Effects of fullerene C60 on liver tissue in liver ischemia reperfusion injury in rats undergoing sevoflurane anesthesia

ABSTRACT

This study aimed to investigate the effects of fullerene C60 on rat liver tissue in a liver ischemia reperfusion injury (IRI) model under sevoflurane anesthesia to evaluate the ability of nanoparticles to prevent hepatic complications. A total of 36 adult female Wistar Albino rats were divided into six groups, each containing six groups as follows: sham group (Group S), fullerene C60 group (Group FC60), ischemia-reperfusion group (Group IR), ischemia-reperfusion-sevoflurane group (Group IR-Sevo), ischemia-reperfusion-fullerene C60 group (Group IR-FC60), and ischemia-reperfusion-fullerene C60-sevoflurane group (Group IR-FC60-Sevo). Fullerene C60 100 mg/kg was administered to IR-FC60 and IR-FC60-Sevo groups. In the IR group, 2 h of ischemia and 2 h of reperfusion were performed. At the end of reperfusion, liver tissues were removed for biochemical assays and histopathological examinations. Hepatocyte degeneration, sinusoidal dilatation, prenecrotic cells, and mononuclear cell infiltration in the parenchyma were significantly higher in Group IR than in all other groups. Thiobarbituric acid reactive substances levels were significantly higher in Group IR than in the other groups, and the lowest thiobarbituric acid reactive substances level was in Group IR-FC60 than in the other groups, except for Groups S and FC60. Catalase and Glutathione-S-transferase activities were reduced in the IR group compared to all other groups. Fullerene C60 had protective effects against liver IR injury in rats under sevoflurane anesthesia. The use of fullerene C60 could reduce the adverse effects of IRI and the associated costs of liver transplantation surgery.

KEYWORDS:

• Fullerene C60
• nanoparticle
• ischemia reperfusion
• sevoflurane
• liver

1. Introduction

Liver ischemia-reperfusion injury (IRI) is a major complication of liver surgery including liver transplantation, liver resection, and trauma [1]. Interruption of blood flow leads to cell death, resulting from the depletion of cellular energy stores and accumulation of toxic metabolites. When the blood flow is restored, injury to the already ischemic liver can occur. This phenomenon is called IRI and remains the main cause of liver dysfunction or functional failure after liver surgery [2]. Therefore, different types of antioxidants and procedures have been developed to reduce the cellular damage secondary to IR.

Sevoflurane, an inhalation anesthetic, was demonstrated to have a protective effect against liver IRI [3]. Sevoflurane protects the liver from IRI by multiple mechanisms that reduce apoptosis, inflammatory response and reactive oxygen species (ROS) [4].

Nanomaterials are currently used as carriers to deliver drugs and other substances to specific cells [5]. Some nanomaterials show strong antioxidant properties and can be used to treat IRI in different organs [6,7]. Among them, C60 fullerene is the most frequent and effective carbon-based antioxidant, which has been demonstrated with the ability to scavenge the mitochondrial ROS to protect cells from apoptosis [8]. C60 fullerene is a spherical molecule whose surface consists of 60 carbon atoms and can act as a scavenger of free radicals in biological systems, resulting in its antitumor, anti-inflammatory, and hepatoprotective properties [9–11]. Since fullerenes are hydrophobic molecules, their solubility in polar solvents is extremely low [12]. Therefore, it must be converted into a highly soluble form. With polyhydroxylation, fullerenes achieved higher solubility and partial stabilization. In experimental models made with this form, the antioxidant, radiation-protective and many protective activities of the nanoparticle were revealed.

Thus, the purpose of this study was to assess the effect of polyhydroxylated fullerene C60 on rat liver tissue in a liver IRI model undergoing sevoflurane anesthesia and to evaluate the ability of these nanoparticles to prevent hepatic complications.

