PROTECTIVE EFFECT OF QUERCETIN AGAINST STATINS INDUCED-HEPATOTOXICITY IN CELL LINE

Objective: Statins are the most prescribed lipid lowering agents and consequently they prevent obstructive cardiovascular events in the world. Severe adverse effects of statins, involving myopathy and hepatotoxicity, sometimes limit their usage as lipid lowering agents. We now investigate the toxicity of statins and prove the protective effect of quercetin against statins toxicity in cell line. 
Methods: Human hepatocellular carcinoma cells HepG2 were used in this study were cultured at 37℃ in 5% CO2, in Roswell Park Memorial Institute medium (RPMI 1640) and were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. HepG2 cells were cultured with some of statins as Atorvastatin, Simvastatin and Rosuvastatin with 10µM concentration with and without pretreatment with 10, 20, 40µM quercetin 4 hours before the addition of statins, then cell line was incubated for 24 and 48 hours. Toxicity of statins was determined by cell viability assay (MTT assay), ALT&AST level assay, lipid peroxidation assay by Malondialdehyde (MDA) and Immunofluorescence staining assay using DAPI. 
Results: ALT&AST levels were significantly increased after the addition of statins in HepG2 cells when compared with the control group (DMSO 0.1%), and ALT&AST levels were significantly decreased with pretreatment of the cells  with quercetin 4 hours before the addition of statins in a dose-dependent manner when compared with statin group. Cell viability percentage (MTT concentration) was decreased significantly after the addition of statins to HepG2 cells when compared with the control group, and MTT concentration was significantly increased with pretreatment of the cells with quercetin 4 hours before the addition of statins in a dose-dependent manner when compared with statin group. MDA concentrations increased significantly after the addition of statins to HepG2 cells when compared with the control group, and decreased when the cells were pretreated with quercetin 4 hours before the addition of statins in a dose-dependent manner when compared with statin group. Statins cause cellular oxidative damage by liberating reactive oxygen species, and the cellular damage was prevented when the cells pretreated with quercetin 4 hours before addition of statins. Conclusions: We found that, quercetin shall protect HepG2 cells from statins-induced hepatotoxicity with 10, 20, 40µM concentrations without significant difference between 20 and 40µM concentrations, and may be developed as a therapeutic agent for possible statins toxicity.


INTRODUCTION
Statins (3-hydroxy-3-methylglutaryl co-enzyme-A reductase inhibitors) are the most prescribed agents and the first-line drugs for treatment of hyperlipidemia and consequently they prevent obstructive cardiovascular events in the world. These pharmaceuticals block the mevalonic acid pathway by inhibition of the rate limiting step in the hepatic de novo cholesterol biosynthesis.
Severe adverse effects, involving myopathy and hepatotoxicity, occurred due to statins and sometimes limited their usage as antihyperlipidemic agents (Beatrice et al.,

2008; James K, 2005).
Many Statins such as Atorvastatin, Simvastatin and Rosuvastatin cause deterioration in liver function tests and liver injury in a predictable and dose-dependent manner, the hepatotoxicity associated with their long-term use remained a significant problem for their clinical use (Rossana Calderonet et al., 2010). The use of statins almost associated with biochemical abnormalities of liver functions, moderate elevations of serum transaminases levels (< 3 times the upper limit of the reference range) have been reported following initiation of therapy and are often transient (Jimmy Jose, 2016).
Different hepatotoxicity mechanisms proposed, some of which completely explained. However, most hepatotoxicity mechanisms have not been entirely understood and therefore researchers keep conducting studies (Bensu Karahalil et al., 2017). The present study focused on statin induced hepatotoxicity and possible hepatotoxicity mechanisms. Many observational studies in North America and Europe have confirmed that that statins increase liver enzymes, risk of myopathy, and risk of diabetes mellitus (Kubatka et al., 2014).
Mitochondrial dysfunction is one of the major factors that explain the mechanism of statin-induced hepatotoxicity. Another major reason for statin induced hepatotoxicity is that statins cause apoptotic cell death (Tolosa et al., 2015). Statins metabolized by the liver cytochrome P 450 enzymes which include a huge number of mono-oxygenases and have a critical function for the catabolism of xenobiotic. Mitochondria or cytochrome P 450 -dependent metabolism act as reactive oxygen species (ROS) generation systems and participate in cell death processes (Sergio Di Meo et al., 2016). During statins usage, hepatocytes produce a significant amount of ROS and cause lipid peroxidation leading to a decrease in the mitochondrial membrane potential and promoting cytotoxicity, the highest amount of ROS produced by simvastatin (Abdoli et al., 2014).
Flavonoids, and specifically flavonols, have been reported to exhibit a wide range of biological activities. The flavonoid used in this study was quercetin, which is a common dietary flavonol which is well characterized in vitro antioxidant activity (A. N. Panche et al., 2016). It is the predominant flavonol found in foods, and intakes of between 6 to 31 mg per day have been reported ( This study aims to analyze the hepato-protective effects of quercetin with special emphasis on its mechanisms of action and provide a broad spectrum review on its hepatic properties. HepG2 is a well differentiated transformed cell line that meets all biochemical requirements for the present study and has been widely used in biochemical and nutritional studies because it is considered one of the experimental models that more closely resemble the human hepatocyte in culture (Lapidot et al., 2002;Narayanan et al., 2001). This work, therefore, designed to investigate the protective effect of quercetin on HepG2 cells against statins-induced hepatotoxicity.

