Hepatoprotective effect of orally applied water-soluble pristine C₆₀ fullerene against CCl₄-induced acute liver injury in rats

Abstract

The development of novel more efficient antioxidants is one of the most perspective approaches for the treatment of the harmful effects of toxins and their metabolites. Due to its remarkable antioxidant properties, pristine C₆₀ fullerene has recently been proposed as a promising candidate for many biomedical applications. The present study aimed to determine whether single oral administration of water-soluble pristine C₆₀ fullerene in small dose (1.5 mg kg⁻¹ bwt) could prevent acute liver injury caused by single intraperitoneal injection of carbon tetrachloride (CCl₄; 1.0 mL kg⁻¹ bwt) in rats. We performed structural examination of the liver and monitored the serum markers relevant to hepatocyte integrity as well as liver functionality. The antioxidative potential of pristine C₆₀ fullerene was assessed in vivo by measuring lipid peroxidation. We also measured the activity of catalase and superoxide dismutase in order to evaluate the antioxidative defense system. Biochemical and pathological results obtained in this study indicate that water-soluble pristine C₆₀ fullerene in a single dose of 1.5 mg kg⁻¹ has a hepatoprotective effect against CCl₄-induced toxicity via its antioxidant properties. According to histopathological examinations and some biological tests, water-soluble pristine C₆₀ fullerene is more effective against liver injury when administered before a toxicant than after one. Moreover, our results show that the low dose of C₆₀ fullerene used has no acute toxicity in rodents. We concluded that water-soluble pristine C₆₀ fullerene can be considered as a powerful liver-protective agent.

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Introduction

The liver is the key organ responsible for maintaining homeostasis within the body. It regulates almost all the biochemical pathways related to metabolism, nutrient supply, energy providing, and formation of immunity.¹ The liver is not only involved in physiological functions but also it is important in protecting our body from the harmful influence of toxins.^2,3 It is well known that the liver plays a central role in transforming and clearing toxic chemicals, medication as well as alcohol but long-term exposure of these xenobiotics could cause liver injury.^4,5 It is widely recognized that impaired liver function may cause serious consequences to the individual.⁶

Risk of xenobiotic-induced liver injury has increased in the past decades.⁷ And nowadays, toxic liver disease is recognized as one of the most common liver disorders in the world while effective pharmacological therapy for this pathological process is absent. The most plausible mechanism of xenobiotic toxicity is an enhancement of free radical production that induces lipid peroxidation and oxidative damage of liver cells.^8,9 Thus, the use of antioxidants as protective agents could be a potential therapeutic strategy for xenobiotic-induced toxicity.

Earlier publications examined the biological activity of C₆₀ fullerene, the unique third natural allotropic form of carbon, to show its great potential for biomedical researches.^10–15 The results of latter research suggest that the water-soluble pristine C₆₀ fullerenes are nontoxic at low concentrations;^16–18 they can penetrate through plasma membrane of cells by passive diffusion or endocytosis^19–21 and demonstrate strong antioxidant properties.^22,23 It is well documented that C₆₀ fullerene, administered intraperitoneally or intravenous, may act as cardio- and hepato-protector in both doxorubicin- and carbon tetrachloride (CCl₄)-induced toxicity.^22,24,25 But there are only few available works about the effects of orally applied C₆₀ fullerene.^26,27 Also there is little information about the protective effects of extremely low dose of water-soluble pristine C₆₀ fullerene against harmful influence of toxins.^28,29

The purpose of this study was to examine the influence of the low dose (1.5 mg kg⁻¹ bwt) water-soluble pristine C₆₀ fullerene in the prevention of acute hepatotoxicity caused by single high dose administration of CCl₄ (1.0 mL kg⁻¹ bwt). In order to know whether it can show equal hepatoprotective activity in pre- and post-treatment experiments, we administered C₆₀ fullerenes both 3 h before and 3 h after intraperitoneal injection of toxicant. These results will help clarify the biochemical mechanisms of C₆₀ fullerene action in biological systems.

Results and discussion

Since the C₆₀ fullerene particles' size directly correlates with their biodistribution and toxicity,^30,31 the atomic force microscopy (AFM) study of pristine C₆₀ fullerene aqueous solution (C₆₀FAS) was performed.