2. Materials and methods

2.1. Experimental animals

This experimental study was conducted at the Gazi University Animal Experiments Laboratory on 2 September 2021, in accordance with the ARRIVE guidelines. The study protocol was approved by the Animal Research Committee of the Gazi University (G.Ü.ET—21.063), Ankara, Turkey. All the animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

A total of 36 adult female Wistar Albino rats (weighing 270–320 g) were used. The rats were kept at 20–21°C in cycles of 12 h daylight and 12 h dark and had free access to food until two hours before the initiation of the study.

2.2. Experimental design

The rats were randomly divided into six equal groups, each containing six rats: sham group (Group S, n = 6), fullerene C60 group (Group FC60, n = 6), ischemia-reperfusion group (Group IR, n = 6), ischemia-reperfusion-sevoflurane group (Group IR-Sevo, n = 6), ischemia-reperfusion-fullerene C60 group (Group IR-FC60, n = 6), and ischemia-reperfusion-fullerene C60-sevoflurane group (Group IR-FC60-Sevo, n = 6). All rats were anesthetized with 50 mg/kg intramuscular ketamine (Ketalar®; 1 mL = 50 mg; Pfizer, Istanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Alfazyne® %2; Ege Vet, Turkey). They were placed on an electric heating pad under warm light.

2.2.1. Sham Group (Group S)

Only midline laparotomy was performed without any additional surgical intervention.

2.2.2. Fullerene C60 group (Group FC60)

Thirty minutes after the intraperitoneal (i. p.) administration of 100 mg/kg polyhydroxylated fullerene C60 (Sigma Aldrich), midline laparotomy was performed without any additional surgical intervention.

2.2.3. Ischemia-reperfusion group (Group IR)

Midline laparotomy was performed, followed by 120 min of ischemia by clamping the porta hepatis by placing an atraumatic microvascular clamp, followed by 120 min of reperfusion.

2.2.4. Ischemia-reperfusion-sevoflurane group (Group IR-Sevo)

Midline laparotomy and IR were performed, and sevoflurane (Sevorane, AbbVie, England) was applied at a 2,3% concentration for the target minimum alveolar concentration (MAC) 1 and a rate of 4 L/min in 100% O₂ concurrently with the onset of the ischemia period and extending for 240 min. The anesthesia protocol was administered in a transparent plastic box. The box was integrated into a semi-open anesthesia device with static hoses.

2.2.5. Ischemia-reperfusion-fullerene C60

Group (Group IR-FC60): Thirty minutes after the administration of 100 mg/kg polyhydroxylated fullerene C60 i.p., IR was performed.

2.2.6. Ischemia-reperfusion-fullerene C60-sevoflurane group (Group IR-FC60-Sevo)

Thirty minutes after the administration of 100 mg/kg polyhydroxylated fullerene C60 i.p., IR was performed, and sevoflurane (Sevorane, AbbVie, England) was applied at a 2,3% concentration for the target minimum alveolar concentration (MAC) 1 and a rate of 4 L/min in 100% O₂ concurrently with the onset of the ischemia period and extending for 240 min.

At the end of the experiments, the rats were anesthetized with intraperitoneal ketamine (50 mg/kg) and sacrificed by exsanguination from the abdominal aorta. Serum samples were drawn for biochemical assays, and liver tissues were removed for histopathological studies.

2.3. Histopathological evaluation

Histopathological assessment was performed at the Department of Histology of Kirikkale University. After fixation, the specimens were prepared as paraffin blocks. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E). Histopathological assessment and scoring were performed using a light microscope. The same pathologist performed histological evaluations in a blinded manner.

In the histopathological examination, each preparation was examined for hepatocyte degeneration, sinusoidal dilatation, prenecrotic cells, and mononuclear (MN) cellular infiltration in the parenchyma. The semiquantitative histological evaluation technique used by Abdel-Wahhab et al. [13] was applied to interpret the structural changes in the hepatic tissues of the control and experimental groups. According to this, (–) (negative point) represents no structural change, while (+) (one positive point) represents mild, (++) (two positive points) represents medium, and (+++) (three positive points) represents severe structural changes.

