Cell lines and culture conditions
HepG2 cells were obtained from the American-Type Culture Collection (ATCC) (Manassas, VA, USA). The complete medium used for the reproduction of cells was prepared using low glucose (1000 mg/L) DMEM (Sigma) including 10% of Fetal bovine serum (Copricorn), 1% of penicillin streptomycin (Sigma), 1% of sodium pyruvate (Sigma), 1% of glutamin (Sigma). All incubations administered to the cells throughout the study were performed in a CO2 incubator (Panasonic) at 37 ⁰C and in a medium containing 5% of CO2.
Thawing and Culturing Cells
HepG2 cells were brought to the laboratory in cryotubes in liquid nitrogen. The frozen cells in the cryotubes were first taken from liquid nitrogen to -80 °C. After waiting for 10 minutes, it was left to dissolve by incubating in a hot water bath at 37 °C for 1-2 minutes. In the meantime, 5 mL of medium was placed on a 15 mL of falcon tube. The cells in the cryotube which were about to dissolve completely in the water bath were taken to the prepared falcon tube (that medium was put into it). It was then gently pipetted and centrifuged immediately. At the end of the centrifuge, the medium was drawn with a pipette without contacting the cells accumulated on the falcon base. A second wash was performed by adding 2 mL of medium onto the cell palette again and gently pipetting. The medium remaining on the cells after the centrifuge was drawn again. HepG2 cells, which were dissolved as explained and purified from dimethyl sulfoxide (DMSO) in 2 washes, were dissolved in 1 mL of medium and then cultured to a 25 mL of flask. When the 25 mL of flask became capacious, the cells continued to be replicated by passaging to the 75 mL of flask.
Counting the Cells by Trypan Blue Method
When the cells are cultured to wellplates or flasks in order to use in analyses, it is essential to culture the same / close number of cells in each flask or wellplate. For that reason, before the cells were cultured in well plates, how many cells per mL were determined and the cells were counted. For this purpose, trypan blue cell counting method was used. Trypan blue is a paint that enables live cells to be seen bright under the light. While live cells painted with trypan blue applied to the slide to be counted are seen as bright under the microscope, dead cells are seen as blue and flat. The cells in four counting zones (A-D) consisting of 16 squares each on the counting slide were counted and averaged and therefore the cell counting was completed. Trypan blue method used in live cell counting was tried to be outlined in Fig 2.
The number of cells per milliliter of medium-cell suspension is calculated using the formula following the cell count. The amount of dilution is important in terms of the reliability of counting. The average number of cells between 50 and 150 is an indicator that the cell suspension was processed to ideal dilution. If the average number of cells is very high, volume of the cells in trypan blue-cell mixture is reduced and more diluted cell suspension is obtained and the count is repeated. In this case dilution rate will also increase. The counting can be performed using the mixture obtained after the volume is completed to 100 µL with trypan blue (dilution rate is 10 or 5) by taking 10 or 20 µL of cell suspension in cases where the number of cells is very high. The following formula is used in order to determine the number of cell per mL:
Number of live cells per mL = (Average Number of Cells) x (104) x (Dilution Factor)
Cells were counted in the presented study as the following. Cells removed from the flask base by trypsinization and purified from trypsin by detripinization were dissolved in 4 mL of medium. 50 µL of cell suspension was taken and mixed with 50 µL of trypan blue (dilution rate is 2). Slides and lamellas (Neubauer counting chamber) to be used in counting were prepared. 10 µL of trypan-blue suspension mixture was taken by pipetting and mounted to the neubauer slide properly. Taking the averages of the cells counted in counting zones, the amount of cells per mL of cell suspension was found with the help of the formula. Afterwards, how many mL of the cell suspension should be taken according to the number of cells to be used in the applications was calculated and manipulations with the cells were initiated.
Cytotoxicity Analyses (MTT analyses)
Cytotoxicity levels of the arbutine isoforms, α-arbutine and β-arbutine, in HepG2 cells were determined by MTT (3-4,5-dimethyl-thiazolyl-2,5-diphenyltetrazolium bromide) method. Cancer cells were cultured in 96 piece-well plates as 5000 cells per 200 µL of medium in each well. Cells were incubated for 24 hours so that the cultured cells could take roots to the flask base. Following the incubation mediums of cells were exchanged with the mediums prepared by dissolving α-arbutine and β-arbutine in appropriate concentrations. The mediums with α/β-arbutines used in MTT analyses were prepared in 7 different concentrations between 0,5 and 200 mM. Each dose application was studied as at least 3 replications. Mediums without arbutines were added into the control wells in the same volume. Then, cells were again left to incubation for 24 hours.