The AFM picture in Fig. 1 clearly demonstrates randomly arranged individual C₆₀ molecules with diameter 0.7 nm and their bulk sphere-like aggregates with a height up to 30 nm. The results obtained are in good agreement with theoretical calculations and experimental measurements^32–36 and demonstrate polydispersity of the C₆₀FAS used in our study.

Fig. 1 AFM image (tapping mode) of C₆₀ fullerenes and their bulk sphere-like aggregates on the mica surface, which were precipitated from C₆₀FAS with initial concentration of 0.15 mg mL⁻¹. Arrows indicate the height of the individual nanoparticles.

CCl₄ is a well-known hepatotoxin.³⁷ The cleavage of CCl₄, catalyzed by liver microsomal cytochrome P450, leads to the formation of trichloromethyl radical (CCl₃˙), which in the presence of oxygen interacts with it to form the more toxic CCl₃O₂˙.³⁸ These highly reactive radicals initiate chain lipid peroxidation (LPO) of the hepatocyte membrane causing functional and morphological changes in the liver cell. CCl₄-induced accumulation of oxidants results in oxidative liver damage which are characterized by increased necrosis, steatosis, and foamy degeneration followed by the progression of fibrosis.^39,40 Liver injury caused by CCl₄ is the best characterized model of xenobiotic-induced hepatotoxicity and has been used extensively for decades in preclinical animal studies for the screening of hepatoprotective activities of different drugs.^41,42 Thus, to evaluate the protective effect of C₆₀FAS against acute xenobiotic-induced hepatic damage, male Wistar rats injected with CCl₄ were used in the present study.

According to previously published papers, several biochemical changes were observed as a result of oxidative stress originating from toxicity induced by CCl₄.^22,43 Our results well agree with these data. The preliminary assessment of integrity and functionality of liver cells in the CCl₄ group was done by determining the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (GGT) activities as well as total and direct bilirubin levels. Effects of C₆₀FAS on these parameters in rats with CCl₄ liver injury model are shown in Fig. 2A–F.

Fig. 2 The influence of a 1.5 mg kg⁻¹ dose of C₆₀FAS on serum markers of liver injuries induced by CCl₄ injection: alanine aminotransferase (A), aspartate aminotransferase (B), alkaline phosphatase (C), γ-glutamyl transferase (D) activities in serum; total bilirubin (E) and direct bilirubin (F) concentrations in serum. Values are expressed as mean ± SEM (n = 6); *p < 0.05 significantly different from the control group; ^#p < 0.05 significantly different from the CCl₄ group.

In our experiments, hepatocytes damage, caused by CCl₄ application, accompanied by a statistically significant increase in ALT and AST activities compared to the control values. The obtained results showed significant decreased of these enzyme activities in the experimental groups that received a 1.5 mg kg⁻¹ dose of C₆₀FAS either under pretreatment or 3 h after CCl₄ treatment condition (Fig. 2A and B). As shown in Fig. 2C and D, activities of ALP and GGT were no significantly changed in CCl₄ treated rats compared to control ones. After 24 h of CCl₄ treatment, the total bilirubin and direct bilirubin levels significantly raised by 3.5 and 5.6 times respectively, compared to control group (Fig. 2E and F). Pretreatment with 1.5 mg kg⁻¹ of C₆₀FAS decreased significantly the CCl₄-induced elevation of serum total bilirubin by 35% as well as direct bilirubin by 45%, whereas treatment with C₆₀FAS after CCl₄ administration caused reduced of these parameters by 60% and 70%, respectively (Fig. 2E and F). When animals were given C₆₀FAS alone no changes in the serum enzymatic activities, neither the concentrations of total and direct bilirubin were observed (Fig. 2A–F).

It is well known that CCl₄ produces liver damage.^37,40 The elevated levels of ALT and AST as well as total and direct bilirubin are indicative for cellular leakage, loss of the hepatocyte membrane integrity and impairment of liver function. In our study, the treatment with C₆₀FAS according to the both scheme of administration suppressed the increment of these parameters induced by CCl₄ injection. We supposed that the reversal of increased serum biomarkers in CCl₄-induced liver damage by the fullerene may be due to the prevention of the leakage of intracellular content and functional improvement of the hepatic cells by its antioxidant effect.^22,23,44 This is confirmed by our findings which showed that C₆₀FAS can overcome oxidative stress caused by CCl₄.

