Cytotoxic and mutagenic capacity of TTO and terpinen-4-ol in oral squamous cell carcinoma

The essential oil of Melaleuca Alternifolia (tea tree oil - TTO) consists of about 100 components, and the highest concentration are terpinen-4-ol. Studies of their cytotoxic capacity have shown effect on malignant neoplastic lineages. The aim of this study was to evaluate the cytotoxic and mutagenic capacity of TTO and main soluble components, terpinen-4-ol and gama-terpinene in cell cultures. Two lineages of oral squamous cell carcinoma and a keratinocyte cell were analyzed: (1) colorimetric analysis Metiltetrazolium (MTT); (2) Micronucleus assay. The results were expressed as susceptibility tests and degree of mutagenicity. The statistical test used in the analysis was one-way ANOVA (Tukey test). The IC50 values obtained from the MTT analysis of cells exposed to TTO were 0.2% for HaCaT, 0.14% for HSC-3, and 0.17% for SCC-25. For exposure to terpinen-4ol, IC50 values were 0.5%, 0.3% and 0.45% for HaCaT, HSC-3 and SCC-25, respectively. The gamma-terpinene didn’t show significant cytotoxic activity, therefore it was impossible to calculate the IC50. TTO and terpinen-4-ol was unable to produce mutagenicity in all the lineages. In conclusion, both the TTO and terpinen-4-ol had cytotoxic capacity on HaCaT, HSC-3 and SCC-25. TTO and terpinen-4-ol wasn’t mutagenic. In this sense, our study provides new perspectives on the potential use of TTO and terpinen-4-ol for the development of new alternative therapies to treat topically locally oral squamous cell carcinoma. This study can be related


Introduction
Oral cancer has been growing day by day and currently occupies 11th worldwide position between the most common carcinomas 1 . Squamous cell carcinoma are responsable for over 90% of oral cancers. Treatment for this cancer may envolve highly mutilating surgeries, chemotherapy and radiotherapy, which are procedures with high cost and low accessibility. As a result, studies try to identify new options for treatment that can improve the survival of patients with oral squamous cell carcinoma (SCS) 2,3 . In this context, experiments with plant-derived components have been performed to identify new drugs of potential use as auxiliaries to the proposed treatment 4,5 . In fact, new chemotherapy drugs originally extracted from plants continue to enter the market and are being analyzed for their usefulness against different cancers, including squamous cell carcinoma.
Melaleuca alternifolia is a herbal medicine that stands out in different studies for medicinal purposes 6 . Melaleuca essential oil (tea tree oil-TTO) is composed of a complex mixture of approximately 100 components 7 , of which the highest concentration components are terpinen-4-ol (39-48%), gamma-terpinene (17-18%) and alphaterpinene (8-13%). The oil is considered non-toxic, with a pleasant odor and is widely used in healing or anti-infectious products. It is extracted from the plant by hydro distillation or steam distillation 6 . Its lipophilic capacity and consequent skin penetration suggest the possibility of topical use 8,9 . The monoterpenes, delivered from TTO, have also been studied for their anticâncer activity. Some of them have shown promising results in the treatment and prevention of skin câncer and leukemia, and others types of cancer such as pancreas, colon and breast in rodents 10 .
This study evaluated the cytotoxicity of TTO and its major soluble componentes: terpinen-4-ol and gamma-terpinene, against head and neck squamous cell carcinoma (SCC) by possible alteration of mitochondrial activity (oxidative respiration, bromine [3-(4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H tetrazolate assay -MTT). MTT assay is based on the effect of compound / extract on glycid cell metabolism through mitochondrial dehydrogenases. The mitochondrial viability and consequently cell viability is quantitated buy cell's ability to convert MTT salt (water-soluble and yellow) in formazan (purple precipitate and water-insoluble) via mitochondrial dehydrogenases 11 .
Subsequently, it was evaluated if the cytotoxic effect causes nuclear damage, at chromosome level, these chromosomal mutations were analyzed by the Micronucleus test (MN). Eventually it is possible that damage to the DNA of the cell doesn't lead to apoptosis or necrosis. These damages can cause chromosomal mutations, which are essential factors to carcinogenesis 12 , therefore, we consider it important to identify the possible mutagenic capacity of our test substances in concentrations lower than those

Tea tree oil and components
The composition of the TTO (Sigma, MO, USA) was analyzed by gas chromatography for oil quality control and to obtain the percentages of its main components. The identification of the relative concentrations of the components was used to calculate the initial concentration to perform the experiments with the two components of higher concentration (terpinen-4-ol and gamma-terpinene).
The ISO 4730 values of the oil components and the composition obtained in the chromatography analysis are expressed in Fig.1. The percentage values for terpinen-4ol and gamma-terpinene were, respectively: 47.66% and 23.58%, based on the chromatography performed.