The MTT solution was prepared as in 5 mg / mL of concentration by dissolving the MTT salt (Sigma) in phosphate buffer (PBS) with pH 7.4. At the end of the incubation, MTT solution in a volume of 10% of the well volume was added to each well. Cells were incubated in this way for 2-4 hours more. Incubation may be limited to 2 hours if there is excessive formation of formazan crystals (purple) in the wells. Because excessive increase in colour density in wells may lead to higher optical densities to be obtained from spectrophotometers. In this case it is possible to get no results from MTT analyses. For that reason, it is important to monitor the colouration in wells after the first two hours as the cells are incubated with MTT solution. If the formation of formazan crystals in the wells in MTT analysis is low, duration of the incubation can be extended to four hours. However, duration of incubation should not be exceed to 4 hours. Because long incubation durations may lead to coloration in dead cells due to the coloration in mediums by formazan crystals occuring in wells. This may cause to get false results from MTT analysis.
The medium in the wells was pipetted without damaging the formazan crystals formed after incubation with the MTT solution. Following this operation, 200 μL of DMSO was added to each well and formazan crystals were dissolved. Finally, optical density of the samples were almost immediately determined at 540 nm using ELISA microtype reader (Biotek, ELx800). Cell viability of the control group without any arbutine was regarded as 100 % and the effect of each dose on cell viability was calculated using the given formula below [24,10].
Cell viability (%) = [ (100 * Absorbancesample)/ (Absorbancecontrol) ]
Lethal doses (such as LD0, LD50) belonging to arbutine isoforms in HepG2 cells were determined using % viability rates that each dose created in cells. Lethal doses were calculated using Epa Probit Analysis Program (Version 1.5). After the lethal doses were determined in the study, experimental groups were created and administrations were initiated. The experimental groups created within the scope of the study were presented in Table 1.
Genotoxicity (Comet Assay and Micronucleus Test) Analyses
In order to determine how high and low doses of β-arbutin affect genotoxicity when administered to HepG2 cells, DNA damage and micronucleus frequencies were determined in the samples. 24 hours after the necessary operations in experimental groups indicated in Table 1, the cells were collected from the flask base by trypsinisation and detrypsinisation. Cell suspensions belonging to the experimental groups created in 1 mL of PBS (phosphate buffer seline) were used in the analyses.
Comet assay was used in order to determine DNA damage. 20 µL was taken from the cell suspension prepared for this purpose and mixed with 100 µL of LMA (low melting agarose) in eppendorf. All of the cell-LMA mixture in eppendorf was taken and mounted to slides prepared by treating with NMA (normal melting agarose) the day before. Each prepared material was painted following the lysis and electrophoresis stages. Painted preparations were scored by counting as 100 cells under fluorescence microscope .
Numbers (frequencies) of micronucleus as a result of arbutine administrations in samples were determined using micronucleus test. For this purpose, the cells belonging to the samples in each group were spread on clean slides. Dried slides were fixed with pure ethanol for 20 minutes. Then, slides were air dried. Cell nuclei were painted by waiting the dried slides for 20 minutes in 5 % of Giemsa solution. Three slides were prepared from each sample in the analyses. 1000 cells from each preparation were counted at 100X . Micronucleus index values were evaluated over an average of 1000 cells.
Biochemical analyzes were performed on cell lysates obtained from cells. Cell lysates were prepared as the following. 24 hours after the mentioned administrations to the experimental groups (Table 1) cells were collected from falcon base following trypsinisation and detrypsinisation. Cells were finally washed with 1 mL of PBS. After the wash, 500 µL of lysis buffer (PBS solution containing 1 % of triton-X-100 and 8 % of protease inhibitor cocktail) was added onto the cells. They were pipetted. Then, they were sonicated (Binder) for 20 seconds in ice. It was waited for 40 seconds. Sonication was repeated for 10 times in this way. As a result of sonication, intracellular / organelle fluids were allowed to pass into the lysis buffer. Insoluble proteins in lysis buffer despite all administrations were precipitated by centrifugation at 8500 rpm at 4 0C for 10 minutes. The supernatants obtained in this way were used as cell lyzates in chemical analyses. Lysis buffer was used as a blank in analyses. Total protein, oxidative stress and proinflammatory cytokine levels were determined through biochemical analyses in the obtained cell lyzates.
Total antioxidant status (TAS) and total oxidant status (TOS) analyzes, which were analyzed as oxidative stress parameters, were spectrophotometrically conducted using commercial kits (Rell Assays). In addition, OSI levels were calculated by considering TAS levels determined as a general expression of antioxidants in the samples and TOS levels determined as a general expression of oxidants in the samples. OSI levels were calculated according to the following formula in accordance with the kit protocols .