LPO in the liver tissue was characterized by measurement of thiobarbituric acid reactive substances (TBARS), which is widely used as a marker of free radical mediated LPO injury. In our experiments, 24 h after a single dose of CCl₄, we observed a significantly increased concentration of TBARS in the liver tissue (Fig. 3) that proved activation of oxidative process in the organ. This data is supported by previous studies. C₆₀FAS abolished the toxic effect of CCl₄ and maintained LPO at a basal level. Applied alone, the 1.5 mg kg⁻¹ dose of C₆₀FAS did not cause any alteration in the TBARS level. These results support the hypothesis that pristine C₆₀ fullerene is a strong antioxidant and non toxic at low concentration.^40,45,46

Fig. 3 The influence of a 1.5 mg kg⁻¹ dose of C₆₀FAS on CCl₄-induced LPO in liver tissue. Values are expressed as mean ± SEM (n = 6); *p < 0.05 significantly different from the control group; ^#p < 0.05 significantly different from the CCl₄ group.

The increased TBARS level in liver of CCl₄-treated rats suggests enhanced LPO leading to tissue damage and failure of antioxidant defense mechanism to prevent formation of excessive free radicals. Superoxide dismutase (SOD, E.C. 1.15.1.1.) has been reported as one of the most important enzymes in the enzymatic antioxidant defense system.^47,48 It is found in the cytosol as a zinc/copper-containing enzyme and in mitochondria as a manganese-containing enzyme. SOD dismutates the superoxide radicals by converting them into hydrogen peroxide and oxygen. Catalase (CAT, E.C. 1.11.1.6.) is an enzymatic antioxidant widely distributed in all animal tissues. CAT is known to break hydrogen peroxide down to water and oxygen and can be found in the peroxisome and mitochondria, especially in liver.⁴⁹

The levels of SOD and CAT in serum as well as in liver tissue were measured for all experimental groups (Fig. 4A–D). High level of CAT in serum was observed 24 h after CCl₄ treatment (Fig. 4B), whereas serum SOD activity was statistically similar with this enzyme activity in serum of control group (Fig. 4A). C₆₀FAS is seems to stimulate the serum SOD activity under CCl₄-treatment conditions because this enzyme level was increased in both groups with CCl₄ injury model treated with C₆₀FAS (Fig. 4A). We have shown that C₆₀FAS did not effect on serum CAT activity of CCl₄-treated rats because the enzyme activity in both C₆₀FAS/CCl₄ and CCl₄/C₆₀FAS groups were approximately equal to this one in serum of group CCl₄ (Fig. 4B). In the case of treatment with C₆₀FAS alone, serum SOD and CAT levels close to control group were observed (Fig. 4A and B).

Fig. 4 The influence of a 1.5 mg kg⁻¹ dose of C₆₀FAS on the activity of antioxidative enzymes in serum and liver tissue of rats treated with CCl₄: superoxide dismutase (A) and catalase (B) activities in serum; superoxide dismutase (C) and catalase (D) activities in liver tissue. Values are expressed as mean ± SEM (n = 6); *p < 0.05 significantly different from the control group; ^#p < 0.05 significantly different from the CCl₄ group.

Effects of C₆₀FAS on hepatic SOD and CAT activities are shown in Fig. 4C and D. Application of CCl₄ significantly elevated the activity of the examined enzymes compared to the control group. Treatment with C₆₀FAS 3 h before the application of CCl₄ as well as 3 h after CCl₄ injection succeeded in preventing oxidative stress in liver tissue and maintained the basal activity of SOD and CAT (Fig. 4C and D). The dosage of C₆₀FAS in this study was not toxic to the liver tissue, and the activity of antioxidative enzymes in the C₆₀FAS group did not reveal any significant changes compared to the control group (Fig. 4C and D).

Previous studies have indicated that CCl₄ can enhance the activities of antioxidant enzymes.⁵⁰ Our present work clearly showed that activities of CAT and SOD in the liver as well as in the serum of rats treated with CCl₄ alone were significantly higher than those in the control group. The elevated these enzyme activities in rats received CCl₄ may indicate a large increase of superoxide anion and hydrogen peroxide after toxicant injection; however, we cannot state in what degree the process of removal of hydrogen peroxide is effective. C₆₀ fullerene was found to be a powerful radical-scavenger for superoxide, hydroxyl and lipid radicals.^22,23,44 In this study, C₆₀FAS was potent in decreasing the SOD and CAT activities in rat liver but not serum, which may be resulted from the biodistribution nature of C₆₀ fullerenes. The uptake of C₆₀ fullerene in liver was relatively high, while the clearance from liver was rather slow.^51,52 This may eventually lead to the relatively long retention of C₆₀ fullerene in liver and consequently be absorbed into liver cells and acted as an antioxidant there.

