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RESEARCH ARTICLE

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Fullerene C60 Nanoparticles Decrease Liver Oxidative Stress through Increment of Liver Antioxidant Capacity in Streptozotocin-Induced Diabetes in Rats

Fariba Namadr1, Farideh Bahrami1, Zahra Bahari1, Bahram Ghanbari2, Shima Shahyad3, and Mohammad Taghi Mohammadi1,3 

1Department of Physiology and Medical Physics, School of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran; 2Department of Chemistry, Sharif University of Technology, Tehran, Iran; 3Neuroscience Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 

Correspondence: mohammadimohammadt@bmsu.ac.ir or mohammadi.mohammadt@yahoo.com 

Namadr F et al. Reactive Oxygen Species 9(26):70–80, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.809

(Received: October 14, 2019; Revised: October 26, 2019; Accepted: October 27, 2019) 

ABSTRACT | Chronic hyperglycemia causes oxidative stress in the liver and enhances the hepatic vulnerability to oxidative damage in diabetes mellitus. Since an excellent antioxidative stress property of fullerene C60 nanoparticles has been demonstrated in a wide range of in vitro and in vivo studies, we examined the effect of fullerene C60 nanoparticles on the oxidative stress markers in the liver of streptozotocin-induced diabetes in rats. Male Wistar rats were randomly divided into 4 groups (n = 8 for each group): normal, treated normal, diabetic, and treated diabetic groups. The rats were made diabetic by a single intravenous injection of streptozotocin (50 mg/kg). Treated (normal and diabetic) rats received orally fullerene C60 nanoparticles (1 mg/kg/day) by a gavage tube for 8 weeks. At termination of the study, the oxidative stress parameters were determined in liver tissues, including malondialdehyde (MDA) levels and the activities of catalase (CAT) and superoxide dismutase (SOD) as well as the content of the reduced form of glutathione (GSH). Treatment with fullerene C60 did not change blood glucose of normal and diabetic rats. Diabetes increased MDA levels and CAT activity but decreased GSH content in the liver. Fullerene C60 administration significantly decreased MDA levels and increased the activities of CAT and SOD as well as ameliorated the histopathological changes in the liver of diabetic rats. Taken together, the results of the present study indicated that fullerene C60 nanoparticles could decrease oxidative stress injury in the liver of diabetic rats likely through potentiation of the hepatic antioxidant capacity. 

KEYWORDS | Antioxidant capacity; Catalase; Diabetes; Fullerene C60; Hepatic injury; Glutathione; Lipid peroxidation; Malondialdehyde; Oxidative stress; Superoxide dismutase 

ABBREVIATIONS | CAT, catalase; GSH, reduced form of glutathione; H&E, hematoxylin-eosin; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; PBS, phosphate-buffered saline 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Animals

2.2. Fullerene C60 Nanoparticles

2.3. Induction of Diabetes

2.4. Experimental Design and Grouping

2.5. Evaluation of Hepatic Oxidative Stress

2.5.1. Hepatic Tissue Homogenates

2.5.2. Determination of the MDA Levels

2.5.3. Determination of the CAT activity

2.5.4. Determination of the SOD Activity

2.5.5. Determination of the GSH Content

2.6. Histopathological Assessment

2.7. Statistical Analysis

  1. Results

3.1. Effect of Fullerene C60 on Blood Glucose Level

3.2. Effect of Fullerene C60 on MDA Level

3.3. Effect of Fullerene C60 on CAT Activity

3.4. Effect of Fullerene C60 on SOD Activity

3.5. Effect of Fullerene C60 on GSH Content

3.6. Effect of Fullerene C60 on Histopathological Changes

  1. Discussion
  2. Conclusion

1. INTRODUCTION 

According to previous studies, the oxidative stress markers are increased in the blood of diabetic patients [1]. Oxidative stress, resulting from reactive oxygen species (ROS) accumulation, causes a detrimental effect on cell macromolecules, including lipid peroxidation, protein oxidation, and nucleic acid damage [2]. The balance between the hepatic production of pro-oxidants and elimination of these reactive species by the hepatic antioxidant systems is disrupted in diabetes [3]. Hence, the vulnerability of the liver to ROS accumulation and oxidative damage is increased in diabetes [3]. It has been demonstrated that pro-oxidant pathways such as the polyol pathway and NAD(P)H oxidase activity are increased in the liver of diabetic patients [4]. On the other hand, the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) as well as the content of non-enzymatic antioxidants such as the reduced form of glutathione (GSH) are decreased in the liver during diabetes [5], which further augment oxidative hepatic injury. 

