Genotoxicity study of 2-methoxyethanol and benzalkonium chloride through Comet assay using 3D cultured HepG2 cells
Article information
Abstract
Though the key data in identifying carcinogenicity is experience in human, long-term carcinogenicity tests using experimental animals are more realistic. Because carcinogenicity tests require much time and cost, performing the test is minimized through pre-screening. Recently, as bioethics has been strengthened, it is required to minimize animal testing in screening tests as well as carcinogenicity tests. The replacement of the micronucleus assay in experimental animal is the beginning, and the ultimate goal is to replace the carcinogenicity test using experimental animals. The micronucleus assay and the comet assay in 3D culture system of human-derived cells is considered as the most applicable practical measures at this stage. This study was conducted to provide more diverse information in the evaluation of carcinogenicity by establishing the comet test method in a three-dimensional cell culture system. In this study, HepG2 cells were cultured for 4 days in hang-in drop method, and then cultured for 7 days on a low adhesion plate to prepare spheroids. The methods were confirmed by d-mannitol (negative control), ethylmethane sulfonate (positive control), and cyclophosphamide (positive control for metabolite). 2-methoxyethanol and benzalkonium chloride were selected as test substances. Though 2-methoxyethanol is positive in in vivo comet assay and in vitro mammalian chromosome aberration test, it is considered negative in the comprehensive genotoxicity evaluation based on negative in bacterial reverse mutation assay, in vitro mammalian cell gene mutation test and mammalian chromosome aberration test. Benzalkonium chloride has been questioned on carcinogenicity because it is a disinfectant ingredient that has become a social issue in Korea. As a result of the Comet assay for 2-methoxyethanol and benzalkonium chloride in the cultured HepG2 cell line, 2-methoxyethanol was evaluated as positive in the metabolic activation system, but benzalkonium chloride was evaluated as negative in both the presence and absence of the metabolic activation system. Therefore, in order to clarify the carcinogenic potential of 2-methoxyethanol, it is judged that additional studies based on mechanistic studies are needed.
Introduction
About 200 million chemical substances have been discovered around the world and are used as medicines, pesticides, and industrial chemicals, etc [1]. Chemicals have brought us many benefits, but they can also pose a risk to us by inducing diseases or environmental pollution during production and use, so it is necessary to identify their hazard in order to prevent and manage them well. Chemicals have been known to cause cancer since the beginning of the 20th century [2], and in 1918, Yamagiwa and Ichikawa [3] first demonstrated using rabbits that coal tar causes scrotal cancer in chimney sweeps [3]. Since then, verification of carcinogenicity has become an important requirement for chemical management, and standardized animal test data have been utilized for carcinogenicity assessment [4–6]. Standardized test methods are uneconomical and unethical tests in which many laboratory animals are sacrificed. Although many efforts have been made to improve these test methods, a test method that can be completely replaced has not yet been developed. However, as an alternative, the genotoxicity test, which is the main mechanism of carcinogenesis, is being used as a screening test [7].
The evaluation of genotoxicity has been mainly performed according to the test guidelines approved by the OECD [8], and many tests have been done in the order of bacterial reverse mutation test (OECD TG No. 471), chromosomal aberration test in cultured cells (OECD TG No. 473), and gene mutation test in transformed cells (OECD TG NO. 476). Most in vivo tests have been performed as micronucleus tests (OECD TG No. 747). In the evaluation of carcinogenicity, a new genotoxicity test method is also being developed because the strategic limitation of the existing genotoxicity evaluation is also confirmed, and improvement of the strategy of carcinogenicity evaluation is also required [9–12].
The Comet assay to confirm genetic damage was developed by Ostling et al. in 1984 and was confirmed as a useful method for predicting carcinogenicity [13–19]. Therefore, the value to the Comet assay is highly suggested in the detection of carcinogenic substances. Kirkland and Speit [20] argued that only 50% of carcinogens that were not detected by the micronucleus test could be confirmed by the gene mutation test (50% positive), but the Comet test was more effective in screening for carcinogens because 90% of them could be identified [20]. In this atmosphere, the OECD developed a standard test method, and in 2016, the Comet assay method using experimental animals was approved, and it is proposed as a final screening data to confirm carcinogenicity along with the micronucleus test [21,22]. Although in vitro testing has not yet been approved as an OECD Test Guideline, research for standardization is continuing because it has advantages in genotoxicity studies on nanomaterials [23–26].
