Journal of Essential Oil Research In vitro Cytotoxicity ...

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In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines a

a

a

Amanda J. Hayes , David N. Leach , Julie L. Markham & Boban Markovic

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a

Centre for Biostructural and Biomolecular Research , University of Western Sydney , Hawkesbury, Richmond, NSW, 2753, Australia b

Department of Safety Science , University of New South Wales , Sydney, NSW, 2052, Australia Published online: 09 Dec 2011.

To cite this article: Amanda J. Hayes , David N. Leach , Julie L. Markham & Boban Markovic (1997) In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines, Journal of Essential Oil Research, 9:5, 575-582, DOI: 10.1080/10412905.1997.9700780 To link to this article: http://dx.doi.org/10.1080/10412905.1997.9700780

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J.Essent. Oil Res., 9, 575-582 (Sep/Oct 1997) RESEARCH REPORT

In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines


Amanda J. Hayes,' David N. Leach and J d e L. Markham Centrefor Biostncctural and Biomolecular Research, Unfversityof Western Sydney Hawkesbuy,Richmond, NSW2753, Australia

Boban Markovic Department of Safety Science, Universityof New South Wales, Sydney, NSW2052, Australia Abstract Cytotoxicity of Australian tea tree oil (oil of Melaleuca al#ernifoliu)and its major oxygenated monoterpenes: terpinen-4-01, 1,8-cineoleand a-terpineol were investigated using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo~metho~phenyl)-2(4-sulfophenyl)-2H-tetrazolium)]assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was a-terpineobtea tree oil>terpinen-4-ol>l,8-cineole and with comparison with the controls used mercuric chloride>tea tree oibaspirin. Antimicrobial activity (MICs) displayed a similar pattern where a-terpineobterpinen4-obtea tree oil>l,8-cineole.The IC,, (a concentration that causes a reduction by half of the activity of mitochondria1dehydrogenase) value of tea tree oil ranged from 0.02 g/L for the Hep G2 cell line to 2.8 g/L for the HeLa cell line.

Key Word Index

Melaleuca alternffoliu,Myrtaceae,tea tree oil, essential oil composition, terpinen4-01, a-terpineol, 1,8-cineol, cytotoxicity, antimicrobial activity, Hep G2, HeLa, MOLT-4, K-562, CTVR-1.

Introduction In recent years there has been a worldwide trend towards the use of natural products, including essential oils, for phytopharmaceutical and phytocosmetic preparations. The broad spectrum antimicrobial activity of Australian tea tree oil has stimulated considerable interest and its incorporation into such preparations is increasing at a rapid rate. Tea tree oil possesses antimicrobial activity against most pathogenic Gram-positive and Gram-negative bacteria as well as a range of yeasts and moulds (1). Tea 'Address for correspondence

Received:May 1996 Revised: October 19%

1041-2905/97/0005-0575$04.00/&@19!97 Allured Publishing Corp.

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HAYIS ETN..


Table 1. Toxicity data of tea tree oll and its components Test protocol

Test species

Acute oral toxicity Acute dermal toxicity Skin sensitisation potential Draize acute dermal 30-Day dermal irritation

Rat Rabbit Guinea Pig Rabbit Rabbit

Ames test Wound repair Acute toxicity for terpenic compounds e.g. a-terpinene, 1 ,I-cineole and terpinolene Acute toxicity for terpenic compounds e.g. a-terpinene, 1 ,&cineole and terpinolene

Salmonella Rabbit

Findings Moderate toxicity Moderate toxicity No effect Some irritancy No visible signs, microscopic changes Negative result No effect

Toxicity data 1.9-2.6 mLkg >2 g/kg

NIA NIA NIA NIA NIA

Rabbit (dermal)

Moderate toxicity

2-5 glkg body weight

Rat (oral)