2.4. Biochemical evaluation

Biochemical examinations were conducted at the Department of Medical Biochemistry at Gazi University. Oxidative stress and lipid peroxidation in liver tissues were evaluated by measuring the levels of thiobarbituric acid reactive substances (TBARS), catalase (CAT), and glutathione S-transferase (GST) enzyme activities.

TBARS assay was performed to determine lipid peroxidation using the thiobarbituric acid method [14]. TBARS measurements were conducted based on the reaction of MDA with thiobarbituric acid (TBA), which forms a pink pigment with an absorption maximum at 532 nm at acidic pH, and 1,1,3,3-tetraethoxypropane was used as a standard MDA solution.

Catalase (CAT) activity is based on the measurement of absorbance decrease due to H₂O₂ consumption at 240 nm, as described by the Aebi H method [15].

Glutathione-S-transferase (GST) enzyme activity was measured using the method described by Habig et al. [16] The GST activity method is based on the measurement of absorbance increase at 340 nm due to the reduction of dinitrophenyl glutathione (DNPG). The results were expressed in international units per milligram of protein.

2.5. Statistical analysis

Statistical Package for the Social Sciences (SPSS, Chicago, IL) 20.0 program was used for statistical analysis. The Shapiro-Wilk test was used for comparisons to determine the distribution of all variable groups. Variations in CAT and GST activities, TBARS levels, and histopathologic parameters were assessed using one-way ANOVA. The Bonferroni post-hoc test was used after a significant ANOVA test to determine the differences among the groups. The results are expressed as mean±standard deviation, and statistical significance was set at p < 0.05.

3. Results

3.1. Histopathological findings

There was a statistically significant difference observed between the groups with respect to the histological changes in the rat liver tissue (hepatocyte degeneration, sinusoidal dilatation, prenecrotic cell, MN cellular infiltration in the parenchyma) determined by light microscopy (p < 0.05). Hepatocyte degeneration was significantly higher in the IR group than in the S and FC60 groups (P < 0.0001 and P = 0.001, respectively). Additionally, hepatocyte degeneration was significantly lower in the IR-Sevo, IR-FC60, and IR-FC60-Sevo groups than in the IR group (p = 0.021, p = 0.001, p = 0.008, respectively) (Table 1, Figure 1–7).

Figure 1. Light microscopic view of liver tissue of group C; normal liver tissue (HL: hepatic lobules; VC: vena centralis; k: kupffer cell hyperplasia; *: sinusoid dilatation; ↓↓: infiltration; →: hepatocyte; c: dikaryotic hepatocytes; e: erythrocyte), H&EX100.

Figure 2. Light microscopic view of liver tissue of group FC60; normal liver tissue (HL: hepatic lobules; VC: vena centralis; e: erythrocyte; con: congestion; *: sinusoid dilatation; →: hepatocyte; c: dikaryotic hepatocytes; ↓↓: infiltration), H&EX100.

Figure 3. Light microscopic view of liver tissue of group IR (HL: hepatic lobules; VC: vena centralis; e: erythrocyte; con: congestion; *: sinusoid dilatation; ↓↓: infiltration; dikaryotic hepatocytes; inf: inflammation; (*): necrotic and apoptotic appearance in hepatocytes; dej: hydrophilic degeneration), H&EX100.

Figure 4. Light microscopic view of liver tissue of group IR-Sevo (HL: hepatic lobules; VC: vena centralis; *: sinusoid dilatation; ↓↓: infiltration; →: hepatocyte; k: kupffer cell hyperplasia; (*): necrotic and apoptotic appearance in hepatocytes), H&EX100.

