In Chemico Evaluation of Tea Tree Essential Oils as Skin Sensitizers

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In chemico evaluation of tea tree essential oils as skin sensitizers. Impact of the chemical composition on aging and generation of reactive species. Cristina Avonto, Amar G Chittiboyina, Mei Wang, Yelkaira Vasquez, Diego Rua, and Ikhlas A Khan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00530 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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Chemical Research in Toxicology

In chemico evaluation of tea tree essential oils as skin sensitizers. Impact of the chemical composition on aging and generation of reactive species. Cristina Avonto,§ Amar G. Chittiboyina,§ Mei Wang,§ Yelkaira Vasquez, § Diego Rua,¥ and Ikhlas A. Khan.§,¤,* §

National Center for Natural Products Research; ¤Division of Pharmacognosy, Department of BioMolecular Sciences; School of Pharmacy, University of Mississippi, University, MS 38677, USA. ¥The Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA.

ABSTRACT: Tea tree oil (TTO) is an essential oil obtained from the leaves of Melaleuca alternifolia, M. linariifolia or M. dissitiflora. Due to the commercial importance of TTO, substitution or adulteration with other tea tree species (such as cajeput, niaouli, manuka or kanuka oils) is common and may pose significant risks along with perceived health benefits. The distinctive nature, qualitative and quantitative compositional variation of these oils is responsible for the various pharmacological as well as adverse effects. Authentic TTOs (especially aged ones) have been identified as potential skin sensitizers, while reports of adverse allergic reactions to the other tea trees essential oils are less frequent. Chemical sensitizers are usually electrophilic compounds, and in chemico methods have been developed to identify skin allergens in terms of their ability to bind to biological nucleophiles. However, little information is available on the assessment of sensitization potential of mixtures, such as essential oils, due to their complexity. In the present study, ten “tea tree” oils and six major TTO constituents have been investigated for their sensitization potential using a fluorescence in chemico method. The reactivity of authentic TTOs was found to correlate with the age of the oils, while the majority of non-authentic TTOs were less reactive, even after aging. Further thio-trapping experiments with DCYA and characterization by UHPLC-DAD-MS led to the identification of several possible DCYA-adducts which can be used to infer the structure of the candidate reactive species. The major TTO components terpinolene, α-terpinene and terpinene-4-ol were unstable under accelerated aging conditions, which led to formation of several DCYA-adducts.

useful tool in pre-screening stages by providing critical information for more rational designs of animal studies. Nonetheless, validated in chemico methods have not yet been successfully applied for the evaluation of complex mixtures. The assessment of the sensitization potential of Tea Tree Oil (TTO) is a classic example of the challenges that toxicologists encounter in the safety evaluation of essential oils and has been used here to test the applicability of a newly proposed in chemico High Throughput Screening method using DansylCYsteAmine (HTS-DCYA) to the rapid evaluation of potential skin sensitization hazard.4 Tea tree oil is defined as the product resulting from “steam distillation of the foliage and terminal branchlets of Melaleuca alternifolia, Melaleuca linariifolia or Melaleuca dissitiflora, as well as other species of Melaleuca provided that the oil obtained conforms to the requirements given in the International Standard”.5-10 The name “tea tree” is quite ambiguous, as plants of the Melaleuca genus (Myrtaceae) share little in common with plants ordinarily used to make tea (Camellia sinensis, Theaceae). The confusion originated after James Cook’s first explorations of the New World in the 18th century, when he noticed that indig-

INTRODUCTION Essential oils are largely used by consumers in a variety of preparations. Due to the frequency of contact with a number of products, the identification and estimation of potential toxicological effects of essential oils is vital.1 Topical formulations (e.g. soaps, perfumes, detergents, personal care and aromatherapy products) represent a major source of contact with essential oils, thus estimation of skin sensitization potential is a major concern in risk assessment.2 Conventionally, contact allergy to fragrances and essential oils is determined based on clinical and epidemiological reports. Such information can be difficult to interpret, as the number of reported dermatological cases mostly depends on the frequency of use and patient history, thus the allergic potential of infrequently used oils can be underestimated.3 So far, animal methods represent the only validated stand-alone approaches for skin sensitization risk assessment. At the same time, determining the skin sensitization potential of essential oils may represent a challenging task, due to the chemical complexity and potential instability of many oils. Such complexity requires extensive studies, with great economic impact and cost in animal lives. To minimize the use of animal tests and maximize the effectiveness of in vivo studies, in chemico alternative methods may serve as a