Moderate toxicity

2-5 gkg body weight

Table modified from Altman (24) and Villar et al. (23)

tree oil has been used to treat acne (21, bites, burns, cuts, coldsores (3,4), tinea (5) and thrush (6) as well as Legionella in air conditioning ducts (7). Tea tree oil quality in Australia has been governed by the Australian Standard AS-2782 (1985) (8) which is currently being superseded by the draft International Standard, ISO/DIS 4730 (9). These standards stipulate a minimum of 30% terpinen-4-01 and a maximum of 15% 1,8-cineole in the oil. Terpinen-4-ol is the major component responsible for antimicrobial activity of the oil (10). Previous work in our laboratory demonstrated the relationship between chemical composition and antimicrobial activity, whereby 1,8-cineole, when present at a concentration of up to 40%, did not destroy the antimicrobial potency of tea tree oil, provided the terpinen-4-ol concentration exceeded 30% (10). The generally accepted view is that 1,8-cineole is thought to be responsible for skin irritation (11-13), although there is little scientific proof to suggest that a 'good oil' is low in l,&cineole and high in terpinen-4-01 [(14); Markham, personal communicationl. Martindale (15) described 1,8cineole as a counter-irritant and tests have shown that applying l,&cineole to the skin of rabbits and humans does not produce irritation or sensitization (16). Conflicting results were obtained using the European patch tests where positive results were reported by Degroot and Weyland (17) and negative results by Knight and Hausen (18). In a more recent report, a skin irritancy trial using patch tests of tea tree oil and 1,8cineole was conducted on 28 human subjects and results showed no irritancy of l,&cineole at concentrations up to 28.1% (Southwell, personal communication). Although tea tree oil is readily available as an Over The Counter (OTC) preparation with a wide range of product lines, only in more recent years has more extensive toxicity data on tea tree oil been reported. These reports encompass contact dermatitis from the application of the oil and oral toxicity resulting in dizziness, disorientation, swelling, dermatitis and/or exacerbation of existing dermatitis (17,19-22). Animal toxicity tests that have previously been performed on tea tree oil are summarized in Table I. In vitro cytotoxicity has been readily accepted as a routine method of non-animal toxicity testing. Many of the tests use color-based viability assays. The MTS [(3-(4,5-dimethylthiazo1-2-y1)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)l (25) cell viability system is a colormetric method for determining the number of functional cells in proliferation or chemosensitivityassays (25). The MTS assay measures the metabolic activity of mitochondria1dehydrogenases, and is a technically simpler method than the standard cytotoxicity testing using M?T (3-(4,5dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide). Korting et al. (26) demonstrated a significant correlation ( ~ 0 . 9 1 ) between ED,,values obtained using the MTT assay and In vim irritancy data for surfactants compared

CYTOTOXICITY OF Am-

TEATREEOIL

577

to a less satisfactory correlation ( ~ 0 . 4 2 )for the Neutral Red Release Assay and in vim irritancy data. The efficiency of MTS has been demonstrated by a number of researchers (27-29). A good correlation between different experimental sources of in vitm cytotoxicity values and fn vlvo LD,,values in rats and mice has been demonstrated (30-32). Studies (3334) have shown a statistically significant correlation (r=0.75) between the in dtm and in vim values of a selection of chemicals from the Multicenter Evaluation of In dtm Cytotoxicity (MEIC) list. The only researchers who have reported on the cytotoxicityof tea tree oil are Soderberg et al. (39, who utilized the neutral red assay on human epithelial and fibroblast cells. These results suggested that tea tree oil possessed a moderately toxic effect. Individual components of the oil were not tested. For the experimentsconducted in this study, cytotoxicity using MTS was adopted as an in vitm testing method to screen the toxicity of tea tree oil and some of it's major components.


