Cytotoxic Effects of Eugenol and α-Terpineol on the Rainbow Trout Gonadal Cells

ABSTRACT


Introduction
Eugenol (C10H12O2) is a volatile monoterpene phenolic aromatic compound found in essential oils obtained from most plant species (Ulanowska and Olas, 2021).Eugenol, which is recognized as a nonmutagenic and safe molecule by the World Health Organization (WHO), is used in various industrial sectors including medicine, pharmaceuticals, dentistry, food sweetening, agriculture, and cosmetics (Nisar et al., 2021).Eugenol's anti-inflammatory, antimicrobial, analgesic, antioxidant, neuroprotective, antidiabetic, and antitumor properties have been scientifically determined.In addition, based on the detection of eugenol's roles in cellular functions, such as triggering apoptosis and stopping the cell cycle, its effectiveness as a therapeutic molecule is being intensively investigated (Zhao et al., 2022;Racea et al., 2023a).Eugenol's therapeutic properties are associated with free radical scavenging (Carvalho et al., 2023).However, it has also been stated that eugenol causes structural and functional damage to tissues in a dose-dependent manner (Carvalho et al., 2022).α-Terpineol (C10H18O) is the first monocyclic monoterpenoid isomer of terpinols (Negreiros et al., 2023).There are reports on the anti-inflammatory, antiproliferative, antitumor, antiulcer, antibronchitis, antimicrobial, antimutagenic, blood pressure-lowering, antidiarrheal, anticonvulsant, and sedative activities of α-terpineol (Sales et al., 2020;Chen et al., 2023).α-Terpineol is extensively used as a flavoring agent in foods, a fragrance in cosmetics and cleaning products, and as a suspension solvent in the production of paints and fuel cells.Additionally, it has uses in the insecticide and miticide sectors, aromatherapy, and pharmaceutical industry (Sales et al., 2020;Chen et al., 2023).However, concentration-and time-dependent toxic effects of α-terpineol have been demonstrated in various model organisms and cancer cells.These toxic effects have mostly been associated with plasma membrane degradation, ROS production, lipid peroxidation, and mitochondrial degradation (Agus, 2021;Negreiros et al., 2023).
Considering the use of eugenol and α-terpineol in food, medical, and agriculture treatments, data on their potential toxicity is very crucial.Fish cells are widely used in vitro toxicology studies.The rainbow trout gonadal cells (RTG-2) from the Oncorhynchus mykiss have advantages such as the ability to metabolize toxic compounds, not requiring an exogenous metabolic system, and showing higher sensititivity than mammalian cells (Yurdakök-Dikmen et al., 2018).The RTG-2 cells have been successfully used for cytotoxicity testing of several materials (Çiçek, 2023).To the best of our knowledge, the concentration-dependent cytotoxic effects of eugenol and α-terpineol have not been investigated on the RTG-2 cells.Thus, the present study aimed to determine the dose and exposure time dependent cytotoxic effects of eugenol and α-terpineol on these cells.

Sulforhodamine B test
The Sulforhodamine B (SRB) assay was proposed in other studies to examine the cytotoxicity of compounds with redox potential in adherent cells (Shakil et al., 2022;Işık and Çiçek, 2024).Therefore, SRB assay was used to assess the cytotoxicity of eugenol and α-terpineol treatments due to their involvement in redox reactions, and because the RTG-2 cells are adherent cells (Bezerra et al., 2017;Agus et al., 2022;Işık and Çiçek, 2024).Briefly, cells were fixed with 100 µL of cold 10% trichloroacetic acid and holded at + 4 °C for 1.5 h.The plates were washed with distilled water (dH2O) five times and were air-dried.Then, the fixed cells were dyed with SRB staining dye by adding 50 µL of a 0.4% (w/v) 1% acetic acid solution and incubated for 30 min in the dark.Later, the plates were washed with a mixture of 5% acetic acid (in a 5:1 ratio) to remove the free staining dye.After the airdrying process, 150 µL of 10 mM Tris base was added to each well, and the plates were agitated at 150 rpm for 10-20 min.The absorbance values were read using a microplate reader (EpochTM, BioTek, USA) at 564 nm (Vichai and Kirtikara, 2006;Shakil et al., 2022).