Figure 5. Light microscopic view of liver tissue of group IR-FC60 (HL: hepatic lobules; VC: vena centralis; con: congestion; *: sinusoid dilatation; ↓↓: infiltration; →: hepatocyte; c: dikaryotic hepatocytes; k: kupffer cell hyperplasia; dej: hydrophilic degeneration; con: congestion), H&EX100.

Figure 6. Light microscopic view of liver tissue of group IR-FC60-Sevo (HL: hepatic lobules; VC: vena centralis; e: erythrocyte; *: sinusoid dilatation; ↓↓: infiltration; →: hepatocyte; k: kupffer cell hyperplasia; (*): necrotic and apoptotic appearance in hepatocytes), H&EX100.

Figure 7. The comparison of histological changes in rat hepatic tissue [mean ± SD], *p <0.05: when compared with group IR.

Table 1. The comparison of histological changes in rat hepatic tissue [mean ± SD].

Histological Change	Group S (n = 6)	Group FC60 (n = 6)	Group IR (n = 6)	Group IR-Sevo (n = 6)	Group IR-FC60 (n = 6)	Group IR-FC60-Sevo (n = 6)	P**
Hepatocyte degeneration	0.33 ± 0.21*	0.50 ± 0.22*	2.00 ± 0.37	1.00 ± 0.37*	0.50 ± 0.22*	0.83 ± 0.31*	0.004
Sinusoidal dilatation	0.33 ± 0.21*	0.33 ± 0.21*	1.83 ± 0.31	0.83 ± 0.31*	0.67 ± 0.21*	0.83 ± 0.31*	0.004
Pycnotic nucleus	0.33 ± 0.21	0.50 ± 0.22	1.17 ± 0.31	0.67 ± 0.21	0.33 ± 0.21	0.50 ± 0.22	0.147
Prenecrotic cell	0.17 ± 0.17*	0.33 ± 0.21*	1.50 ± 0.22	1.00 ± 0.26	0.50 ± 0.22*	0.83 ± 0.31	0.004
Mononuclear cellular infiltration in the parenchyma	0.33 ± 0.21*	0.33 ± 0.21*	1.67 ± 0.33	1.00 ± 0.26	0.67 ± 0.33*	0.83 ± 0.31*	0.021

p**: Statistical significance was set at a p value < 0.05 for ANOVA test.

*p < 0.05: When compared with Group IR.

Sinusoidal dilatation was significantly higher in group IR than in groups S and FC60 (p < 0.0001 and p < 0.0001, respectively). It was also significantly lower in the IR-Sevo, IR-FC60, and IR-FC60-Sevo groups than in the IR group (p = 0.012, p = 0.004, and p = 0.012, respectively) (Table 2, Figure 1–7).

Table 2. Oxidative state parameters in rat hepatic tissue [mean ± SD].

	Group S (n = 6)	Group FC60 (n = 6)	Group IR (n = 6)	Group IR-Sevo (n = 6)	Group IR-FC60 (n = 6)	Group IR-FC60-Sevo (n = 6)	P**
TBARS (nmol/mL)	0.40 ± 0.04*	0.48 ± 0.06*	1.45 ± 0.26	0.76 ± 0.17*	0.53 ± 0.07*	0.62 ± 0.08*	<0.0001
CAT (IU/mg.pro)	2.88 ± 0.50*	2.34 ± 0.41*	0.90 ± 0.15	1.60 ± 0.40	2.40 ± 0.37*	1.88 ± 0.37	0.013
GST (IU/mg.pro)	0.67 ± 0.18*	0.53 ± 0.12*	0.06 ± 0.02	0.32 ± 0.12	0.59 ± 0.15*	0.44 ± 0.11*	0.025

p**: Statistical significance was set at a p value < 0.05 for ANOVA test.

*p < 0.05: When compared with Group IR.

The number of prenecrotic cells was significantly higher in group IR than in groups S and FC60 (p < 0.0001 and p = 0.001, respectively). The number of prenecrotic cells was lower in the Group IR-FC60 than in the IR group (p = 0.005). The number of prenecrotic cells was similar in Group IR when compared with Groups IR-Sevo and IR-C60-Sevo (p = 0.144 and p = 0.055, respectively) (Table 1, Figure 1–7).