The results of our pathohistological analysis of liver tissue conducted in rats after a single dose of CCl₄ are similar to the results that were previously reported.⁵³ Liver histopathological changes after 24 h toxicant treatment were noticeable with light microscopy.

The liver in control group had normal histological structure, typical of this organ (Fig. 5A, C and D).

Fig. 5 Histopathological changes in rat's livers (H&E staining; magnification ×100 (a and b) or ×400 (c–n)): (A) control; (B) CCl₄; (C) control (periportal zone); (D) control (centrilobular zone); (E) CCl₄ (periportal zone); (F) CCl₄ (centrilobular zone); (G) C₆₀FAS/CCl₄ (periportal zone); (H) C₆₀FAS/CCl₄ (centrilobular zone); (I) CCl₄/C₆₀FAS (periportal zone); (J) CCl₄/C₆₀FAS (centrilobular zone); (K) C₆₀FAS (periportal zone); (L) C₆₀FAS (centrilobular zone). Radial structure of the liver cords in control rats, which is damaged after CCl₄ treatment, is shown by black lines. Hydropic degeneration of hepatocytes and infiltration of leukocytes in CCl₄-treated rats are shown by black and white arrows, correspondingly.

After treatment with C₆₀FAS no histopathological changes were found (Fig. 5K and L), only some hepatocytes had more lighter cytoplasm compared to the control group.

The main pathohistological changes in the liver after treatment with CCl₄ were the following: damage of radial structure of the liver cords, hydropic degeneration of hepatocytes, infiltration of inflammatory cells, the appearance of homogeneous eosinophilic content in some sinusoids (seldom), hepatocyte necrosis (seldom) (Fig. 5B, E and F). These changes heterogeneously scattered in the liver (Fig. 5B). Hydropic degeneration of hepatocytes and infiltration of inflammatory cells were significantly greater in the centrilobular zone compared to periportal zone of the liver (Fig. 5B; Table 1).

Table 1 Percentage of hepatocytes with pathological changes

Group	Description	Centrilobular zone	Periportal zone
a p < 0.05 significantly different from the CCl4 group.
I	Control	—	—
II	CCl4	31.0 ± 8.4	13.8 ± 2.6
III	C60FAS/CCl4	14.5 ± 1.7^a	6.0 ± 1.4^a
IV	CCl4/C60FAS	33.2 ± 8.9	15.7 ± 3.0
V	C60FAS	—	—

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Treatment with C₆₀FAS 3 h after the application of CCl₄ did not alleviate all these lesions (Fig. 5G and H). Percentage of hepatocytes with pathological changes no had significant difference compared with the group that received only CCl₄ (Table 1).

After pretreatment with C₆₀FAS 3 h before the application of CCl₄ we observed damage of radial structure of the liver cords, but the leukocyte infiltration was lower. In addition, the number of pathologically altered hepatocytes was significantly fewer (Fig. 5I and J; Table 1). Thus, pretreatment with C₆₀FAS 3 h before the application of CCl₄ protected the liver against injury induced by CCl₄.

These our findings suggest that C₆₀ fullerene more effectively improves liver injury when applied 3 h before toxicant injection. This hypothesis also agree with our results indicating different changes of liver index and liver cytokine profile in rats received C₆₀FAS 3 h before CCl₄ injection and 3 h after one.

It was found, that liver index in CCl₄ treated rats was significantly higher than in control (55.3 ± 2.4 vs. 45.2 ± 1.9). Animals from the group protected by an oral application of C₆₀FAS 3 h before toxicant had liver index similar to the control (47.2 ± 2.8), while in rats treated with C₆₀FAS 3 h after CCl₄ administration liver index was significant higher than in control rats (52.2 ± 3.5) and was comparable to the index of CCl₄ group animals, who received toxicant alone. Elevated liver index may indicate inflammation and pathological changes in the organ.

Our findings suggested that intensity of inflammation process was not the same in liver of rats, whom received C₆₀FAS 3 h before CCl₄ and 3 h after one. This is confirmed by our results of both macro- and microscopic assessment of the liver as well as the results of cytokine profile measurement.