Experimental Test Compounds: Tea tree oil was supplied by Main Camp Plantation (Coraki, NSW, Australia). Standardsincluding terpinen-4-01(97%), 1,8-cineole(99%) and a-terpineol(98%) were purchased from Aldrich Chemical Co (USA). Baseline standards for the f ndtm cytotoxicity assay were mercuric chloride and aspirin (acetyl salicylic acid) purchased from Sigma (USA). Tea Tree 0UComposftJon:The concentration of tea tree oil and its components were analyzed by GC using a Hewlett Packard 5890 Gas Chromatograph equipped with a flame ionization detector. The chromatograph was fitted with a SGE BP5, 25 m x 0.22 mm x 1.0 pm (film thickness) capillary column and data was acquired under the following conditions: initial temperature 60°C for 5 min; program rate 5"C/min; final temperature 250°C for 5 min; injector temperature 220°C; detector temperature 240°C and carrier gas N, at 10 psi. CeU Lfnes:A selection of cell lines were used to represent a range of number of potentially sensitive organdtissue systems for safety assessment. In safety assessment of a product, one always considers the worst case scenario of exposure on the most sensitive tissue and the cells which are in direct contact with the toxicant. The five human cell lines utilized in this study included two adherent cell lines: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; and three suspension cell lines, MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. All cultures were maintained in a color-free medium composed of 50% DMEM: 50% RPMI 1640, (Sigma, USA), supplemented with 5% newborn calf serum (Trace Bioscience, Australia) and containing 2 mM L-glutamine,100 U/mL penicillin, 100pg/mL streptomycin (Sigma, USA) cultures which were maintained at 37°C in a humidified 5% C0,humidified incubator. The adherent cells were removed from the tissue culture surface by the addition of trypsin once the cells reached confluence. Cells were in an exponential phase of growth at the time of testing, with experiments conducted at concentrations of 250,000 to 500,000 cells per mL. The viability of the cells exceeded 95% on all occasions as determined by trypan blue staining (Sigma, USA). Cytotodcffy:A quantity of 10 pL, of each ten-fold serial dilution of test solution, prepared in 96% ethanol and ranging from 0.0001% to lOWh,were added to 990 pL of the desired cell line suspension. The culture was mixed in a fully closed sterile cell culture flask (25 cm*) and incubated at 37OC. These flasks were prepared in duplicate and culture periods of 4 and 24 h were employed. These two time periods served as measures of essentially acute and chronic toxicity. Exposure for 4 h related to an immediate cellular toxicity while any changes in toxicity values at 24 h may be due also to the formation of metabolites of test compounds during the culture period. Three controls were used for each experiment: 1) a blank consisting of ethanol; 2) an LD, consisting of ethanol and cells; 3) an LD,, consisting of ethanol and media. At time zero for the 4 h culture period and 4 h prior to the end of the 24 h culture period, flasks were removed from the incubator and the desired quantities of MTS reagent [from MTS and PMS (phenazine methosulphate) stocks1 was added to each flask in accordance with the

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05

0.45

ICO

0.4

0.35

E

0.3

N

9 0

P


z3

0.25

-

0.2

Terpinen-4-01

0.15

0.1

0.05

I

0 1.OOE-02

I

I

I

1 OOE-01

1 OOE+OO

100E+01

Dose (gramslL)

Example of a MTS cytoxicity assay using Hep-GZ (human liver) cell line (4 h exposure)

manufacturers instruction (25), then incubated for the remaining 4 h of the culture period. After the 4 h reaction period 6 x 100 pL aliquots of the colored product were pipetted into 96 well microtitre plates from each of the flasks. The absorbance of the plates was then read by a TIM-200 plate reader (492 nm). The assay showed an increase in absorbance that was directly proportional to an increase in cell number and that the cell concentrations used were in the optimal linear range. IC,, values (a concentration that causes a reduction by half of the activity of mitochondria1dehydrogenase) using the above listed controls of the test compounds were calculated from the dose response curves (as shown graphically in Figure 1) and expressed as g/L. IC5, absorbance values are obtained as the mean of absorbance between the IC, (cells only control) and IC,, (medium only control) and actual IC,, dose value is then read from the X-axis as the point of intersection of the IC,, absorbance value and the dose response curve. Microbial Cultures: The strains of bacteria used in this study were Staphylococcusaureus NCTC 4163 andBchm’chia colt NCTC 8196. Bacteria were subcultured from nutrient agar slopes into nutrient broth and incubated at 37°C for 1 8 h. Subculturing was performed at least twice. The resulting bacterial broth was used as the innoculum in the microbiological analysis. Cell numbers of the inoculum were standardized at lo6 cells/mL. Minimum Inbibltory Concentration ( M E ) - Agar M e t b o d . The test material was added aseptically to sterile molten agar to give final concentrations of 0.05% to 5%. The agar solutions were then vortexed at high speed for 30 seconds and immediately poured into sterile petri dishes and allowed to set for 30 minutes. The plates were then inoculated by spotting 6 pL of a suspension of the desired microorganism from the prepared inoculum. Plates were incubated at 37°C for 24-48 h. From these results the MIC of the test material was calculated as the lowest concentration at which no growth of the microorganism had occurred. Minimum I n b i b i t o q Concentration (MIC] - B r o t b M e t b o d . A dilution series was carried out over a range of concentrations from 0.05% to 5% of the test material in sterile nutrient broth. A 0.1 mL

Table II. Chemical composition of tea tree oil (Main Camp Batch # 5061) Component

Concentration (%)

a-Pinene a-Terpinene p-Cymene Limonene l&Cineole

Component

Concentration (%)

y-Terpinene a-Terpinolene Terpinen-4-01 a-Terpineol

2.6 8.6 3.6 1.2

19.2 3.5 39.3 3.0

4.3

Table 111. IC,, values (gA) of tea tree oil components and standards of aspirin and mercuric chloride at an incubation period of 4 h against a range of five cell lines