Mononuclear cellular infiltration in the parenchyma was significantly different between groups (p = 0.021). MN cellular infiltration in the IR group was higher than that in the S and FC60 groups (p = 0.002 and p = 0.002, respectively). Furthermore, it was significantly lower in the IR-FC60 and IR-FC60-Sevo groups than that in the IR group (p = 0.017 and p = 0.044, respectively). MN cellular infiltration was similar in the IR and IR-Sevo groups (p = 0.103) (Table 1, Figure 1–7).

When the liver tissue samples were compared in terms of TBARS levels, there was a significant difference between the groups (p < 0.0001). TBARS levels were significantly higher in the IR group than in the S and FC60 groups (p < 0.0001 and p < 0.0001, respectively). TBARS levels in the IR-Sevo, IR-FC60, and IR-FC60-Sevo groups were lower than those in the IR group (p = 0.001, p < 0.0001, p < 0.0001, respectively) (Table 2, Figure 8).

Figure 8. Oxidative state parameters in rat hepatic tissue [mean ± SD], *p <0.05: when compared with group IR.

The enzymatic activities of CAT and GST in the liver tissue were significantly different among the groups (p = 0.013 and p = 0.025, respectively). CAT enzyme activity was lower in the IR group than in the S and FC60 groups (p = 0.001 and p = 0.010, respectively). It was significantly higher in the Group IR-FC60 than Group IR (P = 0.008). However, although CAT enzyme activity was similar in Group IR when compared to Groups IR-Sevo and IR-FC60-Sevo (p = 0.161 and 0.075, respectively), Group IR had the lowest CAT activity (Table 2, Figure 8).

GST enzyme activity was lower in the IR group than in the S and FC60 groups (p = 0.002 and p = 0.014, respectively). GST activity was higher in the IR-FC6O and IR-FC60-Sevo groups than in the IR group (p = 0.006 and p = 0.041, respectively). However, GST activity was similar in the IR and IR-Sevo groups (p = 0.154) (Table 2, Figure 8).

4. Discussion

The present study has shown that fullerene C60 has antioxidant effects in an experimental hepatic IRI model undergoing sevoflurane anesthesia in rats by investigating hepatic histopathological findings (hepatocyte degeneration, sinüsoidal dilatation, prenecrotic cell, MN cellular infiltration in the parenchyma) and biochemical TBARS levels, CAT, and GST enzyme activities.

Liver IRI is a complex pathophysiological process involving multiple factors associated with poor prognosis and multiple organ failure in patients after liver transplantation [17]. Previous studies have demonstrated that liver IR is characterized by oxidative stress responses and the release of ROS, which directly leads to tissue damage and initiates a cascade of deleterious cellular responses, leading to inflammation, cell death, and ultimately organ failure [18,19].

Oxidative stress occurs when cellular levels of ROS exceed the neutralizing capabilities of cellular non-enzymatic and enzymatic antioxidants. CAT is an oxidoreductase that can protect hepatocytes from damage and its levels decrease after IRI [20]. GST catalyzes the conjugation of various endogenous and exogenous compounds to glutathione. Glutathione is the main non-enzymatic cellular antioxidant that protects cells from oxidative injury. GST levels are known to decrease after IR in liver tissue [21]. Similarly, we observed that CAT and GST enzyme activity decreased after liver IRI.

Lipid peroxidation is one of the most important consequences of excessive free radical production and plays a significant role in the mechanism of IRI [22]. The TBARS assay is a method to measure the lipid peroxidation end product MDA, a reactive aldehyde produced by lipid peroxidation of polyunsaturated fatty acids. Previous studies have shown that liver TBARS levels increase after reperfusion [23]. In this study, there was a significant increase in the hepatic levels of TBARS in the IR group compared with the corresponding values in the sham group.