Cytokines are pleiotropic peptides produced by most of all nucleated cells in the body.⁵⁴ In variety tissues, including the liver, constitutive production of cytokines is absent or minimal. There is increasing evidence that cytokines mediate not only hepatic inflammation, apoptosis and necrosis of liver cells but also they mediate the regeneration of liver tissue after injury.⁵⁵

Fig. 6 shows the results of our study of levels of various hepatic inflammatory cytokines under CCl₄-induced acute liver injury in rats with or without C₆₀FAS treatment compared to the control rats. In our study we observed significantly increased pro-inflammatory cytokine (IFNγ, IL-1β, IL-12) levels by 25–50% in liver of rats received CCl₄ compared with control group (Fig. 6A–C). Such results were expected because it is well known that pro-inflammatory cytokines have emerged as a key factor in various aspects of liver disease, such as fibrosis and/or cholestasis. Different changes of pro- and anti-inflammatory cytokine levels were indicated in the case of treatment of CCl₄-administered rats with C₆₀FAS 3 h before toxicant and 3 h after one. When C₆₀FAS was applied after CCl₄ injection, the anti-inflammatory cytokine (IL-4, IL-10) levels significantly decreased by 15–20% (Fig. 6D and E) while pro-inflammatory cytokine IL-12 increased by 40% in liver of rats compared with control group (Fig. 6C). When C₆₀FAS was applied before CCl₄ injection we did not observe any changes in liver cytokine content. Moreover, animals received C₆₀FAS before toxicant had the same cytokine levels as the control group. These results suggests about more intensive inflammation process in liver of rats received C₆₀FAS treatment after toxicant injection: overwhelming expression of pro-inflammatory cytokines can damage healthy tissues, while decreasing of anti-inflammatory cytokines, which can act as inhibitors of inflammation and are necessary for the regeneration of the tissue after the liver has been injured, might inhibit the process of hepatic recovery.

Fig. 6 The influence of a 1.5 mg kg⁻¹ dose of C₆₀FAS on the cytokine profile in liver tissue of rats treated with CCl₄. IFNγ (A), IL-1β (B), IL-12 (C), IL-4 (D), IL-10 (E) contents in liver tissue were expressed as percentage of control (group I); *p < 0.05 significantly different from the control group; ^#p < 0.05 significantly different from the CCl₄ group.

Experimental

Main reagents and kits

C₆₀FAS (0.15 mg mL⁻¹) used in this study was synthesized as previously described.^36,56,57 Briefly, for the preparation of C₆₀FAS we used a saturated solution of pristine C₆₀ fullerene (purity > 99.99%) in toluene with a C₆₀ molecule concentration corresponding to maximum solubility near 2.9 mg mL⁻¹, and the same amount of distilled water in an open beaker. The two phases formed were treated in ultrasonic bath. The procedure was continued until the toluene had completely evaporated and the water phase became yellow colored. Filtration of the aqueous solution allowed to separate the product from undissolved C₆₀ fullerene. Different concentrations of C₆₀ fullerene in aqueous solution (from 0.5 to 0.01 mg mL⁻¹) are obtained by this method.

CCl₄ was purchased from Sigma-Aldrich (St. Louis, MO, USA). CCl₄ solutions for intraperitoneal (i.p.) injection were prepared ex tempore by dissolving the substances in olive oil. Assays kits for the detection of serum ALT, AST, ALP, GGT, direct and total bilirubin were purchased from ELITechGroup (Paris, France). Anti-rat monoclonal antibodies to INFγ, IL-1β, IL-4, IL-10, IL-12 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Horseradish peroxidase-conjugated anti-mouse, anti-goat and anti-rabbit antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Atomic force microscopy study

The state of C₆₀ fullerene particles in aqueous solution was monitored using AFM (“Solver Pro M” system; NT-MDT, Russia). Under the AFM analysis the sample was deposited onto cleaved mica substrate (V-1 Grade, SPI Supplies) by precipitation from an aqueous solution droplet. The sample visualization was carried out in semi-contact (tapping) mode. AFM measurements were performed after complete evaporation of the solvent.

