, S., Sreckovic Dimitrijevic, V., Djordjevic, P.B., Lalic, K., Lalic, N., 2005. Parameters of antioxidative defense in type 2 diabetic patients with cardiovascular complications. Ann. Med. 37, 613–620. Dai, W., Fu, L., Du, H., Liu, H., Xu, Z., 2010. Effects of montmorillonite on Pb accumulation oxidative stress, and DNA damage in Tilapia (Oreochromis niloticus) exposed to dietary Pb. Biol. Trace Elem. Res. 136, 71–78. Dewanjee, S., Sahu, R., Karmakar, S., Gangopadhyay, M., 2013. Toxic effects of lead exposure in Wistar rats: Involvement of oxidative stress and the beneficial role of edible jute (Corchorus olitorius) leaves. Food Chem. Toxicol. 55, 78–91. Diabetes Care, 2010. Excecutive summary, standards of medical care in diabetes. 33, 4–10. Drabkin, D.I., 1946. Spectrophotometric studies. XIV, the crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. J. Biol. Chem. 164, 703–723. Farokhi, F., Farkhad, N.K., Togmechi, A., Band, K.S., 2012. Preventive effects of Prangos ferulacea (L.) lindle on liver
1025
damage of diabetic rats induced by alloxan. Avicenna J. Phytomed. 2 (2), 63–71. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Haleagrahara, N., Jackie, T., Chakravarthi, S., Rao, M., Pasupathi, T., 2010. Protective effects of Etlingera elatior extract on lead acetate-induced changes in oxidative biomarkers in bone marrow of rats. Food Chem. Toxicol. 48, 2688–2694. Kalender, S., Uzun, F.G., Demir, F., Uzunhisarcıklı, M., Aslanturk, A., 2013. Mercuric chloride-induced testicular toxicity in rats and the protective role of sodium selenite and vitamin E. Food Chem. Toxicol. 55, 456–462. Katalinic, V., Modun, D., Music, I., Boban, M., 2005. Gender differences in antioxidant capacity of rat tissues determined by 2,2V-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comp. Biochem. Physiol. 140, 47–52. Kuppusamy, U.R., Indran, M., Rokiah, P., 2005. Glycaemic control in relation to xanthine oxidase and antioxidant indices in Malaysian Type 2 diabetes patients. Diabet. Med. 22, 1343–1346. Lakshmi, B.V.S., Sudhakar, M., Aparna, M., 2013. Protective potential of Black grapes against lead induced oxidative stress in rats. Environ. Toxicol. Pharmacol. 35, 361–368. Liu, C., Ma, J., Sun, Y., 2010. Quercetin protects the rat kidney against oxidative stress mediated DNA damage and apoptosis induced by lead. Environ. Toxicol. Pharmacol. 30, 264–271. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Mehana, E.E., Meki, A.R., Fazili, K.M., 2012. Ameliorated effects of green tea extract on lead induced liver toxicity in rats. Exp. Toxicol. Pathol. 64, 291–295. Nawirska-Olszanska, A., Kita, A., Biesiada, A., Sokol-Letowska, A., Kucharska, A.Z., 2013. Characteristics of antioxidant activity and composition of pumpkin seed oils in 12 cultivars. Food Chem. 139, 155–161. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Orun, I., Selamoglu Talas, Z., Ozdemir, I., Alkan, A., Erdogan, K., 2008. Antioxidative role of selenium on some tissues of (Cd2+ , Cr3+ )-induced rainbow trout. Ecotox. Environ. Safe. 71, 71–75. Ozkan, D., Yuzbasıoglu, D., Unal, F., Yılmaz, S., Aksoy, H., 2009. Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate. Cytotechnology 59, 73–80. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantative and qualitative characterization of glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radical Biol. Med. 26, 1231–1237. Sakr, S.A.R., Mahran, H.A., Nofal, A.E., 2011. Effect of selenium on carbimazole induced testicular damage and oxidative stress in albino rats. J. Trace Elem. Med. Biol. 25, 59–66. Schmatz, R., Mazzanti, C.M., Spanevello, R., Stefanello, N., Gutierres, J., Corrêa, M., da Rosa, M.M., Rubin, M.A., Schetinger, M.R.C., Morsch, V.M., 2009. Resveratrol prevents memory deficits and the increase in acetylcholinesterase activity in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 610, 42–48. Shanmugam, K.R., Mallikarjuna, K., Nishanth, K., Kuo, C.H., Reddy, K.S., 2011. Protective effect of dietary ginger on antioxidant enzymes and oxidative damage in experimental diabetic rat tissues. Food Chem. 124, 1436–1442.
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Sharma, V., Sharma, A., Kansal, L., 2010. The effect of oral administration of Allium sativum extracts on lead nitrate induced toxicity in male mice. Food Chem. Toxicol. 48, 928–936. Suresh, S., Prithiviraj, E., Lakshmi, N.V., Ganesh, M.K., Ganesh, L., Prakash, S., 2013. Effect of Mucuna pruriens (Linn.) on mitochondrial dysfunction and DNA damage in epididymal sperm of streptozotocin induced diabetic rat. J. Ethnopharmacol. 145, 32–41.
Venardos, K., Harrison, G., Headrick, J., Perkins, A., 2004. Effects of dietary selenium on glutathione peroxidase and thioredoxin reductase activity and recovery from cardiac ischemia–reperfusion. J. Trace Elem. Med. Biol. 18, 81–88. Zengin, N., Yuzbasıoglu, D., Unal, F., Yılmaz, S., Aksoy, H., 2011. The evaluation of the genotoxicity of two food preservatives: sodium benzoate and potassium benzoate. Food Chem. Toxicol. 49, 763–769.