Oxidative Stress Index (OSI) = [(TOS/TAS) x 100]
Proinflammatory cytokine (TNF-α, IL-6 and TGF-β) levels were determined at 540 nm in a microplate reader (Biotek ELx800) using human specific ELISA kits (Sun Red). The data obtained from the biochemical analysis were normalized by dividing each sample's own total protein levels. Protein levels of cell lyzates used in biochemical analyses were determined by spectrophotometrical method at 595 nm with the help of a commercial ELISA kit (Fluka) in which Coommassi Brilliant Blue reactive was used.
Determination of RNA Isolation, cDNA Synthesis and mRNA Expression Levels
Bcl-2 and Caspase 3 mRNA expression levels were analyzed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method using total RNA isolated from control and treatment groups in HepG2 cells and cDNAs synthesized afterwards. In the analyses total RNAs belonging to HepG2 cells were isolated by using commercial kit (Gene Matrix). Concentration and quality of the obtained RNAs were determined in nanodrop (BioTek, Epoch 2). Among the isolated RNAs the ones with optical densities (OD260/280) between 1.7 and 2.2 obtained at 260 nm and 280 nm were used in the study.
In order to be used as a template in the PCR reaction, 2 µg of total RNA belonging to each sample was taken and complementary DNA (cDNA) synthesis was first performed through reverse transcriptase (RT). For this purpose, cDNA synthesis kit was used (Thermo). Sybr Green PCR Master Mix (12.5 µL) and primer pair (oligonucleotide) were added in accordance with the protocols using 1.5 µL of the cDNA belonging to each sample. Primers are specific for each transcription analysis and were determined using studies in the literatüre [24,28]. The primers presented in Table 2 were used as 100 ng in each RT-PCR reaction.
With reference to the cycling threshold (Ct) values of the amplification curves of each sample in the analysis performed on the RT-PCR device (Biorad CFX-96), the relative changes in the mRNA expression levels of the target genes were calculated by 2-DDCt method . Calculations were performed using REST 2009 software (Qiagen, Germany). mRNA expression levels were determined as fungible decrease or increase. Beta actin gene was used as the endogenous control. Expression levels of other genes were emended (normalized) according to the beta actin gene level of each sample.
The number of caspase 3 positive (Cas3+) cells and the percentage of the number of p53 (p53+) positive cells in experimental groups were determined in the study through immunocytochemical analyses. For this purpose, the cells detected in 4 % of buffered neutral paraformaldehyde solution for 24 hours were washed 3 times with distilled water at the end of incubation. Routine tissues were monitored using palettes through cytoblock administration. Cells were blocked in paraffine and sections with 4 µm of thickness were cut using a microtome. The samples taken onto the adhesive coated slides were stained using immunocytochemical technique. For this purpose, rabbit anti-caspase 3 (1/200 of dilution, Abcam, ab13847, Cambridge, UK) and Mouse anti-p53 (1/25 of dilution, DAKO, M7001, CA, US) antibodies were instilled on the samples. Avidin Biotin Complex (ABC) method kit (TA-125-UDX, UltraVision Polyvalent HRP Kit, LabVision/ThermoScientific-US) administration was initiated. First, biotinylated igG was dropped. It was incubated for an hour at room temperature. Then, horse radish peroxidase enzyme conjugated avidin was instilled and left to the reaction for 30 minutes at 37 ºC. Slides were washed and tissues were treated with 3-amino-9-ethylcarbazole (AEC) peroxidase substrate (TA-060-HA, AEC Substrate System, LabVision/ThermoScientific-US). Finally, Gill's (III) hematoxylin was used for the background staining and the slides were covered using an aqueous mounting medium. All samples were examined under light microscopes. They were analyzed by using Zeiss Axio Lab.A1 Microscope- AxioCam ICc 5 Camera (ZEN 2, Carl Zeiss Microscopy GmbH) and image J software . The number of caspase 3 positive (Cas3+) cells per mm2 was used when evaluating caspase 3. The percentage of the number of p53+ cells per unit squares (10 largest magnification zones) was used when evaluating p53.
The obtained data were identified as average ± standard deviation (SH) and SPSS 18 package program was used in data evaluation. Whether the data were normally distributed or not was initially tested. One-way analysis of variance (ANOVA), one of the parametrical tests, and Duncan test as post hoc test were applied to the data with normal distribution. Kruskal-Wallis test, one of the non-parametrical tests, was applied to the analysis results without normal distribution to determine whether there was a statistical difference or not. Which groups from the control groups indicated statistical difference in the parameters in which there was a statistical difference between each other was determined by the Mann-Whitney U test.