After the reperfusion period, many histopathologic changes occur, such as hepatocyte swelling, vacuolization, endothelial cell disruption, mononuclear (MN) cell infiltration, and hepatic parenchymal cell necrosis [24–26]. In the current study, pathological changes in liver IRI were simulated by establishing a hepatic IRI model in rats. Hepatocyte degeneration, sinusoidal dilatation, prenecrotic cells, and MN cellular infiltration in the parenchyma in injured liver tissue samples were investigated, and all were identified to be significantly higher after IR.

Previous studies have reported that fullerene derivatives are potent antioxidants and fullerene C60 exerts its protective role by acting as a free radical sponge in the kidney, testes, and lung tissues [27]. Furthermore, Kartal et al. investigated the effects of fullerene C60 on skeletal muscle tissue after lower-limb IRI in streptozotocin-induced diabetic rats. They reported that caspase- 3 activity was decreased after fullerene C60 administration [28].

Fullerenes have hepatoprotective effects against oxidative damage [29,30]. Kuznietsova et al. reported that water-soluble pristine C60 fullerene attenuates acetaminophen-induced liver injury [30]. Fullerene C60 administration significantly normalized liver enzyme levels and MDA, GSH, CAT, and SOD activities that were altered by cyclophosphamide-induced liver toxicity [31]. Injac et al. investigated the potential hepatoprotective effects of fullerene C60(OH)24 in different experimental hepatotoxicity models. They reported that fullerene might have a hepatoprotective effects for liver with antioxidant properties [32,33]. Therefore, in the present study, we investigated the antioxidant properties of fullerene C60 in hepatic IRI. The results of the present study showed that treatment with fullerene C60 increased CAT and GST activities, which is consistent with its protective effect.

It is well known that sevoflurane attenuates the IR-induced increase in MDA and decrease in SOD, CAT, and glutathione, demonstrating its antioxidant effect against liver IR injury [34]. Sevoflurane increased CAT and GST activities, similar to fullerene C60, and might have a positive influence on reducing lipid peroxidation. Similar to the literature, this suggests that sevoflurane might have protective effects against IRI in the liver.

Our study has two limitations. The first limitation was the small number of animals in each group. Second limitation of the present study was the absence of aspartate transaminase (AST) and alanine transaminase (ALT) level measurements. ALT and AST are the standard biomarkers of choice for detecting liver injury.

One of the main concerns about Fullerene C60 is the solution formation problem caused by the hydrophobic structure of this nanoparticle. Although concerns have decreased with the availability of polyhydroxylated fullerenes, the problem does not seem to be over. Because it has been reported that aggregates form after a while after preparing a solution with polyhydroxylated fullerene. Dynamic light scattering of C₆₀(OH)′ solutions revealed the formation of fullerene aggregates at concentrations as high as 40 mg/mL [35]. Despite this high resolution, low stability is a concern. However, with the 100 mg/kg chosen in our study, approximately 25–30 mg of polyhydroxylated fullerene was given to each rat. To minimize the possibility of aggregation, the given fullerene was dissolved in 2 cc of distilled water and administered intraperitoneally.

In conclusion, fullerene C60 has protective effects against liver IR injury in rats under sevoflurane anesthesia. The use of fullerene C60 could reduce the adverse effects of IRI and the associated costs of liver transplantation surgery. Hence, researching nanoparticles, which are the most important raw materials for the future, could have important implications in clinical settings.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

MA, AK, ATT, and OK conceived of and designed the study. CO, AY, EK, and MA performed experiments. MK, TM, and MA collected and analyzed the experimental data. MA, AK, and ATT confirmed the authenticity of the raw data. All the authors have read and approved the final manuscript.

Ethics approval and consent to participate

Ethical approval for this study was obtained from the Gazi University Experimental Animals Ethics Committee (Ankara, Turkey; approval no. G.U.ET—21.063).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.