Data analysis
The study data were analyzed using the GraphPad Prism 9 software (GraphPad Software, Inc., California, USA).Treatments were assessed using one-way analysis of variance and Dunnett's multiple comparison test.All throughout were stated as mean ± SD. Analysis was performed in triplicate (n=6).
Reports on the concentration-dependent cytotoxic effects of eugenol are scarce.High concentrations of eugenol (≥3 mmol/L) have been reported to be cytotoxic to primary oral mucosal fibroblasts.This effect is associated with intracellular glutathione and ATP depletion.However, it has also been stated that low concentrations of eugenol (<1 mmol/L) have a protective effect on cell viability by inhibiting xanthine oxidase activity and lipid peroxidation.It has been reported that eugenol treatment (83 μM) significantly increased the metaphase II rate, first polar body extrusion, cumulus cell expansion, cytoplasmic maturity, viability, glutathione level, division rates, embryo development, expanding blastocyst and blastomere number in bovine oocytes (de Oliveira et al., 2021).It has been demonstrated that treatment with eugenol (50 μM) increased the viability of healthy human keratinocytes (HaCaT) cells over time (Aburel et al., 2021).In addition, it has been stated that eugenol (0.1 mM) did not cause a significant decrease in HaCaT cell viability after 72 h of treatment (Racea et al., 2023b).Some studies have highlighted that the concentration of eugenol matters in inducing cytotoxicity.Besides reports stating that it can only induce cytotoxicity at concentrations in the mM range, there are also some studies showing cytotoxicity at concentrations in the μM range (Bezerra et al., 2017).In this study, eugenol was seen as the only concentration (18.75 μM) that reduced cell viability compared to the control group at 48 h.This effect may be attributed to the dual nature of eugenol, which can act as both an antioxidant and a pro-oxidant agent in response to oxidative stress (Bezerra et al., 2017).Figure 2 demonstrates that both higher and 12.5 μM concentrations of α-terpineol showed highly toxic effects on the RTG-2 cells after 24 h and 48 h of treatment.While 3.125 μM of α-terpineol exhibited a toxic effect after 24 h of exposure, it non-significantly increased cell viability after 48 h of treatment.
In addition, 6.25 µM of α-terpineol non-significantly improved the viability of the RTG-2 cells relative to the control group after 24 h of treatment, but decreased the cell viability after 48 h of treatment.3.125, 6.25, 12.5, 25, 50, and  It has been reported that α-terpineol can inhibit cellular division, have a mutagenic effect with bridge formation and delayed anaphase stages, and have a cytotoxic effect on cells through apoptosis depending on the dose and exposure time (Negreiros et al., 2023).In addition, considering the penetration effects of α-terpineol, the cytotoxicity of α-terpineol can be associated with apoptosis resulting from in cell permeability, increased fluidity, and cell cycle rate.It has been reported that αterpineol caused changes in cell membrane function and leakage of intracellular materials by destabilizing the cell membrane, showing a toxic effect against antibiotic-resistant bacteria (e.g., Escherichia coli O157:H7) (Zengin and Baysal, 2014).In another study, it has been reported that the hydroxyl group of α-terpineol can form glycosidic bonds and hydrogen bonds with PO 2 -and COO-, transforming the polar head groups of phospholipids into a tight package.This process reduces membrane fluidity, disrupts electron transport in the membrane, precipitates the membrane pH gradient, and accumulates reactive oxygen species (Yang et al., 2023).

Conclusion
In this study, it was shown that all eugenol treatments (≥37.5 μM) significantly increased the RTG-2 cell viability compared to the control group cell viability (100%) at 24 and 48 h of treatment.In addition, it has been understood that α-terpineol (≥12.5 μM) had a toxic effect on the RTG-2 cells.
Based on this data, it can be concluded that eugenol may have the potential to be used as a healing agent, particularly in wound care, while α-terpineol may have the potential to serve as an antibiotic agent or a toxic agent against microorganisms and pests.However, it should not be forgotten that results obtained from in vitro studies may differ from responses obtained from in vivo models.
Therefore, 3D cell culture techniques, which yield results closely resembling in vivo studies, and molecular-based toxicogenomic research that relies on the correlation between concentration and treatment time, are necessary to comprehend the behavior and identify the pathways, reactions, and environmental conditions of eugenol and α-terpineol at the cellular level.Future studies may focus on these compounds and provide the data needed for these two monoterpenes.

Statement of Conflict of Interest
The author declares that there is no conflict of interest regarding the study.

Author's Contributions
The author declares that she has contributed 100% to the article.