On the other hand, genotoxicity studies using a 3D cell culture system to replace in vivo tests are being actively conducted [27–29]. The verification of the micronucleus test and the Comet test using human skin cells proposed with the support of the European Cosmetics Group has been completed [27–30]. HepG2 cells derived from human hepatocytes are being studied as a tool for the Comet test. A 2D cell culture system has also been proposed [25], but studies in a 3D cell culture system are also being actively conducted [31–34].
2-methoxyethanol is used in large quantities as inks, varnish coatings, textile dyes, anti-icing additives, and photosensitizers in the semiconductor industry. 2-methoxyethanol was negative in most tests confirming gene mutagenicity, including microbial reversion mutagenicity test, and showed weak positive in sister chromosome exchange test. In addition, most of the in vivo tests were negative, and it was evaluated as a material that does not cause genotoxicity [35]. However, it causes reproductive toxicity and was reported as positive in a short-term in vivo Comet test [36]. Benzalkonium chloride is commonly used as a disinfectant in food, cosmetics and industry. Many disinfectant ingredients, including Benzalkonium chloride, have been receiving a lot of attention in Korea since the humidifier disinfectant incident, and the possibility of carcinogenicity of the disinfectant ingredients is also suspected. According to the known genotoxicity information of Benzalkonium chloride, both in vitro gene mutations and cytogenetic effects, including microbial reversion mutation tests, were predicted or confirmed as negative. It was also confirmed as negative in the in vivo micronucleus test [37]. However, as Benzalkonium chloride is a substance of great social interest, additional confirmation of its carcinogenic potential was required. Therefore, this study evaluated the genotoxicity of two substances through the Comet assay using a 3D cell culture system.
Materials and Methods
Test substances
d-mannitol (Sigma M4125) was used as a negative control, and ethylmethane sulfonate (Sigma M0880) and cyclophosphamide monohydrate (Sigma C0768) were used as a positive control. 2-methoxyethanol (Sigma 000E0399) and benzalkonium chloride (Sigma 12060) were used as test substances. The physicochemical properties of these test substances are described in Table 1. Test substance were prepared using DMSO (Sigma D8414) as a solvent. For S9, a metabolic activation system, Moltox S9 (part id 11-05L.2) was prepared with cofactor III (Genogen C06-003) at a concentration of 30%, and then treated to a final concentration of 3%.
Physicochemical properties for 2-methoxyethanol and benzalkonium chloride.
Cell lines and cell subculture
Cell line HepG2 (ATCC No. HB-8065) isolated from a hepatocellular carcinoma of a 15 years old, white, male youth with liver cancer and CHL/IU (ATCC CRL-1935™), which is mainly used for genotoxicity tests such as chromosomal aberration tests, were used in this study. In addition to the advantage that HepG2 cells are derived from humans, HepG2 cells respond well to direct mutants and have the advantage of having p53, a tumor suppressor gene.
The cell lines were subculture at intervals of 1 week into about 1×106 cells in 10 mL of medium (minimum essential medium, Gibco 11095-080) containing 10% fetal bovine serum (Hyclone, SH3008403) and 100 unit/mL of Penicillin/Streptomycin (CytivaHyclone SV30010) in a 75 cm2 flask (Thermo 156499). Passaging was performed as follows: First, the supernatant was removed and then washed once with phosphate buffered saline (Gibco 10010-23) without calcium/magnesium. Cells attached to the bottom of the flask were treated with 2 mL of Trypsin-EDTA (Gibco 10010-23) in a CO2 incubator (Thermo 51030303-TIF) for about 5 minutes to decompose the intercellular binding protein, 8 mL of the cell culture medium was added, and single cells were formed by pipetting several times. The single-celled cells were placed in a 15 mL tube and centrifuged at 4 °C, 1500 rpm for 10 minutes (Hanil Combi S14R), the supernatant was removed, washed once again with 5 mL of phosphate buffered saline, and 1 mL of fresh culture medium was added. The cells obtained in a 15 mL tube were counted, and about 1×106 cells were placed in a 75 cm2 flask and cultured in a CO2 incubator. During cell culture, every 2–3 days, Trypsin-EDTA was not treated and the supernatant culture medium was freshly changed. CHL/IU was subculture according to HepG2 with a passage cycle twice a week.
3D cell culture
After preparing HepG2 and CHL/IU cells were prepared at 1.25×105 cell/mL, 5 mL of medium was put in a culture dish with a diameter of 100 mm, and 20 μL of cell solution was dispensed inside the lid (about 55 sites) and cultured by the hang-in drop method for 4 days. Then, the cells were transferred to a low adhesion plate (96 well plate, Corning 4515), filled with 100 μL of medium, and cultured for 1 week. During the culture, the culture medium was exchanged with fresh medium every 2–3 days to form spheroids. Cells cultured as spheroids were used for cytotoxicity test and Comet assay.
