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Byline: S. Mushtaq and L. Naz

Keywords: Quercetin, CCl4, hepatotoxicity.


Liver being one of the vital organs in humans, plays the most significant role in storage, enzymatic metabolism, secretion, detoxification and elimination of drugs and xenobiotics, which has made liver more susceptible for the detrimental effects produced by the xenobiotics (Bedi et al., 2016). Cellular degeneration, inflammatory responses along with fibrotic alterations contribute to hepatic functional decline as evident in cirrhotic liver. Instability of reactive free radicals-mediated oxidative stress has found to be the major mechanism in causing hepatotoxicity. Prevalence of cirrhosis has always been under-estimated in European countries due to difficulties in accessing data from individual countries (Blachier et al., 2013), yet findings proposed approximately 1,70,000 deaths per annum (0.1% mortality rate) in Europe (Zatonski et al., 2010).

In Pakistan, Hepatitis C Virus, Hepatitis B Virus or both co-exist with cirrhosis in 73% of patients; furthermore, HCV sero-prevalence in 60% of cirrhotic patients were also documented in Karachi (Umer and Iqbal, 2016). Widespread use of Carbon Tetrachloride (CCl4) as organic solvent in insecticides, resin, wax, oil products, rubber products, cooling equipment, dry cleaning agents and as furniture and carpet stain remover, has made it a great concern as a frequent environmental toxin (Altug et al., 2007). Owing to the hepatotoxicity-producing properties of carbon tetra chloride, its widespread use is done by many researchers for in-vivo and in-vitro toxicity model development (Coballase-Urrutia et al., 2013; Khuon, 2012; Salem et al., 2015; Jaramillo-Juarez et al., 2008; Ogeturk et al., 2005). Thus, CCl4 administration specifically affects hepatocytes causing cellular necrosis, fatty deterioration, fibrotic changes, and Cirrhosis.

Quercetin (QC) is one of the abundantly found dietary phytochemicals (Prior, 2003) especially in tea, fruits and vegetables (Terao et al., 2008; Kelly, 2011). Onions (Allium Cepa L.) are found to be the richest among other QC containing vegetables (i.e. 200-600 mg/kg) (Hertog et al., 1993), whereas other potentially high amounts of QC were quantified in asparagus, green pepper, and red leaf lettuce (Nishimuro et al., 2015), tomatoes broccoli, apple, lettuce (Uzun et al., 2010; Nishimuro et al., 2015). Several studies have suggested protective role of QC through its antioxidative, anti-inflammatory (Hassan et al., 2019; Al-Harbi, 2019; Afifi et al., 2018), chemo preventive (Russo et al., 2014), anti-viral, anti-ischemic, and anti-allergenic potential in diversified life-style related diseases (Morales et al., 2012; Nishimuro et al., 2015).

To the best of our knowledge, data regarding the hepatoprotective effects of QC is scarce. The intended purpose of the current investigation was therefore aimed to construct an experimental rat model of chronic liver disease (liver cirrhosis) through repeated doses of CCl4 and to investigate the potential of QC against CCl4 induced chronic hepatotoxicity.


Animal Selection and Acclimatization: Male Wistar rats (n=24), obtained from Dow University of Health Sciences, Karachi, Pakistan, were weighed and housed in clean, dry, propylene cages. The animals were exposed to 12 hours dark and light cycle at controlled temperature (25+-2AdegC). The rats were provided with standard lab diet and water was provided ad libitum. Acclimatization was done for one week prior to start of the experiment. The animals were maintained as per the guidelines and regulations of the local animals' ethical committee of the University of Karachi.

QC Extraction: QC was extracted via lyophilized onion and onion skin with methanol using Waring blender (5 minutes) and filtered via Whatman no. 1 filter paper. Concentrated filtrate was obtained via rotary evaporation (Velioglu and Mazza, 1999; Shin et al.,2010).

Experimental groups: The animals were randomly divided into four experimental groups (n=6).

Group I: Control group; given normal saline (2 ml/kg of BW); Group II: CCl4 treated group; CCl4 (0.8 ml/kg BW.) administered subcutaneously, twice a week; Group III: CCl4+QC treated Group; received CCl4 via subcutaneous injection (0.8 ml/kg BW) and 1ml QC extract/100g BW p.o. daily; Group IV: QC treated Group; received 1ml quercetin extract/100g BW p.o. daily.