Fullerene C60 nanoparticles, made purely from carbon atoms in hollow sphere-shapes, have been demonstrated to afford cytoprotective actions in biological systems [6]. The antioxidant property is the main cytoprotective role of fullerene C60 that has been reported in a wide range of in vitro and in vivo studies [7–10]. These nanoparticles exert antioxidative effects likely through passing the cell membrane and localizing preferentially in the mitochondria [7]. Fullerene C60 derivatives protected the cells against photo-induced cytotoxicity as well as photo-induced oxidative stress [11]. In this regard, repeated oral administration of fullerene C60 prolonged the survival in an experimental model of CCl4 intoxication in rats [6]. Fullerene administration in ischemic stroke decreased the injury of cerebral ischemia by inhibition of oxidative stress and inflammation [12–14]. Treatment with fullerene C60 also decreased the axonal loss and neurodegeneration in a mouse model of progressive multiple sclerosis [15]. Moreover, the potential hepatoprotective functions of fullerene C60 have been reported in cyclophosphamide-induced acute hepatotoxicity in rats [16]. 

Since there is no data about the effects of fullerene C60 on the oxidative stress parameters in diabetic liver, the present study examined the effects of oral repeated administration of the nanoparticles on the oxidative stress injury in the liver of experimentally induced diabetes in rats.

2. MATERIALS AND METHODS 

2.1. Animals 

All experimental protocols used in the present study were approved by the Institutional Animal Ethics Committee of the University of Baqiyatallah Medical Sciences (Tehran, Iran). Adult male Wistar rats (220 ± 20 g; 8‒10 weeks old) were employed in the current study. The rats acclimatized in the institutional animal house and were given standard chow and water ad libitum. The animals were housed under controlled conditions of light exposure (12 h light/dark cycles), temperature (22‒24oC), and humidity (40‒60%). 

2.2. Fullerene C60 Nanoparticles 

Fullerene C60 with a purity of > 90% was obtained from Sharif University of Technology (Iran). Fullerene C60 was dissolved in sesame oil and administered via oral gavage at a dose of 1 mg/kg/day according to the previous studies [6, 17]. 

2.3. Induction of Diabetes 

Rats were allowed one week to acclimatize and then diabetes was induced by a single intravenous dose of freshly prepared streptozotocin (STZ; 50 mg/kg dissolved in normal saline; Sigma-Aldrich, St. Louis, MO, USA) through the lateral tail vein. Non-diabetic rats received the same volume of normal saline. Diabetes was confirmed by determination of the blood glucose level and the rats with a blood glucose level above 450 mg/dl were selected as diabetic animals. Animals were housed in the same room but in separate cages (1 per cage). 

2.4. Experimental Design and Grouping 

The rats were randomly divided into four groups in equal numbers (n = 8 for each group). The first group was considered as normal (control group) and the animals of this group received orally sesame oil per day without fullerene C60 in the same volume of the fullerene C60-treated rats for 8 weeks. The second group was considered as fullerene C60-treated group and the rats of this group received daily fullerene C60 (1 mg/kg), dissolved in sesame oil, by gavage for 8 weeks. The third group was used as control diabetic group and the diabetic animals of this group like the normal group received orally sesame oil per day without fullerene C60 in the same volume as for the fullerene C60-treated rats for 8 weeks. The fourth group was considered as fullerene C60-treated diabetic group and these diabetic rats received daily fullerene C60 (1 mg/kg), dissolved in sesame oil, by gavage for 8 weeks. Treatment of diabetic rats started at day 5 after STZ injection and continued for 8 weeks. Blood samples (500 μl) were collected from the tip of the snipped tail at day 5 after STZ injection and the end of study under light anesthesia with ether. Then, blood samples were centrifuged (4,500 g) and serum was collected and stored in a freezer (–80oC) for the determination of serum glucose. 

2.5. Evaluation of Hepatic Oxidative Stress 

2.5.1. Hepatic Tissue Homogenates 

The liver tissues were quickly removed under deep anesthesia for the determination of the oxidative stress parameters. First, the liver tissues were weighed and washed in ice-cold phosphate-buffered saline (PBS). After homogenization of the hemispheres in ice-cold PBS (1:10), the homogenates were centrifuged (14,000 g) at 4oC for 15 min. Then, the supernatants were used to determine the levels of GSH and malondialdehyde (MDA) as well as the activities of SOD and CAT. The protein content of the samples was measured according to the method of Bradford [18]. 

2.5.2. Determination of the MDA Levels 

The MDA levels were determined by the method of Satoh [19]. First, 1.5 ml of trichloroacetic acid (TCA, 10%) was added to 0.5 ml of the tissue homogenate. The mixture was vortexed and incubated at room temperature for 10 min. Then, 2 ml of thiobarbituric acid (0.67%) was added to 1.5 ml supernatant and incubated in a boiling water bath for 30 min in sealed tubes. After cooling the samples to room temperature, 1.25 ml of n-butanol was added and vortexed. The samples were finally centrifuged at 2000 g for 5 min and the supernatants were separated. The absorbance of the supernatants was recorded by a spectrophotometer at 532 nm. 1,1,3,3-Tetraethoxypropane was used as a standard to determine the levels of MDA. The MDA levels of the liver tissues were calculated as nmol/mg protein. 

2.5.3. Determination of the CAT activity 

The method of Aebi was used to determine the activity of CAT in the tissue homogenate [20]. First, the homogenate was incubated in the reaction mixture that contained 0.1 ml homogenate and 0.85 ml potassium phosphate buffer (50 mM, pH 7.0) at room temperature for 10 min. Then, the reaction was started by adding 0.05 ml H2O2 (30 mM prepared in 50 mM potassium phosphate buffer, pH 7.0). A decrease in the absorbance was recorded by a spectrophotometer at 240 nm for 3 min. The specific activity of CAT was calculated as U/mg protein. One U is defined as the amount of enzyme required to decompose 1 μmol of H2O2 per min. 

2.5.4. Determination of the SOD Activity 

The SOD activity was determined according to the method of Winterbourn et al. [21], based on SOD-mediated inhibition of the reduction of nitroblue tetrazolium (NBT) by superoxide. The reaction mixture contained, in potassium phosphate buffer (67 mM, pH 7.8), 0.1 M EDTA, 0.3 mM sodium cyanide, 1.5 mM NBT, and 0.1 ml of the sample. To initiate the reaction, riboflavin (0.12 mM) was added to each sample. After 12 minincubation, the absorbance of samples was recorded by a spectrophotometer at 610 nm for 5 min. The amount of enzyme needed to induce 50% inhibition was taken as one U. The SOD activity was calculated as U/mg protein. 

2.5.5. Determination of the GSH Content 

The GSH content was determined according to the method of Tietz [22]. First, the cellular protein was precipitated by adding sulfosalicylic acid (5%). After centrifugation of the sample at 2,000 g for 10 min, the supernatant was removed for measuring the GSH content. Briefly,the reaction mixture contained 100 μl of the protein-free supernatant, 100 μl of 0.04% 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in 0.1% sodium citrate, and 800 μl of 0.3 mM Na2HPO4. After 5 min incubation at room temperature, the absorbance was recorded at 412 nm with a spectrophotometer. The GSH content was calculated as nmol/mg protein. 

2.6. Histopathological Assessment 

Histopathological evaluation was performed following hematoxylin-eosin (H&E) staining. The livers were fixed, dehydrated (by 70%, 80%, 96%, and 100% ethanol), and cleared (by xylene). Afterward, the liver samples were paraffin-embedded and coronal serial sections (5-μm thickness) were prepared for staining. The samples were treated twice with a xylene solution (each time for 15 min) for clearing. The histopathological images were observed under a light microscope (Nikon, Japan) connected to a digital camera (CMEX, Netherlands) for capturing the micrographs. 

