CCl₄-induced hepatotoxicity: protective effect of rutin on p53, CYP2E1 and the antioxidative status in rat

Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl₄) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2‐4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl₄ metabolites and cytokines, which propagate inflammatory response [6]. CCl₄ is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7, 8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl₃) and peroxy trichloromethyl radicals (·OOCCl₃). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl₄ exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9, 10].

An increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11, 12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl₄ administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].

Flavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18, 19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20, 21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl₄-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.

The fields of dietary modification and chemoprevention show considerable effective approaches against oxidative stress and are the focus of research these days [36]. Various studies have shown that several mutagens and carcinogens cause generation of oxygen-free radicals, which play a major role in the emergence of cancer and other health disturbances [37, 38]. The present study revealed that CCl₄-induction in rats remarkably increased the level of ALT, AST, ALP and γ-GT. CCl₄ causes acute hepatocyte injuries, altered membrane integrity and as a result enzymes in hepatocytes leak out [39]. However, after treatment with rutin, the pathological increases in ALT, AST, ALP and γ-GT were significantly restored. These results indicate that rutin has the ability to protect against CCl₄-induced hepatocyte injury, which is in agreement with a previous study [40] that reported the protective consequence of polyphenolic compounds against CCl₄-induced liver cirrhosis. Importantly, the increased serum concentrations of triglycerides, total cholesterol and LDL, and the decreased level of HDL, were restored to normal values with rutin co-treatment. This may be explained on the basis that rutin has a strong ability to chelate multivalent metal ions, especially zinc, calcium and iron. Indeed, its ability to chelate minerals has been reported to have some protective effects, such as decreasing iron mediated free radical formation and lowering serum cholesterol, triglycerides and lipid peroxides in experimental animals [41]. Similar findings were reported in another study [42] that investigated the hepatoprotective effects of plant bioactive compounds against CCl₄-induced hepatic injury in rats.

ROS formed during the biotransformation process of CCl₄ are more reactive and toxic than the parental compound. Biotransformation of CCl₄ occurs in the endoplasmic reticulum and the isoenzyme implicated in this process is CYP2E1 [43, 44]. Our results showed that the active free radical/intermediate of CCl₄ caused a reduction in CYP2E1, which was markedly restored by rutin treatment. Our results showed conformity with previous investigations, which demonstrated that the polyphenolic natural product is responsible for its protective, effects [45, 46].

Results of the present study revealed that exposure of rats to CCl₄ resulted in depletion of antioxidant activities. In consonance with our results, Szymonik-Lesiuk et al.[1] reported that CCl₄ intoxication leads to changes in antioxidant enzymes and reactive intermediates involved in the bioactivation of CCl₄ that may truss to those enzymes to prevent their inactivation. Furthermore, our results correspond with [11, 12], and are in agreement with an investigation following CCl₄ intoxication [47].

Glutathione provides a first line of defense and scavenges free radical oxygen species (ROS). The decreased concentration of GSH in liver may be due to NADPH reduction or GSH utilization in the exclusion of peroxides [48]. GSH-dependent enzymes offer a second line of protection as they primarily detoxify noxious byproducts generated by ROS and help to avert dissemination of free radicals [49]. GSH-Px detoxifies peroxides by reacting with GSH and converting it into GSSG, which is reduced to GSH by GSR [50]. Our study revealed that CCl₄ treatment in rats markedly changed the activity of antioxidant enzymes, which was reverted by the co-administration of rutin. Thiobarbituric acid reactive substances (TBARS), the final metabolites of peroxidized polyunsaturated fatty acids, are considered as a late biomarker of oxidative stress [51]. In our experiments, major decrease in lipid peroxidation and consequent reduction in TBARS were obtained by treatment with rutin. The increment in lipid peroxidation, as assessed by the elevated levels of TBARS following CCl₄ administration, has been well documented [12]. Data of the present study indicated that lipid peroxidation induced by oxidative stress caused DNA damage. TBARS react with the DNA strand to form the M₁G adduct, the mutagenic pirimedopurinone adduct of deoxyguanosine [52]. Administration of rutin markedly reduced the DNA damage, which is in close agreement with a previous study [11]. This level of DNA damage decreases the expression of p53 and blocks cells in the G phase of the cell cycle, which gives the cells additional time to repair the DNA damage. However, severe DNA damage may elicit apoptosis [53]. The data revealed that CCl₄-induction caused marked reduction in p53. This result may be explained on the basis that CCl₄ acts as a tumor promoter through increasing the intracellular concentration of ROS necrosis/regeneration and cell proliferation and/or may be due to mutation of p53. Our results regarding p53 are in agreement with previous studies [54, 55].

These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress.