In vitro evaluation of Rheum ribes induced genotoxicity in HepG2 cell lines

Rheum ribes is a perennial herbaceous plant belonging to the Polygonaceae family that grows more on rocky and gravelly slopes in high altitude areas of Levant and Turkey. Rheum ribes is consumed as food and widely used in folk medicine against nausea, constipation and for different diseases including diabetes and hypertension. Unfortunately, the research on Rheum ribes toxicity is insufficient. In our study, the human hepatocellular carcinoma (HepG2) cell line was used in a cytotoxicity evaluation of Rheum ribes water, methanol and chloroform extracts by MTT and NRU tests. Comet assay was used to investigate the genotoxicity potentials of the plant extracts. Our results show that all extracts cause cell death in a concentration dependent manner at 5-50 mg/mL concentrations. The IC50 values are 14.29-31.94 mg/mL by MTT and 21.15-27.66 mg/mL by NRU assay. The highest concentration (25 mg/mL) of methanol extract causes significant DNA damage (8.7-folds). In conclusion, similar to a lot of plants used in folk medicine the risk of Rheum ribes is still unknown. The uncontrolled use of this plant could cause harm to the patients. Our results indicate the possible cytoand gentoxicity effects of Rheum ribes, these results should elevate concerns about the safety of Rheum ribes and other folk herbs.

Although the therapeutic, pharmacognostic, antibiotic and some of biochemical characteristics of Rheum ribes have been well studied, the toxic potential studies are very few and insufficient. For this reason, in this study the cytotoxic and genotoxic potential of methanol, chloroform and water root extracts were evaluated in HepG2 human hepatocarcinoma cells used previously as models of in vitro conditions to study the apical uptake, metabolism and absorption of nutrients and the toxicity of chemicals and drugs (Martin et al. 1997;Brand et al. 2000;Goya et al. 2015).

Materials
Human hepatocarcinoma HepG2 cell line (HB-8065) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell culture medium (Eagle's minimum essential medium, EMEM), Phosphate buffer solution, Fetal bovine serum (FBS), Trypsin-EDTA solution and the antibiotic solution (100 IU/mL penicillin and 100 mg/mL streptomycin) were obtained from Wisent Bioproducts (Montreal, Canada), and all the other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Dried roots of Rheum ribes [Rhizoma Rhei ribi] were purchased from herbalists and spice sellers in Istanbul. Plant samples were tested and identified by Prof. Dr. Emine Akalin (Istanbul University, Department of Pharmaceutical Botany).

Plant extractions
Three different extracts of Rheum ribes were prepared using water, methanol and chloroform. For this, the roots were pulverized, 2.5g of this powder was then treated with 25 mL methanol or chloroform for 30 minutes in a water bath shaker at 25°C. A rotary evaporator and steam from nitrogen gas (40°C) were used to concentrate and dry the extracts. After dissolving the solid residue in 1 mL of dimethyl sulfoxide (DMSO), the solutions were filtrated using 0.45 µm filters. For the water extract, the infusion method was performed. 25 mL of hot (90°C) sterile water was added to 2.5 g of root powder and stirred for 30 minutes at a fixed temperature (90°C). After cooling, the mixture was filtered with filter paper and 0.45 µm filters. The methanol and chloroform extracts concentrations were 2500 mg/mL, the water extract concentration was 100 mg/mL (Abudayyak et al. 2015).

Cell culture and exposure
Human hepatocarcinoma cells (HepG2) were cultured in EMEM medium supplied with 10% heat inactivated FBS and 1% antibiotics. The cells were incubated at 37°C, 90% humidity and 5% CO 2 (confluence 60-80%). 96-well plates were used for the cytotoxicity assays and 6-well plates for the genotoxicity assay. The cell density was 1x10 3 -5x10 3 cell/ well for the cytotoxicty assays and 1x10 5 cell/ well for the genotoxicty assay. The final exposure concentrations were 0.25-50 mg/mL, the exposure period was 24 hours.

Cytotoxicity evaluation
The cytotoxic effects of Rheum ribes root extracts were evaluated using MTT and NRU assays. After the incubation with different extract concentrations (5-50 mg/mL), the exposed cells were treated with MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide) for 2 hours, the succinate dehydrogenase enzyme in the viable cells metabolized the yellowish water soluble MTT to water non-soluble formazan violet crystals. In the NRU assay, the weak red cationic dye accumulates in integral and healthy cells by creating electrostatic hydrophobic bonds with the lysosomal matrix. The cellular enzyme activity in the MTT assay and the integrity of the cells in the NRU assay are evaluated as a sign of cells' viability. A Microplate spectrophotometer system (Epoch, Germany) at 540 nm and 590 nm was used to measure the optical densities (ODs) for NRU and MTT, respectively. The nonexposed cells and cells exposed to DMSO (1%) were evaluated as negative and solvent controls respectively. The concentration -cell death (%) curves were used to calculate the median inhibitory concentrations (IC 50 ) that were responsible for the death of 50% of the cells (Mosmann 1983; Borenfreund et al. 1985).

Genotoxicity evaluation
The Comet assay was used to evaluate the genotoxic potential of Rheum ribes rhizomes extracts in the HepG2 cells. The concentrations of exposure were 6.25; 12.5 and 25 mg/mL for both methanol and chloroform extracts and 0.25; 0.5 and 1 mg/mL for water extract. For this, the exposed cells were trypsinized, washed with PBS 1X and mixed with prewarmed low-melting point agarose. Cells were layered on agarose pre-coated microscope slides, covered with a cover slip and allow to solidify. After fixation on the slides, the cells were treated with lysis solution for one night. The slides were washed and incubated for 20 min in fresh a cold electrophoresis buffer before electrophoresis for 20 min, and treated with a neutralization buffer for 15 minutes. Before evaluation under a fluorescent microscope (Olympus BX53, Olympus, Tokyo, Japan) the cells were stained with ethidium bromide. The Comet analysis and scoring program (Comet Assay IV, Perceptive Instruments, Suffolk, UK) was used to image and score at least one hundred cells per sample. DNA damage to individual cells was expressed as a percentage of DNA in the comet tail (mean tail intensity %) (Singh et al. 1988;Abudayyak et al. 2017). The nonexposed cells and cells exposed to DMSO (1%) were evaluated as negative and solvent controls respectively. For genotoxicity evaluation hydrogen peroxide (H 2 O 2 ; 100 µmol/L) was used as a positive control.