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In the present work, ten different oils from different tea tree species have been analyzed for their chemical composition and relative chemical changes over an 18 month period. The aging stage of the oils has been estimated by quantifying the variation of major TTO components over time along with the increased content of peroxides and p-cymene. The formation of electrophilic and reactive species after aging has been evaluated using a High Throughput Screening method (HTS-DCYA) recently developed for the rapid identification of potential skin sensitizers using a fluorescent thiol as trapping agent.4 Main TTO components were also evaluated individually for stability and reactivity, in order to extrapolate their contribution as a hazard source. Ultra High Performance Liquid Chromatography coupled with Diode Array Detector and Mass Spectrometer (UHPLC-DAD-MS) analysis on DCYA adducts provided additional information in regard to the potential reaction products. As a result, the major DCYA adducts were isolated and the structures were confirmed with NMR spectroscopy. Based on the DCYA-adduct structure, the nature and potential origin of the parent reactive molecule is also discussed and the results are herein presented for the first time.

enous people used the leaves of native shrubs as substitution for tea.11 The name was further extended to indicate various plants of the genera Leptospermum, Melaleuca, Kunzea and Baeckea due to a similar translation of different local names. The use of the same vernacular name for taxonomically distinct species can result in substitution of authentic TTO with other “tea tree” products, in spite of their remarkable dissimilarity in terms of botanical, chemical features and geographical distribution.12 The composition of commercial TTOs is regulated by international guidelines ISO 4730 (2004) and the Australian standard AS 2782-2009 ("Oil of Melaleuca, Terpinen-4-ol type").10,13 Essential oils from L. scoparium and K. ericoides (both occasionally designated as New Zealand tea trees) are also identified as manuka and kanuka oil, respectively.14 Melaleuca cajuputi Powell, commonly known as “swamp tea tree” or “paperbark tea tree”, is used as a source of cajeput oil. Niaouli oil may refer to the essential oil obtained from M. viridiflora Sol. ex Gaertn. (common throughout northern Australia) or M. quinquenervia (Cav.) S.T.Blake (distributed along the East coast of Australia, in New Guinea and New Caledonia).15 The three species were previously treated as different varieties under Melaleuca leucadendra (L.) hence the commercial niaouli and cajeput oils can have very different compositions depending on their botanical source.16 The chemical diversity of the various oils is responsible for the different beneficial and adverse effects commonly observed.17,18 Melaleuca alternifolia oil has been reported to cause allergic reactions after topical exposure. The oil is classified as a moderate sensitizer by the Local Lymph Node Assay.5 The chemical compounds present in M. alternifolia oil lack the requisite mechanistic domains to act as skin sensitizers,19 but most constituents have been classified as potential pre- or pro-haptens.20 TTO rapidly oxidizes after exposure to light, oxygen or heat and the changes in the chemical composition have been correlated to an increased sensitization potential of TTO after aging.21 Aged oils have been found to contain increased amounts of p-cymene along with decreased content of total terpinenes.5 It has been postulated that the degradation of non-oxygenated monoterpenes to the more stable endproduct, p-cymene may occur via formation of unstable oxygenated intermediates (epoxides, hydroperoxides, endoperoxides etc.) Although, p-cymene was considered as nonsensitizer, some of these elusive species may be responsible for the clinically observed allergic reactions to aged TTOs. Several of these potential sensitizers are also found in nonauthenticated TTOs, but the report of adverse effects are less frequent compared to authentic TTOs. Skin sensitizers are usually small lipophilic molecules containing reactive electrophilic sites capable of covalent binding to nucleophilic sites on skin proteins to form immunogenic complexes.22 Several in chemico methods have been proposed to characterize potential sensitizers by exploiting the reactivity of the candidate compounds toward model nucleophiles.23,24 In chemico methods have proven to be useful tools to estimate the reactivity of potential sensitizers but less suitable for complex mixtures such as essential oils. So far, only the cell-based KeratinoSensTM assay has been applied to the investigation of botanical extracts25 but the applicability of such in vitro methods may be limited in the presence of cytotoxic components.