Following 60 days of treatment, the animals were sacrificed, hepatic tissues were excised, washed with saline, weighed and stored at -80AdegC. Plasma and serum samples were collected via heparinized tubes and serum separating tubes, respectively and stored at -80AdegC for biochemical estimations.

Blood Collection and Processing: Blood was collected from each experimental group animal after 48 hours of the last dose via decapitation according to the standard protocol and then transferred in heparinized sample tubes and serum separating tubes. Plasma and serum thus obtained was stored at -80 AdegC until analysis was done.

Liver sample Preparation of Liver Homogenate: Liver lobes were made free of the associated fascia, followed by saline rinsing, drying then 10% Formalin immersion for histopathological specimen's preparation and consequent storage at -80AdegC for further analytical estimations. Liver parenchymal-lysate was made via tissue perfusion with saline in chilled (1.17%) potassium chloride using homogenizer, the resultant homogenate thus procured was subjected to centrifugation (8000g, 5 minutes, 4AdegC) for nuclear-fragments' isolation. Post-mitochondrial supernatant (PMS) was obtained by centrifuging the post-nuclear supernatant (10500g, 20 minutes, 4AdegC). Hepatic antioxidants and Malondialdehyde estimation were done using the homogenate.

Histopathological Examination: The hepatic tissues were fixed in 10% formalin and paraffin embedded. 4 um thick sections were then made and staining with Hematoxylin and Eosin was done for microscopical examination. The Hepatic-lesion's extent was evaluated from the histological sections via grading and quantitative scores (French et al., 1988). The grading system is as follows:


Score 0###no conspicuous impairment

Score 1###confined hepatocytes' impairment on less than 25%


Score 2###confined hepatocytes' impairment on 25-50% area

Score 3###extensive, rather localized hepatocytes' lesion

Score 4###pervasive hepatocytes' necrosis

Analytical Procedures

Assessment of Hepatotoxicity and Hepatic Oxidative Stress: Alanine Transaminase (Reitman and Frankel, 1957), Aspartate Transaminase (Reitman and Frankel, 1957), Plasma Alkaline Phosphatase (Rec, 1972), and Serum Total Bilirubin (Jendrassik and Grof, 1938) levels were spectrophotometrically analyzed using commercially prepared kits from Randox Laboratories to assess liver damage. Catalase (Sinha, 1972), Superoxide dismutase (SOD) (Kono, 1978), Glutathione Reductase (GSH) (Carlberg and Mannervik et al, 1985) were also analyzed.

Results were represented as mean +- standard deviation (SD). The experiment was laid out under completely randomized design. The data were analyzed by SPSS 16 version through one-way Analysis of Variance (ANOVA) to evaluate differences among treatments followed by the Least Significant Test (LSD) post hoc multiple comparison test at [alpha] a$? 0.05.

Table 1. Comparison among Body weight, Liver weight, and Relative liver weight of Control and different Experimental Groups of rats.

###Group I###Group II###Group III###Group IV

###(Control)###(CCl4 1)###(CCl4+ QC 1,2)###(QC 1,2,3)

Initial Body Weight (g)###124+-1.095###181.5+-23.132c###169.83+-16.91c,n###143.33+-11.76n,a,a

Final Body Weight (g)###162+-7.669###209.5+-23.67b###214+-35.89b,n###177.3+-22.54n,n,n

Liver Weight (g)###5.35+-0.537###7.92+-1.312a###4.9+-0.2n,a###5.86+-1.102n,a,n

Relative Liver Weight###2.619+-0.323###6.986+-1.321b###2.86+-0.0173b,a###3.306+-0.372n,b,n

Table 2. Comparison among Various Plasma Enzymes and Bilirubin Concentration of Control and different Experimental Groups of rats.

###Group I(Control)###Group II (CCl4 1)###Group III (CCl4+ QC1,2)###Group IV(QC 1,2,3)

ALT (U/L)###20.917+-4.05###32.36+-4.52c###25.78+-3.59 a,b###24.3+- 2.84 n,a,n

AST (U/L)###67+-5.24###88.4+-6.98a###42.33+-12.48 c,c###70.25+-11.15 n,a,c

ALP (U/L)###96.45+-7.49###127.7+-11.59c###115.27+-6.62 a,n###110.69+-16.41 a,a,n

Bilirubin (mg/dl)###0.69+-0.06###2.38+-0.44c###1.233+-0.36 b,c###0.922+-0.2117 n,c,n

Table 3. Comparison among Various Liver Antioxidant Enzymes and MDA activity of control and different Experimental Groups of rats".