MATERIALS AND METHODS
HepG2 cells which were used in this work were purchased from (VACSERA, Egypt), the cells were grown in Roswell Park Memorial Institute medium (RPMI 1640) in 75 cm 3 tissue culture flask and were supplemented with 1% of 100 mg/ml of streptomycin, 100 units/ml of penicillin, 2 μmol/ml of L-glutamine, 250 ng /ml Fungizone and 10% of heat-inactivated fetal bovine serum (all from Lonza ® , Verviers, Belgium). The cells were incubated in a humidified 5% (v/v) CO 2 atmosphere at 37 ºC and the culture medium was changed every other day and the cells were usually split 1:3 when they reached confluence. The confluent cultures were passaged every four days by trypsinization using 1.5ml trypsin/EDTA solution for 5 min at 37 ºC in the cell culture laboratory at Faculty of Pharmacy, Al-Azhar University (Boys), Egypt.

The groups design:
In order to fulfill our proposed aims we classified the groups as follows:

Determination of Cytotoxicity by cell viability assay (MTT assay):
HepG2 cells were seeded in a 96-well plates (5000 cells/well) and then were treated with quercetin at different concentrations (10, 20 and 40µM) 4 hours before the addition of statins. After incubation of the cells for 24 and 48 hours at 37˚C and 5% CO 2 , 1ml of MTT solution was added to each well of a micro titer plate and incubated for 2 to 4 hours at 37˚C. MTT assay based on the principle that viable cell converts MTT into formazan crystals (D. M. Chung et al., 2015). The resultant formazan product dissolved in 1ml of acidic isopropanol/well, and its concentration was measured at 570nm by a micro plate reader (Beckman ® , UV DU 640 spectrophotometer).
All viability assays were performed in triplicate; its percentage growth inhibition was calculated by the following formula:

Determination of ALT & AST:
Assay of ALT activity was performed by kinetic method using Spinreact ® reagent kits (H. U. Bergmeyer et al., 1986).
After 24 and 48 hours of incubation, cell culture media from each three wells were collected in eppindorf tube, centrifuged at 3000×g for 10 min and stored at -80°C until assay. Enzymes activities were calculated by the following equation: Lipid peroxidation biomarker (Malondialdehyde MDA): MDA as a marker for lipid peroxidation, it was estimated according to a colorimetric method (Antonio Ayala et al., 2014). Briefly, 0.5ml of the supernatant was added to 3ml of 1% O-phosphoric acid and 1ml of 0.6% thiobarbituric acid. The mixture was boiled in a water bath at 95℃ for 30 minutes. After cooling the mixture, 4 ml of n-butanol was added to each tube and mixed vigorously. The n-butanol phase (the upper layer) was separated by centrifugation at 2000 rpm for 10 minutes and the absorbance of the pink color was measured at 534nm. MDA concentration was calculated using the following equation: MDA concentration expressed as nmol/ml. Immunofluorescence assay using DAPI staining: Cellular oxidative stress was quantified by the dichlorofluorescein assay to determine the effect of quercetin on the intracellular generation of reactive oxygen species ROS. After being oxidized by intracellular oxidants, Dichloro-dihydrofluorescein diacetate (DCFH-DA) converted to dichlorofluorescein (DCF) (Wang and Joseph, 1999). To assess the expression of lipogenic enzymes by immunofluorescence, cells were seeded on coverslips in 6-well plates in a humidified 5% CO 2 atmosphere at 37 °C for 24 and 48 hours, cells were