Statistical analysis
Cytotoxicity assays were done in triplicate, experiments were also repeated four times on different days (n=12). The genotoxicity evaluation was done in triplicate. Data is expressed as mean ± standard deviation (SD). The significance of the differences between negative control and exposed cells was evaluated using one-way analysis of variance (ANOVA) and Dunnett t-test by The Statistical Package for the Social Sciences (SPSS) version 23.0 for Windows (IBM Corp.; Armonk, NY, USA). P values of less than 0.05 were selected as the levels of significance.

RESULTS AND DISCUSSION
Rheum ribes is used as a raw and cooked food in Middle East culture (Oktay et al. 2007;Özcan et al. 2007), it is also widely used as a medical herb (Abu-Irmaileh et al. 2003;Alaadin et al. 2007;Özcan et al. 2007;Naqishbandi et al. 2009). The research related to Rheum ribes has mainly focused on the ethnopharmacological relevance (Afifi et al. 2000;Abu-Irmaileh et al. 2003;Cakilcioglu et al. 2010;Nabati et al. 2012;Polat et al. 2013;Kaval et al. 2015;Korkmaz et al. 2015), pharmacognostic characterization (Munzuroglu et al. 2000;Tosun et al. 2003;Özcan et al. 2007;Andiç et al. 2009;Naemi et al. 2014;Amiri et al. 2015), therapeutic (Otoom et al. 2006;Gholamhoseinian et al. 2009;Naqishbandi et al. 2009;Sayyah et al. 2009;Sindhu et al. 2010;Kasabri et al. 2011;Hadjzadeh et  the IC 50 to be 400 µg/mL in MCF-7 breast cancer cells. An in vivo study evaluated the acute and sub chronic toxicity (for 60 days) of Rheum ribes aromatic water in the Wistar rat, an increases of some enzymes like lactate dehydrogenase, abnormality in heart with tissue hemorrhage, hypertrophy and infiltration of inflammatory cells were noticed, the not observed adverse effect level (NOAEL) was calculated to be 250 and 500 mg/kg b.w./day for male and female rats, respectively (Mojarrab et al. 2015) In this work exposure to Rheum ribes extracts for 24 hours caused a decrease in the viability of HepG2 cells dependent on concentration manner. The MTT assay results show that IC 50   values are 14.29; 33.67; and 31.94 mg/mL for the extracts of water, chloroform and methanol, respectively (Figure 1). The IC 50 values for the NRU assay for water and methanol were 21.15 and 27.66 mg/mL, respectively (Figure 2). At the highest concentration (50 mg/mL) of chloroform extract, cellular death was 32.8%. The differences between the cytotoxicity assays were discussed previously and the different results obtained in these different assays were reported (Weyermann et al. 2005;Fotakis & Timbrell 2006). There were many factors including the interaction between the tested xenobiotics and the chemicals of the assay (Wang et al. 2010). In the case of NRU some chemicals decrease the lysosomes account in the cells, leading to negative false results. Additionally, some chemicals may increase the activity of succinate dehydrogenase enzymes causing false positive results in the MTT test. Similarly, chemicals affecting cellular adherence could also cause cells loss leading to false positive results. This could explain the difference in chloroform extracts between MTT and NRU assays.
To the best of our knowledge the genotoxicity of Rheum ribes has not been evaluated previously. Comet assay results show that Rheum ribes water and chloroform and the low concentrations of methanol extracts did not cause any significant DNA damages after 24 hours exposure. Only the highest concentration of methanol extract (50 mg/mL) causes significant DNA damage (8.7-folds) in HepG2 cells (Figure 3).
Unfortunately, there is very little research which discusses the difference in toxicity between different herbal extraction methods. According to this research, it is also difficult to argue that methanol extracts are more toxic than chloroform ones or vice versa. In a previous study, the toxicity of the extracts of ten herbs was evaluated with the MTT test. The results showed that the chloroform extracts of all the test herbs were more cytotoxic than the methanol extracts and none of the water extracts showed any cytotoxicity. The chloroform extracts (in general) possessed more mutagenic activity with the Ames test than methanol and water extracts (Abudayyak et al. 2015). Similar results were also found by Chan et al. (2015). In contrast, previous studies showed that the chloroform and water extracts of Tribulus terrestris were less cyto-and genotoxic than methanol extracts (Abudayyak et al. 2015 B). However, in this study, the results indicate that while the methanol extracts were less cytotoxic than the water and chloroform extracts, they possessed the highest genotoxicity.

CONCLUSION
Contrary to the popular belief that herbs are safe because they are natural products, some herbs can cause significant toxic effects, drug interactions, and even morbidity or mortality. It would be beneficial to evaluate at the very least the cytotoxicity, genotoxicity and carcinogenicity of these herbs in order to assess the associated risks to our health. Rheum ribes is one of herbs that are consumed frequently in Turkey and Middle East countries yet the data concerning the safety of Rheum ribes is still insufficient. There is a need for in vivo and in vitro studies to evaluate its toxic effects. Our results conclude that Rheum ribes can have some negative effects on human hepatocytes by causing cell death and DNA damage.