###Group I###Group II###Group III###Group IV

###(Control)###(CCl4 1)###(CCl4+ QC 1,2)###(QC 1,2,3)

Catalase (umol/g tissue)###15.81+-1.68###3.331+-0.18b###12.932+-9.1n, b###7.879+-3.63a,n,n

GSH (unit/g tissue)###0.711+-0.06###0.02+-0.0013c###0.035+-0.02n,a###0.03+-0.004c,n,n

SOD (unit /g tissue)###1.036+-0.593###0.022+-0.23a###1.073+-0.58n,b###0.819+-0.31n,b,n

MDA (umol/g tissue)###0.605+-0.23###1.4+-0.65a###1.04+-0.17a,a###0.67+-0.12n,a,n


The functional importance of liver in xenobiotics' metabolism renders the hepatic tissues to be crucially vulnerable to the oxidative deterioration. CCl4 is a well-known hepatotoxic agent. In several studies, researchers have used CCl4 for developing in-vivo and in-vitro toxicity animal model owing to its hepatotoxicity-producing potential (Ogeturk et al., 2005; Jaramillo-Juarez et al., 2008; Khuon, 2012; Coballase-Urrutia et al., 2013; Salem et al., 2015). Thus, specific target of CCl4 administration appeared to be the hepatocytes, causing cellular necrotic alterations, fatty deterioration, fibrosis and cirrhosis.

The obtained liver weight and relative liver weight among the experimental groups is depicted in Table 1.

CCl4 has shown significant alteration (augmentation) in the hepatic weight in comparison with the control group (pa$?0.05) indicative of the hepatic injury leading towards fibrosis; whereas the CCl4+QC group had a significant reduction in the hepatic weight when compared with CCl4 group (pa$?0.05) suggesting the reversal of the hepatic lesion towards alleviation of the disease-succession. The body weights, initial and final, were found to be increased among the experimental groups; however, the gain in weight was less and non-significant in the CCl4 group as compared with the control group and CCl4+QC group (Table 1).

Potential of QC on Modification of Plasma Enzyme Levels and Bilirubin: CCl4 induced hepatotoxicity was evidenced by the significant elevation in the plasma enzymes' concentration, ALT, ALP (pa$?0.001), AST (pa$?0.05), and Bilirubin (pa$?0.001) in CCl4-treated group as compared to the control group.

Significant amelioration in ALT, AST and Bilirubin (pa$?0.05, 0.001, 0.01) was unveiled in the CCl4+Quercetin-treated group in comparison with the CCl4-treated group (Table 2). Significantly elevated levels of plasma enzymes were indicative of the hepatic damage produced by chronic CCl4 administration (Table 2). QC has shown to revert the deteriorative alterations of the liver parenchyma induced by CCl4, as indicated by the significant reduction of AST (pa$?0.001), ALT (pa$?0.01), and Bilirubin (pa$?0.001) levels in the CCl4+QC treated group in comparison with the CCl4 treated group (Table 2). Comparable findings were reported by Hassan and associates (Hassan et al., 2019), Kebieche and associates following the Epirubicin-induced acute liver injury (Kebieche et al., 2009), sodium fluoride (NaF)-induced deleteriousness of hepatocytes (Nabavi et al., 2012) and thioacetamide-induced hepatic necrosis (de David et al., 2011).

Potential of QC on Modification of Hepatic GSH, SOD and Catalase Activities: QC has shown to have strong antioxidant scavenging potential as reported by several studies (Hassan et al., 2019; Al-Harbi, 2019; Afifi et al., 2018).

GSH, SOD and Catalase provides the major mechanism of defense against hepatic oxidative damage. GSH possess lipid and hydrogen peroxide neutralizing capability, that chiefly contributes to its antioxidative potential (Han et al., 2006). In hepatocytes, abundance of GSH as a major reducing molecule also provides protection to the cellular proteins against oxidative damage. Proteins containing cysteine residue are especially vulnerable to redox modifications. Transformation of the cysteine's sulfhydryl form to thiolate form make it more sensitive against oxidative damage. GSH contributes in maintaining the redox state of the proteins' sulfhydryl groups (Yuan and Kaplowitz, 2009). In our study, significant reduction (pa$?0.001) in the activity of GSH was observed in CCl4 treated group in comparison to the control group.