Cytotoxicity test
A cytotoxicity test was performed to determine the treatment concentration in the Comet assay. For cytotoxicity in the 2D cell culture system, 100 μL of HepG2 aCHL/IU cell lines prepared at 100 cell/mL were put in 96 well plate, incubated for 24 hours, treated with the test substance for 24 hours, and washed twice with PBS (Phosphate Buffered Saline) without calcium/magnesium. Then, 110 μL of CCK-8 (R&D CK4-11) prepared as a fresh medium was added and incubated in an CO2 incubator for 3 hours, and then absorbance was measured at 450 nm (Biotec Synergy H1). Cell viability was calculated by dividing the absorbance value of the test substance-treated group by the absorbance value of the solvent control group and then multiplying by 100. The highest concentration in the Comet assay was set as the IC20 concentration at which the survival rate decreased by 20%, for substances with low cytotoxicity (substances with IC20 of 10 mM or more), 10 mM was set as the highest concentration. Cells cultured with three-dimensional spheroids were also carried out like cytotoxicity in two-dimensional cultured cells.
Comet assay (single cell gel electrophoresis assay)
The cells treated with the test substance for 24 hours were treated with Trypin-EDTA (Gibco, 10010-23) to make them cell separation, and then the Comet assay was performed. After washing the 96-well plate treated with the test substance once with PBS without calcium and magnesium, the cells made into single cells by treatment (2D cultured cells for 5 minutes, 3D sphenoids within 10 minutes while checking the state of cell differentiation through a microscope) with 50 μL of Trypin-EDTA were mixed with LMAgarose (Trevigen, 4250-050-02) in a 1:10 ratio, and then 80 μL was dropped on a comet slide, and then cells were lysed by placing them in the lysis solution (Trevigen, 4250-050-01) at 4 °C for 30 minutes. The lysed cells were placed in an alkaline solution (Sodium hydroxide 0.6 g, 200 mM EDTA 250 μL, deionized water 49.75 ml) for 20–60 minutes to unwind and denaturate the DNA, and then electrophoresis was performed at 15 V for 30 minutes. After electrophoresis was completed, the cells were washed twice with distilled water, fixed in 70% ethanol for 30 minutes, dried at 40 °C for 30 minutes, and then stained with SYBR™ Gold Nucleic Acid Gel Stain (Thermo Fisher, S11494).
The Comet definition was determined by working out the percentage of DNA in the tail, dividing this by the integrated tail intensity and multiplying this by 100. The figure achieved was then divided by the total tail integration cell intensity. The percentage of tail DNA intensity was analyzed by the fluorescence image analyzer (Panoramic scan, 3dHistech, NFEC-2015-11-206063).
Statistical analysis
The percentage tail DNA intensity was measured for about 100 cells in each test group and expressed as the median, upper 25%, and lower 25% values of each value. For differences between the test groups, the Kruskal-Wallis and Mann-Whitney U tests were performed. In addition, trend analysis was performed through linear regression analysis.
Results
Determination of cell culture method
As a result of confirming the morphology of HepG2 cells cultured by the hang-in drop/low adhesion plate complex culture method and the low adhesion plate single culture method, it was confirmed that HepG2 cells formed more spherical spheroids in the hang-in drop/low adhesion plate complex culture method than in the low adhesion plate alone culture on the 11th day of culture (Figure 1). Therefore, the hang-in drop/low adhesion plate complex culture method was selected in this Comet assay.
Cell morphology (400×) of HepG2 cells according to the hang-in drop and the low adhesion plate culture only culture methods.
Confirm the adequacy of the comet assay
For HepG2 and CHL/IU cells, the suitability of the Comet assay was confirmed by using d-mannitol as a negative control and ethylmethane sulfonate as a positive control. There was no significant difference in the cytotoxicity of the positive and negative controls according to the HepG2 and CHL/IU cell culture methods (Figure. 2 and 3).
Confirmation of comet assay using negative and positive control substances in CHL/IU cells. (A~C), negative control, d-mannitol. (A) cytotoxicity test; (B) comet assay in 2D culture; (C) Comet assay in 3D culture. (D~F), positive control, ethylmethane sulfonate. (D) cytotoxicity test; (E) comet assay in 2D culture; (F) Comet assay in 3D culture. *: significance level by a test <0.05, Box: Top 25% and 75% of median. Number of test repetitions: 1 time.