Concentration of MDA = (absorbance of sample/absorbance of standard) × concentration of standard
treated firstly with 10, 20 and 40µM quercetin for 4 hours, then statins were added (atorvastatin 10µM, rosuvastatin 10µM and simvastatin 10µM). Control samples were treated with media and equivalent concentration of DMSO (0.1%). After incubation for 24 and 48 hours, the wells were incubated with dichlorofluorescen (DCF) for only 30 minutes, then media was removed and cells were washed 3 times in 2 minutes with PBS. Nuclei was stained with 4', 6-Diamidino-2 Phenylindole Dihydrochloride (DAPI) which was added in a concentration of 5μg/ml for only 3 minutes at room temperature, and excess of DAPI was washed with PBS three times (10 minutes for each). Slides were mounted in the mounting medium (Fluoromount ® ). Microscopic examination was done by LEICA ® fluorescence microscope (model: Leica DM5500 B from Leica Microsystems, USA) under red, blue and green channels. Fluometric analysis: fluometric intensity of microscopic fields was measured for each cultured cell using ImageJ/NIH software (National Institute of mental health, Bethesda, Maryland, USA) for statistical analysis and was represented as a ratio [green (fluorescence intensity) / blue (DAPI for nucleus staining)].

Statistical analysis
Statistical analysis was performed using Graphpad prism (version 5) software; all data were expressed as the mean ± standard deviation (SD) of at least three experiments performed in triplicate. Differences between group means were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons, and statistical significance was established as p<0.05.

RESULTS
Determination of IC 50 of quercetin in HepG2 cell line was done by Cell Viability Assay and the following concentrations were used (0, 5, 10, 20, 40, 50, 100, 200μM). IC 50 of quercetin was obtained by computerized Graph Pad Prism-6 program from dose response curve and listed in Table 1.  1). Pretreatment of HepG2 cell line with 10, 20 and 40µM quercetin for 4 hours reduced cell damage after 24 hours incubation and cell damage was further reduced after 48 hours (fig 1).

Determination of ALT & AST:
Cells were treated with statins for 24 and 48 hours then ALT and AST were measured, the enzyme levels increased. The data shows that toxicity after 48 hours of incubation is higher than the toxicity after 24 hours as shown in figure 2 and figure 3, ALT and AST levels decreased after the addition of 10, 20 and 40µM quercetin in a dose-dependent manner.    Immunofluorescence assay: Fluorescence intensity was measured in the cells treated with statins after 24 and 48 hours incubation which evoked a significant increase in fluorescence intensity, which means elevation of reactive oxygen species (ROS). Pretreatment of the cells with (10, 20 and 40µM) quercetin 4 hours before the addition of statins decreased fluorescence intensity that indicates reduction in ROS levels in response to the addition of quercetin in a dose-dependent manner. After 24 hours incubation:
As shown in figure 5, the addition of 10µM atorvastatin, 10µM rosuvastatin and 10µM simvastatin significantly elevated ROS in HepG2 cells, which can be determined by measurement of the fluorescence intensity compared with the control group. Pretreatment of HepG2 cells with quercetin 4 hours before the addition of statins showed significant decrease in ROS in a dose-dependent manner. 40µM querctin is slightly more protective than 20µM quercetin and is extremely more protective than 10µM quercetin. We observed that, simvastatin elevated ROS more than atorvastatin, and atorvastatin elevated ROS more than rosuvastatin.

After 48 hours incubation:
Control (  A shown in figure 6, the addition of 10µM atorvastatin, 10µM rosuvastatin and 10µM simvastatin significantly elevated ROS in HepG2 cells, which can be determined by measurement of the fluorescence intensity compared with the control group. Pretreatment of HepG2 cells with quercetin 4 hours before the addition of statins showed significant decrease in ROS in a dose-dependent manner.
In case of atorvastatin and simvastatin, there was no significant difference between 20µM quercetin and 40µM querctin but they offered more protection than 10µM quercetin. But in the case of rosuvastatin, 40µM querctin was slightly more protective than 20µM quercetin and was extremely more protective than 10µM quercetin. We concluded that, simvastatin elevated ROS more than rosuvastatin and rosuvastatin elevated ROS more than atorvastatin.