MATERIALS AND METHODS Chemicals and Reagents. Specimens of all essential oils samples are deposited at the botanical repository, National Center for Natural Products Research (NCNPR, The University of Mississippi, USA) and documented with NCNPR accession code. Sample T1 (ID #13159), T2 (#13061), T3 (#13103), T5 (#13026), T7 (#13105), T8 (#13104) and T10 (#13070) were purchased from online sources. Non-commercial essential oils T6 (#13146), T9 (#13149) and commercial oil T4 (#13145) were kindly provided by the Australian Tea Tree Industry Association (ATTIA, Casino NSW, Australia). Pure compounds including cinnamaldehyde (CAS# 1437110-9), p-cymene 99% (99-87-6), α-terpinene >89 FCC (99-865), α-terpineol >96% mixture of isomers (98-55-5), ɣterpinene >97% (99-85-4), (+)-terpinen-4-ol ≥98.5% (sum of enantiomers, 2438-10-0), δ-terpinene (terpinolene) >90% (586-62-9), sabinene 75% (3387-41-5), polymer-bound triphenylphosphine (P-TPP, 1-3 mmol P/g resin) and Quantofix® peroxides test sticks (0-25 mg/L) were purchased from Sigma. The fluorescent compounds DCYA and DCYA disulfide were synthesized as described by Chittiboyina et al.26 Gas Chromatography Instruments and Conditions. Characterization of essential oils and pure TTO components were performed by gas chromatography coupled with mass spectrometry (GC-MS) using an Agilent 7890 GC instrument equipped with an Agilent 5975C mass detector and an Agilent 7693 autosampler (Agilent Technologies, Santa Clara, CA, USA). An Agilent J&W HP-1 fused silica capillary column (60 m × 0.25 mm i.d.) coated with a 0.25 µm film of crosslinked 100% dimethyl-polysiloxane was used. Helium was used as the carrier gas at a constant pressure of 24 psi. Analysis of essential oils was performed by temperature programming set up as follows: 2 min at 50 °C and then programmed at 2.0 °C/min to 180 °C. The injector temperature was 250 °C (split ratio was set to 25:1). Duplicate injections were made for each sample. Mass scan spectra were recorded at 70 eV from m/z 35 to 450. The compounds were identified by comparison of the spectra with the databases (Wiley and NIST)

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Chemical Research in Toxicology mM HCl and 1 mL of water. The solution was extracted twice with hexane (5 mL) to remove the non-dansylated components. The aqueous layer was collected, excess of acetonitrile was removed under vacuum and the remaining solution was re-extracted in CHCl3. The organic layer was dried using anhydrous MgSO4, filtered and concentrated under vacuum. The sample was then loaded onto a Sephadex LH-20 column (2 x 37 cm), eluted using MeOH as the mobile phase and collected fractions were analyzed by UHPLC-DAD-MS. UHPLC-DAD-MS Analyses. Characterization of potential DCYA adducts was performed using an Agilent 1290 Infinity UHPLC-UV system equipped with an Agilent 6120 quadrupole mass detector (Agilent Technologies, Santa Clara, CA, USA) and a UHPLC BEH C18 column (1.7 µm 2.1x 100 mm, Waters Corporation, Milford, MA, USA). The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.05% formic acid. An eluent gradient was performed as follows: 15 % to 65% B (0.4 ml/min) in 25 min then to 95% B in 5 min and hold for 5 min. UV absorption was monitored at 254 nm and 340 nm. Fluorescence emission was monitored at 520 nm (excitation wavelength 350 nm). Positive and negative ESI scans were recorded in the range m/z 100-1000. Accelerated aging conditions. One milliliter of each pure compound was put in a clear vial with a septum. Oxygen atmosphere was maintained with a balloon filled with oxygen and the solution was constantly mixed with magnetic stirrer under daylight conditions. Aliquots of each sample (5 µL) were collected at various time points and diluted to 5 mg/mL in hexane. The samples were immediately analyzed by GCMS by diluting 50 µL of the hexane stock solution in 150 µL of hexane containing undecane (500 µg/mL) as the internal standard. The remaining stock solution (5 mg/mL in hexane) was stored at -20 °C for fluorescence reading experiments.