Confirmation of comet assay using negative and positive control substances in HepG2 cells. (A~C), negative control, d-mannitol. (A) result of cytotoxicity test; (B) comet assay in 2D culture; (C) Comet assay in 3D culture. (D~F) positive control, ethylmethane sulfonate. (D) result of cytotoxicity test; (E) comet assay in 2D culture; (F) Comet assay in 3D culture. *: significance level by a test <0.05, Box: Top 25% and 75% of median. Number of test repetitions: 1 time.
As a result of the Comet assay, a dose-dependently significant increase in % tail DNA value was observed when 2D cultured CHL/IU cells were treated with d-mannitol (Figure 2 B), and no trend was observed in 3D cultured cells (p<0.611), a significant increase in the % tail DNA value were observed at 0.31 and 2.5 mM showed (Figure 2 C). When ethymethane sulfonate was treated to 2D and 3D cultured cells, the % tail DNA value showed a dose-dependent increase (Figure 2 E and F). From the above results, no increase in the % tail DNA value was observed in HepG2 cells cultured in 2D and 3D treatment with d-mannitol, but a significant increase in the % tail DNA value was observed in ethylmethane sulfonate treatment. Therefore, it was found that the predictive value of genotoxicity was higher in HepG2 cells than in CHL/IU cells.
Using cyclophosphamide, the effect on the metabolic activation system was confirmed. As a result of treatment with cyclophosphamide in 3D cultured HepG2 cells with or without metabolic activation system, all % tail DNA values increased significantly (Figure 4). However, HepG2 cells cultured without metabolic activation system showed a significant increase at the concentrations treated with 2.5 and 5 mM, but did not show a significant increase at the concentrations treated with 10 mM, so it was evaluated as negative (p<0.966) in the trend evaluation. However, in HepG2 cells cultured with metabolic activation system, an increase in % tail DNA values were observed in a concentration-dependent manner.
Comet assay of cyclophosphamide in 3D cultured hep G2 cells with or without metabolic activation system. (A) cytotoxicity; (B) % tail DNA without metabolic activation system; (C) % tail DNA with metabolic activation system. *: significance level by a test <0.05, Box: Top 25% and 75% of median. Number of test repetitions: 1 time.
The comet assay of 2-methoxyethanol and benzalkonium chloride
As a result of performing the Comet assay for 2-methoxyethanol and benzalkonium chloride using 3D cultured HepG2 cells cultured, a significant increase in the % tail DNA value was observed in HepG2 cells with metabolic activation system during 2-methoxyethanol. However, benzalkonium chloride did not show a significant increase in HepG2 cells with or without metabolic activation system (Figure 5).
Comet assay of 2-methoxyethanol and benzalkonium chloride in 3D cultured HepG2 cells. (A~C), 2-methoxyethanol; A, cytotoxicity assay; without (B) or with (C) metabolic activation system. (D~F), benzalkonium chloride. (D), cytotoxicity; without (E) or with (F) metabolic activation system. *: significance level by a test <0.05, Box: Top 25% and 75% of median. Number of test repetitions: 1 time.
Discussion
Cancer is a disease that is not easy to cure, and it is important to identify the carcinogen first in order to block the carcinogen in a cost-effective and sustainable way [38]. Therefore, IARC is making international efforts to identify carcinogens, such as providing carcinogenic information on about 1,000 chemical substances based on human evidence and carcinogenicity test results [39]. In the evaluation of carcinogenicity of chemicals, human evidence data is preferentially used, but if there is no available human evidence, carcinogenicity test data using animals has to be used [6,40]. Carcinogenicity test using laboratory animals is uneconomical because it consumes a lot of time and money and is unethical due to the use of many laboratory animals. However, there is no alternative test method established so far, and the chemical substances evaluated for carcinogenicity can only be known through human evidence or limited to those that have been tested for carcinogenicity in advanced countries such as the United States and Japan. It is expected that no more than 1,000 substances have been tested. Recently, due to the efforts to minimize animal testing and animal ethical issues, the performance of the 2-year carcinogenicity test has been decreasing. Therefore, as an alternative, there is a trend to study carcinogenesis-based mechanisms.
In the case of Korea, effects on genes (mutations) and cytogenetic effects were confirmed for carcinogenicity screening of chemicals for the reasons described above. The effect on the gene was confirmed by the reverse mutation test using microorganisms, and the cytogenetic effect was confirmed by the micronucleus test using experimental animals. The reverse mutation test is the most used screening test and has the advantage of easily and directly confirming the mutation of a gene but has been pointed out that the effect in humans is not sufficiently reflected and the false positive is high. In addition, micronucleus testing has been required to be replaced by bioethics due to the use of laboratory animals.