Cells

HeLa K562 CTVR-1 Molt-4 Hep G2

Tea tree oil

1,&Cineole

Terpinen-4-ol

a- Terpineol

Mercuric chloride

Aspirin

2.8m0.90 2.8M0.40 0.56f0.08 0.6M0.10 2.8M0.30

3.8M0.40 4.0M0.40 2.9M0.30 4.2M0.80 3.0M0.65

2.7M0.30 1.2M0.40 0.54k0.06 0.8M0.10 0.5M0.09

1.1M0.20 0.5M0.20 0.39f0.10 0.3MO.10 0.8M0.22

0.05f0.01 0.05f0.01 0.08f0.05 0.16f0.02 0.05-10.01

2.4M0.42 4.5M0.30 1.2M0.12 1.5M0.10 2.1M0.40

IC.. values expressed as Mean fSD, where a minimum of two repeats with six measurementsper dilution were performed ~

~~~

~~

~

~

Table IV. IC, values (gA)of tea tree oil components and standards of aspirin and mercuric chloride at an incubationperiod of 24 h against a range of five cell lines Cells

Tea tree oil

1,&Cineole

Terpinen-4-ol

a-Terpineol

HeLa 2.7M0.07 K562 0.27f0.05 CTVR-1 0.31f0.09 Molt-4 0.3M0.10 Hep 6 2 0.02f0.006

2.5f0.50 0.94f0.03 6.7kl.60 1.2f0.80 0.14k0.06

0.14f0.012 0.25k0.05 0.33k0.08 0.29f0.05

0.1~0.01 0.16i0.03 0.07f0.03 0.32f0.08 0.02M.008

0.06+0.01

Mercuric chloride Aspirin 0.05f0.01

0.08f0.008 0.01k0.003 0.01f0.005 0.01*O .005

1.4M0.27 1.4M0.40 0.10f0.002 0.1M0.05 0.25*0.03

IC.. values exoressed as Mean +SD, where a minimum of two reDeats with six measurementsDer dilution were Derformed

aliquot of the prepared inoculum of test organism was added to each dilution. The inoculated dilutions were incubated at 37°C for 24-48 h. From these results the MIC of the test material was calculated as the lowest concentration at which there was an absence of turbidity. Results and Discussion The composition of the Main Camp Tea Tree oil is summarized in Table 11. This tea tree oil is characteristic of an Australian tea tree oil comprising approximately 40% terpinen-4-01, 3% a-terpineol and 4% 1,8-cineolewhich conforms to the Australian Standards for tea tree oil (8,9). The results of the 4 h MTS assay indicate that the decrease in cell viability is a close measure of the acute effect of the test compounds on the cellular activity of the cell lines tested, while MTS with 24 h exposure showed further cumulative toxicity on the cells. Of all cell lines tested, Hep G2 was the most affected by each of the compounds tested after 24 h of exposure. Results of the cytotoxicity study for IC,,values at 4 and 24 h incubation periods for 100%tea tree oil and its major oxygenated monoterpenes terpinen-4-01, a-terpineol and 1&cineole are summarized in Table I11 and Table IV,respectively. Aspirin and mercuric chloride were chosen as the controls from which to determine the relative toxicity of tea tree oil and its major components and as baseline controls between experiments. Aspirin is available as an OTC drug and is regarded as a moderately safe compound with extensive toxicity data

H A m P.T AL.

580

Table V. MIC % (mU100 mL) of major components of tea tree oil using broth and anar methods with comDarisons of micro dilution MlCs ~

Staphylococcus aureus

Escherichia coli Test compounds

Agar

Broth

Micro-dilutlona

Agar

Broth

Tea tree oil Terpinen-4-01 alpha-Terpineol 1.&Cineole

0.2 co.1 1 .o

0.3 0.3 0.2 >1.o

NKested 0.06 0.06 0.25

0.3 0.15 0.1 >1.o

0.5 0.5 0.3 >1.o

Micro-Dilutiona NKested 0.25 0.25 0.5


‘Results from Carson and Rilev (38)

available on its usage. It has an LD,, value of approximately 1.3 g/kg. Mercuric chloride is described as a ‘violent poison’ and ‘highly toxic’ with 1-2 g often resulting in fatalities (36). The results indicate that a-terpineol is the most cytotoxic of the tea tree oil components tested. Tea tree oil and terpinen-4-01 have similar toxicity with the exception of their affect on Hep G2 cells at 4 h, but at 24 h the IC,, values are again similar. It is of interest to note the toxicity of terpinen-4-01 as it is the major component of tea tree oil (approximately 40%) and is responsible for the major antimicrobial activity of the oil (10). 