Etanol ve Dimetil Sülfoksidin Çeşitli İnsan Hücre Hatları Üzerindeki in vitro Sitotoksik Etkisi

Although the cytotoxic effects of ethanol and dimethyl sulfoxide (DMSO) on some cell lines have been shown in the literature, there is no study about the cytotoxic effects of these solvents on common used human cell lines, such as melanoma (VMM917), lung cancer (A549), colon cancer (WiDr), normal colon (CCD 841 CoN) and fibroblast (BJ) cells. The aim of this study was to determine the cytotoxic effects of ethanol and DMSO on human breast (MCF-7), liver (HepG2) and cervix (HeLa) cancer cells in addition to above mentioned cells. For this purpose, the cells were treated with different concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, and 4%, v/v) of ethanol and DMSO and then subjected to MTT assay. According to the results, ethanol and DMSO exhibited dose-dependent cytotoxicity in all cells studied. The most DMSO and ethanol sensitive cells were WiDr and VMM917, while the most resistant cells were determined as BJ and A549, respectively. The results revealed that the concentration range in which ethanol and DMSO exhibited cytotoxic effect in each cell line is different. In order not to cause false positive and negative results, the concentration range in which the solvents used in cell culture studies do not have cytotoxic effects should be determined. Research Article Article History Received : 12.03.2020 Accepted : 24.04.2020


INTRODUCTION
For scientific, economic and ethical reasons, cell culture systems have become a widely used laboratory tool in determining the cytotoxicity of a variety of compounds of drug candidate (Forman et al., 1999). These systems allow the investigation of the effects of candidate compounds on cellular functions using stable homogeneous cells (Timm et al., 2013). While it is relatively possible to determine the effects of watersoluble compounds on cells, it is an important problem that the organic solvents used to dissolve apolar compounds may have independent cytotoxic effects on the studied cells (Forman et al., 1999). Organic solvents, such as ethanol, acetone, dimethylformamide and dimethyl sulfoxide (DMSO), are often used to dissolve hydrophobic compounds in cell culture assays due to their strong solubility properties (Jamalzadeh et al., 2016). However, these organic solvents are reported to have cytotoxic effects on various cell types (Forman et al., 1999;Timm et al., 2013). Some studies even show that various cell lines have different sensitivity to the same solvent (Jamalzadeh et al., 2016;Hajighasemi and Tajik, 2017;Yi et al., 2017). Therefore, it is reported that the type and concentration of the used solvent must be determined in such a way as to ensure the solubility of the material being examined without adversely affecting the experimental procedures such as cell growth (Jamalzadeh et al., 2016).
Ethanol is a two-carbon alcohol compound and is frequently used in the dissolving of hydrophobic compounds and preparation of natural product extracts in experimental studies (Jamalzadeh et al., 2016;Misir et al., 2018;Misir et al., 2020). At the cellular level, ethanol is well known to have long-term effects, such as DNA damage and then carcinogenesis, as well as pleiotropic effects that can lead to short-term cytotoxicity (Kade et al., 2016). Dimethyl sulfoxide [DMSO, (CH3)2SO] is a highly polar organic liquid, widely used as a solvent (Yi et al., 2017). DMSO can dissolve a large number of non-polar and polar small components, increase cell membrane permeability, prevent free radical formation and increase the penetration of pharmaceutical mediators into cells (Hajighasemi and Tajik, 2017). DMSO is generally considered a low toxicity solvent. However, it has been reported that DMSO can induce cytotoxicity in various cells based on the type of cell. The mechanism proposed for DMSO cytotoxicity is explained by its effect on the physical properties of phospholipids in the membrane (Hebling et al., 2015;de Abreu Costa et al., 2017).
Various studies have investigated the cytotoxic effect of ethanol and DMSO on some cell lines. Yeo et al. (2000) demonstrated that ethanol exhibits a dosedependent cytotoxic effect on Swiss 3T3 cells by inhibiting DNA synthesis and protein tyrosine phosphorylation, while Wu et al. (2010) showed that the DMSO concentrations higher than 4% (v/v) exhibits cytotoxic and apoptotic properties in the pheochromocytoma (PC-12) cell line. Timm et al. (2013) reported that ethanol and DMSO have a statistically significant cytotoxic effect on five different human white blood cell lines depending on the cell type. Human melanoma (VMM917), lung (A549) and colon (WiDr) cancer cell lines are frequently used in in vitro experiments as melanoma, lung and colon cancer models, respectively (Gazdar et al., 2010;Berg et al., 2017;Liu et al., 2017). Colon normal (CCD 841 CoN) and fibroblast (BJ) cells are also frequently used cell lines in in vitro genotoxicity, aging and cancer models Schäuble et al., 2012;Ferreira et al., 2019). However, to the best of our knowledge, there is no study that determines the concentration-dependent cytotoxic effects of ethanol and DMSO on these five cell lines. The aim of this study was to determine the concentration-dependent cytotoxic effects of ethanol and DMSO on these cell lines for the first time.

Chemicals
All chemicals used in cell culture studies were purchased from Lonza (Verviers, Belgium) and Biological Industries (Kibbutz Beit Haemek, Israel). All the chemicals used in the analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents used were HPLC grade.