In this study, the comet assay was conducted on 3D cultured cells, which are being actively studied recently as a tool for carcinogenicity screening, to confirm the carcinogenicity information of 2-methoxyethanol and benzalkonium chloride. As HepG2 cell line, human-derived cells were used. A variety of methods have been used to culture the cells as 3D spheroids. Ramaiahgari et al. [32] used a Stampar et al. [31] also used a rotating bioreactor. In this study, the hang-in drop method used by Elje et al. [33] and the direct culture method on the low adhesion plate used by Mandon et al. [34] were reviewed and the hang-in drop method was selected and used. In both methods, spheroids were formed, but under the conditions of this study, they grew into more clear spherical cells in the methods of culture with hang-in drop (Figure 1).
In order to establish the appropriate conditions for the Comet assay, HepG2 cells, which are often used for the Comet assay, and CHL/IU cells, which are generally used for the genotoxicity test, were selected. In the 2D culture of CHL/IU cells, the median % tail DNA of the untreated control group was 3.19, and in the groups treated with ethylmethane sulfonate 0.31, 0.63, and 1.25 mM, it clearly increased to 19.8, 48.5, and 79.7, respectively, and a similar trend in the 3D cell culture showed. However, when d-mannitol was treated, the % tail DNA showed a high value of 13.4% even at 0.31 mM. Even in the 3D cell culture system, it was higher than 3.69% of the untreated control group, but no statistically concentration-dependent trend was observed. There was no difference between the d-mannitol-treated group and the untreated group in HepG2 cells cultured in 2D and 3D cell cultures, and a clear increase was seen in the case of with ethylmethane sulfonate-treated (Figure 3). From these results, we judged that the HepG2 cell line was more suitable for the Comet assay than the CHL/IU cell line under these study conditions. A standardized comet test using the TK-6 cell line is being performed to confirm genotoxicity in a 2D cell culture system [26], but the HepG2 cell line is also being studied for genotoxicity in a 2D cell culture system [25,41,42]. In addition, we studied as a representative cell line in a 3D culture system to replace animal testing [31–34]. Therefore, it was decided to use the HepG2 cell line in the Comet assay for this study. As a result of the assay, 2-methoxyethanol induced gene damage in the presence of metabolic activation. These results support the results of Anderson et al. [36] and are considered to have potential to affect genes.
Despite about 1,000 carcinogenicity tests performed using laboratory animals, the carcinogenicity of chemicals identified so far through carcinogenicity tests is less than 5% of the total [43]. Despite this, the number of substances tested has recently been decreasing due to various reasons such as bioethical viewpoints [44,45]. However, as an alternative, Smith et al. [46] suggested 10 main characteristics related to carcinogenesis, suggesting that more diverse studies including genotoxicity in carcinogenicity evaluation are needed. 2-methoxyethanol had low carcinogenic potential in the genotoxicity tests identified so far. However, as a result of this study, it was confirmed that gene damage could be induced in the Comet assay treated with metabolic activation system. Therefore, it suggests a potential for cancer related to non-genotoxicity rather than a carcinogenic potential by established genotoxicity test system. On the other hand, benzalkonium chloride did not induce gene damage in the presence or absence of metabolic activation. It is judged that this can be used as data to lower the carcinogenic potential of benzalkonium chloride.
Conclusions
The appropriateness of the Comet assay was verified using d-mannitol as a negative control, ethylmethane sulfonate as a positive control, and cyclophosphamide as a positive control for metabolic activity through a 3D cell culture system using the HepG2 cell line established through this study. Then, as a result of confirming the effects of 2-methoxyethanol and benzalkonium chloride on the gene using the 3D cell-cultured HepG2 cell line, 2-methoxyethanol was evaluated as positive in the metabolic activation system, but benzalkonium chloride in the presence or absence of the metabolic activation system was evaluated as negative. Therefore, in order to clarify the carcinogenic potential of 2-methoxyethanol, it is judged that additional studies based on mechanistic studies are needed.
Acknowledgement
The author thanks to the Occupational Safety and Health Research Institute for providing financial support.
Notes
Conflict of interest
The author declares no conflict of interest.
CRediT author statement
CHL: Project administration, Supervision, Conceptualization, Methodology, Visualization, Formal analysis; KMS: Investigation, Data Curation, Visualization, Writing-Original draft Preparation; SDS: Visualization, Writing-Reviewing & Editing.