MTT cell viability assay
This analysis served as a parameter for producing the IC 50 , which was used as the standard concentration for subsequent experiments 15 . Thus, this first analysis had more dilutions of TTO and their soluble portions. This experiment also served to determine the inclusion of soluble portions, the component that could not calculate the IC 50 was not included in the next experiment. In addition, as TTO is the initial viability analysis product, none of the component was analyzed at a higher concentration than that found in TTO (proportional relative concentration tested in TTO).
Cells were seeded in 96-well plates (1x10 5 cells/wells) in complete medium. On the following day, different concentrations of TTO and soluble portions were added to the cells in triplicates. It was exposed to TTO at decreasing concentrations (1/2) from the initial concentration of 2% (1%, 0.5%, 0.25%, 0.125%, 0.062%, 0.031%) for 24 hours, in all cases, combining 0.4% dimethyl sulfoxide diluent (DMSO) into the culture medium.
For the main soluble portions, we use the chromatography result to calculate the relative concentrations of each product and adjust for the experiments. In the case of terpinen-4-ol the initial concentration used was 1% (0.5%, 0.25%, 0.125%, 0.062%, 0.031%, 0.015%), for the same time as exposure to TTO and for gamma-terpinene was 0.5% for significance (* p <0.05 ** p <0.01 *** p <0.001).

This study was approved by the Ethics Committee on Human Research (CEP), by
Araraquara School of Dentistry -UNESP.

TTO and terpinen-4-ol mutagenic capacity
TTO and terpinen-4-ol were not mutagenic (Fig.3). IC 20 , IC 10 and IC 05 were statistically different from PC, and the three concentrations tested showed no statistical difference among themselves or against the NC and VC groups, showing that the concentrations were not mutagenic against any of the analyzed lines.

Discussion
In recent decades, much has been said about the use of medicines from natural extracts 6 . The alarming rise in cancer cases around the world, and the limitations of current treatments, have made increased the search for treatments with natural medicines that have antitumor action.
Doll-Boscardin et al. 16 (2012), confirmed in an in vitro study, the cytotoxic and antitumor potential of Eucalyptus benthamii essential oil and some related terpenes (alpha-terpinen, terpinen-4-ol and gamma-terpinene) in three different tumor lineages.
The results indicated that the cytotoxic effect caused cell death by apoptosis. In addition, there was inhibition of cell proliferation in consequence of cytotoxicity. Krifa et al. 17 (2015), studied the essential oil (EO) extracted from the Pituranthos Torturous plant and main components, sabinene, alpha-pinene, limonene and terpinen-4ol, on melanoma lineages. The results showed that EO was able to inhibit tumor growth associated with apoptotic features, including nuclear condensation, suggesting a potential drug for cancer treatment. The cytotoxic assay used for our study was colorimetric analysis of Metiltetrazolium (MTT). Based on this assay, we found that both TTO and terpinen-4-ol were able to reduce viability in all strains. The initial TTO concentration used was 2%, followed by seven dilutions (1/2). While for terpinen-4-ol, the initial concentration was half, 1%, according to its relative percentage found by gas chromatography.
Increased cytotoxicity was observed at higher concentrations, while at lower concentrations, cell death percentages were low, suggesting a dose-dependent effect for both TTO and terpinen-4-ol.
The IC 50 found for HaCaT line was 0.2%. This value was higher than those found for squamous cell carcinoma lines, HSC-3 and SCC-25, which were 0.14% and 0.17%, respectively, suggesting greater susceptibility of carcinoma lines. The same occurred after exposure to terpinen-4-ol, where the IC 50 values of the three lineages doubled, being 0.5% for HaCaT, 0.3% for HSC-3 and 0.45% for SCC-25. This fact occurred because the relative percentage of terpinen-4-ol in the oil is approximately 50%. The gamma-terpinene soluble portion, present in approximately 24% of the TTO, was not able to reduce viability in 50% of the cells, so it was not used in the other analysis.
Differently to the concentrations found in the present study, Hammer et al. 18  Calcabrini et al. 4 (2004) also reported using lower TTO concentrations, 0.005% to 0.03%, for MTT analysis in human melanoma cells. The authors reported there was no difference in cell viability at a concentration of 0.005% and until to 0.01%, there was no interference on the growth of human melanoma line tested by them. From 0.02% and 0.03% concentrations was possible to observe large inhibition of cell growth. This study also does not mention the exposure time of TTO and terpinen-4-ol concentrations used.
In study by Greay et al. 5 (2009), the tested concentrations of TTO, terpinen-4-ol and gamma-terpinene, on mice mesothelioma, melanoma and on fibroblast line, ranged from 0 , 01% to 0.15%, exposed to treatments for 24 to 72 hours. The IC 50 found for TTO

Conclusion
In conclusion, both the TTO and terpinen-4-ol had cytotoxic capacity on HaCaT, HSC-3 and SCC-25. TTO and terpinen-4-ol wasn't mutagenic. In this sense, our study provides new perspectives on the potential use of TTO and terpinen-4-ol for the development of new alternative therapies to treat topically locally oral squamous cell carcinoma.