2.7. Statistical Analysis 

The statistical analysis software, SPSS (V.21, Chicago, IL, USA) was used for statistical analysis. All data were presented as mean ± SEM. Kolmogrov–Smirnov test showed normal distribution of data, so one-way variance (ANOVA) and Tukey post-hoc test were used to compare data between groups. Statistical significance was set at p < 0.05.

3. RESULTS 

3.1. Effect of Fullerene C60 on Blood Glucose Level 

As illustrated in Figure 1, rats with STZ-induced diabetes exhibited a significantly increased blood glucose level, which was not affected by fullerene C60 treatment. Treatment with fullerene C60 did not affect the blood glucose level in normal rats either.

 

FIGURE 1. Blood glucose levels in the rats at the end of study. All values are expressed as mean ± SEM. ***, p < 0.001 compared to normal group. 

3.2. Effect of Fullerene C60 on MDA Level 

As shown in Figure 2. rats with STZ-induced diabetes showed a significantly increased hepatic MDA level,which was completely prevented by fullerene C60 treatment. Treatment with fullerene C60 did not affect the hepatic MDA level in normal rats.

 

FIGURE 2. Hepatic MDA levels in the rats at the end of the study. All values are expressed as mean ± SEM. ***, p < 0.001 compared to normal group; †††, p < 0.001 compared to diabetic group. 

3.3. Effect of Fullerene C60 on CAT Activity 

As shown in Figure 3, rats with STZ-induced diabetes exhibited a significantly increased hepatic CAT activity, which was further increased by fullerene C60 treatment. Treatment of normal rats with fullerene C60 also led to a significant increase in the hepatic CAT activity.

 

FIGURE 3. Hepatic CAT activity in the rats at the end of study. All values are expressed as mean ± SEM. **, p < 0.01 and ***, p < 0.001 compared to normal group; p < 0.05 compared to diabetic group. 

3.4. Effect of Fullerene C60 on SOD Activity 

As shown in Figure 4, induction of diabetes by STZ ledtoa29%reductioninthehepaticSODactivity, but this reduction is not statistically significant. However, treatment of the diabetic rats with fullerene C60 significantly increased the hepatic SOD activity.

 

FIGURE 4. Hepatic SOD activity in rats at the end of study. All values are expressed as mean ± SEM. , p < 0.05 compared to diabetic group. 

3.5. Effect of Fullerene C60 on GSH Content 

As illustrated in Figure 5, rats with STZ-induced diabetes exhibited a significantly decreased hepatic GSH content. However, treatment of the diabetic rats with fullerene C60 did not affect diabetes-inducedGSH reduction. Treatment of normal rats with fullerene C60 did not affect the hepatic GSH content either.

 

FIGURE 5. Hepatic GSH levels in rats at the end of the study. All values are expressed as mean ± SEM. *, p < 0.05 and ***, p < 0.001 compared to normal group.

3.6. Effect of Fullerene C60 on Hepatic Histopathological Changes 

As shown in Figure 6, in the livers of both control and fullerene C60-treated normal animals, hepatocytes are regularly arranged around the hepatic sinusoids. Hepatic injury was observed in the diabetic livers in the form of dissociation of the hepatocytes with pyknosis and disintegrated nuclei along with deep basophilic staining of the cytoplasm as well as sinusoidal dilatation and hepatocytes disarrangements. Treatment of the diabetic rats with fullerene C60 significantly improved the above histopathological changes.

 

FIGURE 6. Representative photomicrographs of H&E-stained livers of normal (N), fullerene C60-treated normal (NF), diabetic (D) and fullerene C60-treated diabetic (DF) rats. The diabetic liver is characterized by the hepatocytes with pyknosis and disintegrated nuclei (arrows) as well as deep basophilic staining of cytoplasm. These changes largely disappeared in C60-treated group (Scale bars = 20 μm; magnification = 400×).