Cytotoxicity Experiments
MTT assay with a 72-h treatment time was employed to measure the cytotoxic effects of DMSO and ethanol on various cell lines (Mosmann, 1983). All cancer and CCD 841 CoN cells were seeded into a flat-bottomed 96-well cell culture plates at 1×10 4 cells per well, while BJ cells were seeded at 2×10 3 cells per well (Demir et al., 2019a;Demir et al., 2019b). The cells were then treated with varying concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, and 4%, v/v) of DMSO and ethanol for 72 h (Casañas-Sánchez et al., 2016). Cisplatin was used as a positive control in cytotoxicity experiments to show that the experimental setup was working correctly (Turan et al., 2017;Demir et al 2018b). Subsequently, 10 μL of MTT dye (0.25 mg mL -1 ) was placed inside each well. The crystals that formed were then dissolved in DMSO. Finally, absorbance was measured using a microplate reader (Molecular Devices Versamax, California, USA) at 570 nm. Optical densities were employed to calculate percentage viabilities in treated cells compared to untreated control cells. Log-concentrations versus %cell viabilities were plotted with a logarithmic graph, which was then used to determine the IC50 values (Aliyazicioglu et al., 2019;Demir et al., 2020).

Statistical Analysis
All experiments were performed four times, the results being expressed as mean±standard deviation. Normal distribution was determined using the Kolmogorov-Smirnov test. One-Way ANOVA was used to analyze intergroup differences. p<0.05 was regarded as significant.

RESULTS and DISCUSSION
Cell culture systems are widely used to determine in vitro effects of drugs and natural product extracts (Timm et al., 2013). Drugs or natural products whose effects are investigated are usually made soluble with organic solvents. Therefore, when working with waterinsoluble compounds and it is imperative to determine the range of concentration in which the organic solvent used for each cell group has a cytotoxic effect (Forman et al., 1999). DMSO and ethanol are two of the most used organic solvents in cell culture studies (Jamalzadeh et al., 2016), and there are limited studies about their own cytotoxicity on only some cell lines, such as Swiss 3T3, PC-12, Mono Mac 6, HL-60 and RAW 264.7 (Yeo et al., 2000;Wu et al., 2010;Timm et al., 2013). While VMM917, A549, WiDr, CCD 841 CoN and BJ cell lines are common used cell lines in in vitro experiments as cancer, aging and genotoxicity models, (Gazdar et al., 2010;Schäuble et al., 2012;Aliyazicioglu et al., 2017;Berg et al., 2017;Liu et al., 2017;Ferreira et al., 2019), there is no study that determines the concentration-dependent cytotoxic effects of ethanol and DMSO on these cell lines. In this study, it was therefore aimed to determine the concentration-dependent cytotoxic effects of DMSO and ethanol on these cell lines in addition to MCF-7, HepG2 and HeLa cell lines. The cytotoxic effect of ethanol and DMSO was evaluated using MTT assay, which is a is a non-radioactive, quick, and affordable method (Demir et al., 2018b) and cell viability percentages are shown in Figures 1 and 2.
The results showed that DMSO and ethanol had cytotoxic effects on the all studied cells in a dosedependent manner. When all cells are evaluated together, statistically significant cytotoxic effect of DMSO and ethanol was emerged starting at a concentration of 0.2% (v/v) and 0.4% (v/v), respectively. Interestingly, the statistically significant cytotoxic effect of ethanol on A549 cell line was only seen starting at a concentration of 2% (v/v). In order to make the results more understandable, the IC50 values of DMSO and ethanol in all studied cell lines were calculated and presented in Table 1. Şekil 2. Etanolün insan hücre hatları canlılığı üzerindeki etkisi. *İstatistiksel olarak anlamlı farkı gösterir, p<0.05.  (Yeo et al. 2000;Clave et al., 2014;Casañas-Sánchez et al., 2016;Jamalzadeh et al., 2016). The mechanism of this cytotoxic effect of ethanol is explained by its property to increase the amount of reactive oxygen species (ROS), rate of apoptosis and to modulate the amount of many proteins, such as adenylate cyclase, protein kinase C, protein tyrosine kinases and phospholipase C and D (Mikami et al., 1997;Yeo et al., 2000;Clave et al., 2014;Casañas-Sánchez et al., 2016). There are various report about the cytotoxic effect of DMSO on various cell lines, such as colon cancer (CaCo-2), retinal ganglion (RGC), astrocyte, human leukemic (THP1, U937, Jurkat, Molt-4) cell lines and it is reported that the IC50 values of DMSO on these cell lines vary between 0.5% to 3% (v/v) (Da Violante et al., 2002;Galvao et al., 2014;Yuan et al., 2014;Hajighasemi and Tajik, 2017;Singh et al., 2017). The mechanism of this cytotoxic effect of DMSO is explained by its property to interact with cell membrane and to modulate the metabolism, apoptosis and cell cycle (Singh et al., 2017;Yi et al., 2017).

CONCLUSION
This study is the first to demonstrate the cytotoxic concentration range of ethanol and DMSO in the VMM917, A549, WiDr, CCD 841 CoN and BJ cell lines.
The results reported here show that DMSO and ethanol have cytotoxic effects even at very low concentrations in these cell lines. Therefore, we believe that solvent concentrations should always remain the same when testing serial dilutions of compounds analyzed on these cell lines. It should also be noted that the response of each cell type to the solvent is different and that results obtained by solvent interactions in a cell type cannot be transferred to other cell types.

Researchers Contribution Rate Declaration Summary
The authors declare that they have contributed equally to the article.

Conflicts of Interest Statement
None of the authors had any financial or personal relationships with other individuals or organizations that might inappropriately influence their work during the submission process.