Significant elevation in the GSH enzyme activity (pa$?0.05) was observed in the QC+CCl4 treated group as compared to CCl4 treated group, suggesting its radical scavenging potential (Table 3). Comparable findings were reported by El-Sayed and associates following CCl4 mediated hepatotoxicity (El-Sayed et al., 2015) and Afifi and associates after thioacetamide induced hepatotoxicity (Afifi et al., 2018). Dismutation of superoxide anion into H2O2 is catalyzed by SOD; eventually, the biotransformation of H2O2 into water is done by GSH and Catalase. The interplay among SOD, Catalase and GSH causes the reduction of unstable radicals into water. In our study, significant curtailment (pa$?0.05) was observed in the SOD activity in CCl4 treated group in comparison to the control group suggesting accumulation of superoxide anion in the hepatocytes.

Significantly increased activity (pa$?0.01) of SOD was observed in CCl4+QC group as compared to the CCl4 treated group that may be due to the improvement in the radical scavenging potential of SOD activity (Table 3).

Human catalase (hydroperoxidase) is a heme containing enzyme with NADPH being important cofactor for its activity. NADPH prevents catalase inactivation by H2O2. Abundantly found in the cells, this enzyme efficiently scavenges H2O2. Hepatocytes and erythrocytes possess the highest activity of Catalase (Young and Woodside, 2001). In our study, significant curtailment was observed in the Catalase enzyme levels (pa$?0.01) in CCl4 treated group in comparison with the control suggesting catalase activity reduction that may be due to the reduced levels of NADP available to convert inactive catalase into active form. Treatment with QC and CCl4 provided significant elevation in the Catalase enzyme activity (pa$?0.01) (Table 3). Comparable results indicating protective effects of QC has previously been reported in several studies (Al-Harbi, 2019; Kebieche et al., 2009; Nabavi et al., 2012; de David et al., 2011).

Effects of CCl4 and QC administration on Hepatic concentration of MDA: MDA, a major marker indicating lipid peroxidation which is the fundamental component of oxidative stress. The elevated levels of MDA in CCl4 treated rats were indicative of the oxidative damage produced to the liver cell membrane polyunsaturated fatty acids via CYP2E1-mediated activation of CCl4 and consequent transformation into tricholoromethyl and trichloromethyl peroxy radicals. The hepatic MDA concentration was significantly increased (pa$?0.05) in CCl4 treated group as compared to the control group. In our study, CCl4+QC treated group showed significant reduction (pa$?0.05) in the MDA levels as compared with the CCl4 group (Table 3). Our findings are consistent with that of other studies done to elucidate the effects of QC (Afifi et al., 2018; Al-Harbi, 2019; Nabavi et al., 2012).

Reversal of the initially CCl4-induced decreased antioxidant enzymes to near standard levels was observed in our study following QC administration (Table 3). The histopathological findings in CCl4 treated rats included dilatation of the central vein, congested sinusoidal expansion with infiltration of the inflammatory cells suggesting peri-acinar necrotic alterations in hepatic tissues (Figure 1). Approximately 35 to 45% of decayed hepatic cells were seen. In comparison with the control group of rats, periportal-and portal fibrosis as well as inflammation along with portal lobulitis were evident in the CCl4 intoxicated group. QC treatment reduced the extent of the portal-as well as periportal fibrotic features from severe to mild form. Hepatocytic decay was absent in the QC treated rats.

Conclusion: The deleterious effects produced by CCl4 exhibited by the observed fibrotic alterations of the hepatic parenchyma, elevation of plasma enzymes levels and the reduction in the hepatic-antioxidant enzymes, was shown to be significantly reverted attributed to QC-administration which suggests the potential protective role of QC against CCl4-induced hepatotoxicity.

Acknowledgements: We are thankful to the National Research Program for Universities-HEC (NRPU-6598) for funding the research.


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Author:S. Mushtaq and L. Naz
Publication:Journal of Animal and Plant Sciences
Geographic Code:4E0EE
Date:Dec 31, 2020

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