1,8-Cineole is the least toxic in most cell lines tested and is comparable to that of aspirin in its toxic rating. Aspirin was slightly more toxic than 1,8-cineole for Hep G2 cells at 4 h possibly due to the metabolism of aspirin by liver cells. All test compounds other than 1,8-cineole at 4 h showed an increase in cell death when compared to that of the standard‘control aspirin, while mercuric chloride at both 4 and 24 h time periods with respect to all components tested had the most cytotoxic effect on all cell lines. The overall rating for toxicity of tea tree oil and its components was a-terpineobtea tree oil>terpinen-4-01>1&cineole and with reference to tea tree oil and the controls used the toxicity rating was mercuric chloride>tea tree oibaspirin. The IC, range of tea tree oil in the human cell lines tested was 0.02-2.8g/L. In comparison with this value the LD,, of tea tree oil in vivo of rats (Table 1) has been determined as 1.9-2.6 mL/kg (24). The IC,, range of l,&cineole in the cell lines tested was 0.14-4.20 g/L compared to the published acute toxicity (rabbit dermal and rat oral) value for 1,8cineole which was determined as 2-5 g/kg body weight (23). These results indicate that human cells show a greater sensitivity to tea tree oil than rats. This is probably due to the greater susceptibilityof cells in a closed experimental culture system compared to the metabolic dynamics of an experimental animal, where losses by evaporation and excretion occur very rapidly (23,24). Of the five cell lines tested, epithelial like cells (HeLa) at a time period of 4 h were the most robust cells with respect to the monoterpene alcohol based tea tree oil componentswhile the liver derived cells (Hep G2) at 24 h were the most susceptible cells to damage by tea tree oil and its components. HeLa cells have been used extensivelyas a good model for toxicity studies (30,34) and are phenotyped similar to human skin fibroblasts. More elaborate tests are possible with in uitro skin models to study the irritancy potential of test compounds (37). The results of these skin models correlateswell with tn vitro cytotoxicity assays (26). This, supports the use of in vitro cytotoxicty assays as potentially the first test to run before more detailed investigations using tn uitro skin models (37). Our cytotoxicity results support the use of tea tree oil in topical applications but not for ingestion purposes. These differences in cytotoxicityof cell lines could indicate a metabolic difference between the HeLa and the Hep G2 cell lines. An example of a MTS cytotoxicity assay using H e w 2 (human liver) cell line can be seen in Figure 1. As the liver is the major organ for metabolism these observations for increased cytotoxicity of the oxygenated monoterpenes terpinen-4-01, 1&cineole and a-terpineol could be a result of direct inhibition of liver cells by these compounds or by their resulting metabolites. The antimicrobial activity (MICs) of tea tree oil and its major components is summarized in Table V with comparisons made to published results of Carson and Riley (38). a-Terpineol displayed the greatest

antimicrobialactivity in our results while Carson and Riley (38) report comparable antimicrobialactivity for a-terpineol and terpinen-4-01.1,8-Cineoledisplayed the weakest antimicrobialactivity in all studies. The overall trend indicates that the antimicrobial activity of a-terpineobterpinen-4-ol~ea tree oil>l,8cineole which is similar to the pattern of cytotoxicity activity where a-terpineobtea tree oibterpinen4-01>1,8-cineole. The correlation between MIC and cell toxicity data of tea tree oil and its components indicates the similarities of toxicity in prokaryotic and eukaryotic cells. The potential exists to extend this toxicity study to determine whether a correlation exists between irritancy and toxicity data using epithelial cell lines and advanced cellular function assays for particular interest in topical applications, such as cosmetic and therapeutics. The results of this study indicate the usefulness of the in vitro cytotoxicity assay MTS as an excellent rapid technique for elucidating the toxic properties of essential oils such as tea tree oil and its components without the ethical problem of using experimental animals.


Acknowledgments AJH wishes to thank UWS-Hawkesbury for a PhD Scholarship andBlackmores Ltd. for financial support of the project. We also thank Dr. 2.H. Wu Department of Obstetrics and Gynecology, Westmead Hospital, Westmead, for supplying the cell cultures.

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