Animals and experimental design

A total of 30 white Wistar male rats weighing 175 ± 10 g were kept in standard laboratory conditions with free access to water and rodent chow. The animals used in this study were treated in accordance with international principles of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 1986). The study was conducted after obtaining Institutional Animal Ethical Committee clearance.

The rats were randomly divided into five experimental groups, namely (Table 2): the control rats (group 1) were treated with olive oil (1.0 mL kg⁻¹) by i.p. as vehicles for CCl₄; II–IV groups were treated with 50% CCl₄/olive oil (1.0 mL kg⁻¹) by i.p. injection, at once;^26,41 C₆₀FAS in a single dose of 1.5 mg kg⁻¹ was given by oral gavage (o.g.), 3 h before (C₆₀FAS/CCl₄ group) and 3 h after (CCl₄/C₆₀FAS group) administration of CCl₄; group V was treated with C₆₀FAS only in accordance to the scheme described above. All animals of I–IV groups were sacrificed 24 h after CCl₄/vehicle injection by urethane anesthesia. Animals of V group were anesthetized and sacrificed 24 h after C₆₀FAS treatment.

Table 2 Animal groups and experimental design

Group	Description	n	Administration-agenda	50% CCl4/olive oil (1.0 mL kg^−1)	C60FAS (1.5 mg kg^−1)
I	Control	6	i.p. olive oil	−	−
II	CCl4	6	i.p. in single dose	+	−
III	C60FAS/CCl4	6	o.g. before 3 h/i.p.	+	+
IV	CCl4/C60FAS	6	i.p./o.g. after 3 h	+	+
V	C60FAS	6	o.g. in single dose	−	+

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It is important to note that a dose of 1.5 mg kg⁻¹ C₆₀FAS applied in our experiments does not present any acute or subacute toxicity in animals: it was significantly lower than the maximum tolerated dose of C₆₀ fullerene, which was found to be 5 g kg⁻¹ both for oral or i.p. administration to rats.²²

Finally, it was established that during 48 h the water-soluble C₆₀ fullerene derivatives can be localized in the liver of mice.⁵⁸

Samples collection

Blood samples were collected in routine biochemical test tubes. Serum for the determination of biochemical parameters was prepared by centrifugation at 1000g for 30 min of previously incubated (for 30 min at 37 °C) blood samples. The sera separated and kept at −20 °C until analysis.

Livers were immediately collected after the animals sacrificed and weighted for calculation of the liver index, which was expressed as mg (wet weight of organ) per g (dead body weight). Liver tissue samples were taken from the left liver lobe and cut into two pieces. One piece was fixed in 10% formalin for histopathological examination. The other piece (1 g) was homogenized in 10 mL ice-cold 50 mM Tris–HCl buffer (pH 7.4) using an automatically homogenizer. The homogenate solution was centrifuged at 12 000g for 30 min at 4 °C. The supernatant was collected and stored at −80 °C for further biochemical analysis. The protein concentration was determined by the Bradford method⁵⁹ using crystalline bovin serum albumin (BSA) as a standard.

Biochemical assays of serum

The serum activities of ALT, AST, ALP, GGT, as well as levels of total and direct bilirubin were measured by the biochemical analyzer Microlab 300 (Elitech, France) using commercial kits from Elitech diagnostic (France) according to the standard protocols provided by manufacturers. Enzyme activities were presented in international unit per liter (U L⁻¹). Bilirubin concentration was expressed as μmol L⁻¹.

Measurement of lipid peroxidation

LPO in the liver tissue was characterized by the spectrophotometric assessment of TBARS.⁶⁰ An aliquot of 0.4 mL of liver tissue homogenate was added to 1.6 mL an aqueous solution of 25 mM Tris–HCl and 175 mM KCl (pH 7.4). Total protein fraction was separated from the mixture by precipitation with 20% trichloroacetic acid and further centrifugation for 15 min at 5000g. After addition of 1 mL of 0.8% aqueous solution of thiobarbituric acid to 2 mL of obtained supernatant, the samples were heated for 30 min in a boiling water bath. After cooling, the optical density of the samples was determined with a spectrophotometer (Smart Spec™ Plus, BioRad, USA) at 532 nm. The amount of LPO products was calculated using the molar extinction coefficient ε₅₃₂ = 1.56 × 10⁵ M⁻¹ cm⁻¹. The TBARS level was expressed as nmol mg⁻¹ of protein.