DISCUSSION
Statins are the most commonly prescribed drugs for treatment of cardiovascular diseases and hyperlipidemia and atorvastatin, rosuvastatin and simvastatin are the most frequently prescribed statins. Hepatotoxicity is one of the most important side effects of statins therapy (Abdoli et al., 2014). However, the mechanism of hepatotoxicity induced by statins is not clearly understood yet and the process of apoptosis is either aberrant or inhibited in cancer cells (M. Schuler and D.R. Green, 2001). However, the effect of statins on cancer cells have been studied before, the molecular mechanism involved in their apoptotic effects remains a major focus of investigation.
In our study, we tried to recognize the toxic effect of statins on hepatic cells in vitro using HepG2 cell line and identify the protective effect of quercetin against statins-induced hepatotoxicity.
Our study showed that statins induced hepatocellular toxicity by different ways, by decreasing cell viability% in agreement with (Janine Kah et al., 2012), we observed that atorvastatin decreased cell viability% more than rosuvastatin more than simvastatin after 24 and 48 hours when compared with the control group (DMSO 0.1%), and 10, 20 and 40µM quercetin protect the cells by increasing cell viability % when added 4 hours before statins without significant difference in protection between 20 and 40µM quercetin. We observed that statins increased alanine transaminase level (ALT level), simvastatin increased ALT level more than rosuvastatin more than atorvastatin after 24 and 48 hours when compared with the control group, pretreatment with 10, 20 and 40µM quercetin 4 hours before statins could protect the cells by decreasing ALT levels without significant difference in protection between 20 and 40µM. Also we observed that statins increased aspartate transaminase (AST) level, simvastatin increased AST level more than atorvastatin more than rosuvastatin after 24 and 48 hours when compared with the control group, pretreatment of the cells with 10, 20 and 40µM quercetin 4 hours before treatment with statins could protect them by decreasing AST levels without significant difference between 20 and 40µM quercetin. But there is a little significant difference in protection between 20 and 40µM quercetin when added with 10µM simvastatin after 24 hours incubation. We concluded that statins increase ALT level more than AST level.
In order to investigate the mechanism of statins hepatotoxicity, we determined malondialdehyde MDA as a lipid peroxidation parameter (Giuliana Cighetti et al., 2002). We observed that, statins increased MDA concentration after 24 hours incubation which was in accordance with (Taskeen Fathima et al., 2016), simvastatin increased MDA level more than atorvastatin more than rosuvastatin after 24 and 48 hours when compared with the control group, pretreatment of the cells with 10, 20 and 40µM quercetin 4 hours before addition of statins could protect the cells by decreasing MDA levels without significant difference between 20 and 40µM, there is no significant difference in protection with 10µM quercetin when added with rosuvastatin, and 10µM rosuvastatin does not affect MDA level when compared with the control group after 24 hours incubation. But after 48 hours incubation, 10µM rosuvastatin significantly increased MDA when compared with the control group.
It is very important to investigate the effect of statins on hepatic cells by determination of reactive oxygen species (ROS) and investigation of the protective effect of quercetin by immunofluorescence staining assay using DAPI. We observed that statins increased ROS in HepG2 cells when added and incubated for 24 and 48 hours, simvastatin increased ROS level more than atorvastatin more than rosuvastatin after 24 hours when compared with the control group which was in accordance with (Abdoli N et al., 2014). But after 48 hours incubation, rosuvastatin toxicity is more than atorvastatin toxicity, and pretreatment of the cells with 10, 20 and 40µM quercetin 4 hours before addition of statins decreased ROS level according in a dose-dependent manner without significant difference in protection between 20 and 40µM quercetin with simvastatin toxicity after 24 hours incubation. And after 48 hours incubation there is no significant difference in protection between 20 and 40µM quercetin with atorvastatin and rosuvastatin.

CONCLUSION
Atorvastatin, rosuvastatin and simvastatin have a toxic effect on HepG2 cells with a dose of 10 µM. Quercetin could protect the HepG2 cells from statins inducedtoxicity with 10, 20 and 40µM doses without significant difference between 20 and 40 µM concentrations.