4. DISCUSSION 

The results of present study indicated that treatment of diabetic rats with fullerene C60 decreased the main index of oxidative stress (MDA) and considerably improved the histopathological changes in the diabetic livers. Fullerene C60 also augmented the antioxidant capacity of the liver in diabetic rats by increasing the activity of catalase and SOD. Hence, fullerene C60 is proposed as an excellent antioxidant for protecting against diabetes-induced hepatic oxidative stress (Figure 7).

 

FIGURE 7. The probable mechanism of hepatoprotection by fullerene C60 nanoparticles. As illustrated, increased ROS generation and weakened antioxidant defense contribute to hepatic injury in diabetes. Fullerene C60 protects against diabetic liver injury likely via both directly scavenging ROS and augmenting the hepatic antioxidant defense. 

The results of present study showed that the MDA level, as a valuable index for ROS accumulation and oxidative stress, increased in the livers of diabetic rats. Oxidative stress in the tissues causes cell apoptosis and death through detrimental effects on the cellular macromolecules [2]. In the present study, the histopathological assessment also showed these damaging effects of oxidative stress on hepatocytes in the diabetic livers. Oxidative stress develops as a result of an imbalance between pro-oxidant and antioxidant systems in diabetes [23, 24]. Studies have demonstrated that activation of pro-oxidant system mediates ROS overproduction in cells and tissues during diabetes [2]. Weakening the antioxidant defense system is another mechanism that leads to ROS accumulation and oxidative stress in diabetes [23]. In this regard, our results indicated that diabetes decreased the GSH levels in the liver of diabetic rats. GSH as a non-enzymatic antioxidant protects the cells against oxidative stress by neutralizing the free radicals as well as preserving the cellular levels of other antioxidants [23]. According to our results, diabetes also increased the CAT activity of the liver. A significant change in the activity of antioxidant enzymes such as CAT and SOD has been reported in diabetic patients [25]. The activities of antioxidant enzymes are sensitive to oxidative stress, and both enhancement and reduction have been demonstrated in various pathological conditions with ROS accumulation [26]. 

Our results showed that fullerene C60 administration during diabetes significantly decreased ROS accumulation and oxidative stress (decreased MDA levels) in the liver of diabetic rats. Fullerene C60 also improved the histopathological changes of the liver in diabetic rats. It is suggested that fullerene C60 may ameliorate the histopathological damage through inhibition of the harmful effects of ROS on hepatocytes. Fullerene C60 acts as a free radical sponge and eliminates the various oxygen and nitrogen free radicals in biological environments [7, 8, 12]. Fullerene C60 also behaves as an excellent antioxidant through passing the cell membrane and localizing preferentially in the mitochondria [7]. Our results also indicated that administration of fullerene C60 enhanced the antioxidant capacity of the liver in diabetic rats. Fullerene C60 significantly increased the activities of antioxidant enzymes, CAT and SOD, in diabetic rats as well as CAT activity in normal rats. It was reported before that fullerene C60 increased the antioxidant capacity of the cells and tissues [16, 17]. In this regard, fullerene C60 improved the MDA levels and recovered the antioxidant system in cyclophosphamide-induced acute hepatotoxicity [16]. Also, fullerene C60 could act as the SOD and CAT mimetics in an in vitro system [7, 27]. In the present study, using fullerene C60 did not change the GSH content of the liver in normal and diabetic animals. Although several studies have reported the altered GSH content by fullerene C60 [16], others have shown that fullerene C60 has no effects on tissue GSH content [28]. It is suggested that the type of pathological states as well as fullerene C60 dosage may be the causes of these inconsistent results.

5. CONCLUSION 

It is concluded that fullerene C60 may protect against ROS-induced hepatotoxicity in diabetic states. The protective effects of fullerene C60 may be due to the potentiation of the hepatic antioxidant defense systems. Hence, it is suggested that administration of fullerene C60 can be useful for protecting against hepatic oxidative injury in diabetes. 

ACKNOWLEDGMENTS 

The authors are thankful to the Vice Chancellor for Research of Baqiyatallah University of Medical Sciences, Tehran, Iran. Data presented in this manuscript is a part of a PhD thesis. The authors declare no conflicts of interest. 

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