Antioxidative enzyme assays

SOD activity was assayed by the method of Sirota T. V.⁶¹ based on the capability of SOD to inhibit the autooxidation of adrenaline: 10 μL of serum or liver homogenate was added to a cuvette containing 2 mL of 0.2 M carbonate buffer, pH 10.65, and then 0.1 mL of 0.1% adrenaline was added. Specific enzyme activity (relative unit, U) was calculated as the relation of an optical density value of experimental sample to control one (without biological material) measured at 347 nm at 3 min since adding of adrenaline expressed per 1 min and total protein content in experimental samples (U mg⁻¹ min⁻¹).

CAT activity was measured by the method of Korolyuk et al.:⁶² 0.1 mL serum or liver homogenate (each sample was previously 10-fold diluted with the Tris–HCl buffer, pH 7.4) was incubated in 2 mL freshly prepared 0.03% H₂O₂ at room temperature for 5 min. Control sample included 0.1 mL buffer instead biological material. The enzymatic reaction was stopped by addition of 1 mL of 4% ammonium molybdate and the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm against blank (mixture of 0.1 mL buffer, 2 mL distillate water and 1 mL ammonium molybdate). The enzyme activity was calculated as the difference of H₂O₂ content in control and experimental samples by using calibration curve previously prepared with standard H₂O₂ solutions. Activity of CAT was expressed as μg mg⁻¹ of protein.

Cytokine immunoassay

Cytokine measurements in liver tissue were done by enzyme-linked immunosorbent assay according to the standard instructions. ELISA plates were coated overnight at 4 °C with samples of liver homogenate previously diluted with Tris–HCl buffer, pH 7.4 to obtained concentration of proteins 10 μg mL⁻¹. After being washed, plates were blocked with 5% nonfat dry milk for 1 h at 37 °C and washed again. After that plates were incubated for 1 h at 37 °C with specific primary antibodies against the cytokines such as IFN-γ, IL-1β, IL-12, IL-4, IL-10. Plates were washed and incubated for 1 h at 37 °C with corresponding secondary antibodies conjugated to horseradish peroxidase. After washing, substrate (OPD and hydrogen peroxide) was added. The reaction was stopped by addition of 2.5 N H₂SO₄. Plates were read at 492 nm by a microplate reader (μQuant™, BioTek Instruments, Inc). The different cytokine concentration in the control group of animals (group I) was set at 100%, and changes in cytokine concentration are given as percentage of controls.

Pathomorphological study

Livers were fixed in 10% formalin solution, containing 4% (wt/vol) formaldehyde. After fixation the samples were dehydrated and embedded in paraffin. 5 μm-thick sections were cut using a microtome Microm HM325. The sections of liver were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol, stained with hematoxyline and eosine, dehydrate, clear, and mount with mounting medium. These sections were analyzed by microscope Olympus BX41 at magnification ×100 or ×400 to detect pathological changes. Additionally percentage of hepatocytes with pathological changes was calculated. The digital microphotographs of stained sections of liver were taken using a computer-assisted image analyzing system (microscope Olympus BX41 and Olympus digital camera C5050).

Statistical analysis

The data of biochemical estimations were reported as mean ± SEM for six animals in each group. Statistical analyses were performed using one-way analysis of variance (ANOVA). Differences were considered to be statistically significant when p < 0.05.

Conclusions

In this study, CCl₄-induced hepatotoxicity was used as a model of liver injury. Our results showed that CCl₄ significantly increased serum hepatotoxicity markers and altered MDA level, antioxidant enzyme activities, cytokine profile in liver tissues. Considering that the mechanism of CCl₄ toxicity are predominantly based on free radical production we can conclude that oxidative stress plays an important role in xenobiotic-induced liver damage process. Treatment with water-soluble pristine C₆₀ fullerene in final dose of 1.5 mg kg⁻¹, given orally 3 h before as well as 3 h after toxicant, can normalize serum markers levels, improve the antioxidant ability, maybe dye its strong antioxidant and free-radical scavenging capabilities. According to the results obtained in our pathohistological and biochemical examinations, orally applied C₆₀FAS 3 h before CCl₄ injection more effectively protect liver against chemical induced toxicity than C₆₀FAS administered 3 h after toxicant. Our results complement a previously conducted investigations indicating high antioxidative and cytoprotective potential of C₆₀ fullerene without any recorded side effects. Further investigations should be directed to determination of the pathways of its activity.

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