using a probability-based matching algorithm. Further identification was based on the relative retention indices compared with the literature or with reference standards purchased from commercial sources. Investigations of chemical stability of pure TTO components were performed by GC-MS using the following temperature program: 50 °C for 2 min, then 1.5 °C/min to 110 °C, followed by 6 °C/min increment to 220 °C. The injector temperature was 250 °C (split ratio was set to 30:1). Duplicate injections were made for each sample. Mass scan spectra were recorded at 70 eV from m/z 50 to 400. HTS-DCYA method. The presence of potential reactive compounds was investigated using a modified version of the protocol described by Avonto et al.4 For the analysis of samples collected during the accelerated aging, test stock solutions prepared for GC-MS analysis (5 mg/mL in hexane) were used and diluted to final concentration for the HTS-DCYA assay. Briefly, 500 µL of a 2.5 mM solution of DCYA was mixed with 500 µL of a solution 5.0 mM of electrophile in acetonitrile (ACN, for pure compounds), or 500 µL of an essential oil stock solution (10 mg/mL). For positive/negative controls (PC and NC), 500 µL acetonitrile was added to the DCYA solution in place of the electrophile. Three hundred µL of the DCYAsample solution was then split in two microcentrifuge tubes. Sixty microliters of aqueous pH 10 buffer was added to the reaction (R) tubes and negative controls, while 60 µL of acetonitrile was added to the blanks (Bl) and positive controls. The solution was incubated for 20 min at room temperature. Sixty mg of SiliaBond® (Maleimide, ≥ 0.64 mmol/g, SiliCycle) was then added, the suspension was vortexed and incubated for 90 min with vigorous shaking. The samples were centrifuged at 10,000 rpm (10 min), then 60 µL of the supernatant was diluted to a final volume of 1 mL with ACN. Two hundred µL (in triplicate) of the diluted solutions was put into a 96 well solvent-resistant microplate. Each plate was read in triplicate using fluorescence end-point readings. Fluorescence end-point readings. The experiments were performed on a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Data were acquired and processed using SoftMax Pro 5 (Molecular Devices, Sunnyvale, California, USA). The fluorescence readings were recorded at emission wavelength 520 nm (excitation 350 nm, cutoff 420 nm), temperature 23 °C, 50 readings (high sensitivity). The Reactivity Index (RI) was calculated based on the average of 9 readings per sample (3 readings/well in triplicate) as follows: − −

= 100 − −

RESULTS AND DISCUSSION Pharmacological and toxicological profiles of essential oils can be very difficult to extrapolate from the mere sum of the profiles of their main, individual components as required today.1 The presence of specific components and their abundance deeply affect the overall chemical, physical and biological properties of an oil. To add even more complexity, numerous constituents are often chemically unstable and undergo rapid changes over time forming complex mixtures of degradation products. Non-oxygenated monoterpenes are known to form numerous oxidized by-products upon air exposure.27 These processes usually follow free-radical mechanisms and can generate a large number of potential reactive organic radicals, hydroperoxides, endoperoxides, hydrogen peroxide and other electrophilic hydrolytic products.28 Many of these products are intrinsically unstable, thus the reactions could proceed further until stable end-products are formed and accumulate in the oil. The identification of such degradation products may be elusive and reactive intermediates may not be stable enough to analyze under commonly used analytical conditions and instrumentation. In the case of authentic TTO, aged oils have been linked to potential adverse effects, with increased skin sensitization potential after topical application. Previous studies on aged oils identified an indirect correlation between the risk of sensitization potential and the increased content of pcymene and total peroxides in the oils. A number of potential

Reduction of peroxides. A 10 mg/mL solution of essential oil in acetonitrile was filtered through 300 mg of polymerbound triphenylphosphine (P-TPP) in a capillary column. The resulting eluate was then used for reactivity assays as explained in the HTS-DCYA method section. Isolation of DCYA adducts for UHPLC-DAD-MS analysis. To a solution of T7 essential oil (950 mg) in acetonitrile (10 mL), an equal volume of DCYA (25 mM in acetonitrile) was added, followed by addition of 3.85 mL of pH 10 buffer. After 3 h, the pH was adjusted to 7 by addition of 1 mL of 50

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culprits has been proposed, but the exact identification of all potential sensitizers is still elusive.5 In the present work, ten different oils from different tea tree species (Table 1) have been analyzed for their chemical composition and relative chemical changes over an 18 month period. Chemical composition and classification of “tea-tree” oils. The sample set chosen for the study included four tea tree oils and six oils from different species of ‘tea trees’ (Table 1). The essential oils’ initial chemical composition was characterized by GC-MS (Figs. S1-S4, Supporting information). The samples were divided into “authentic TTO” or “non-authentic TTO” groups based on the quantification of 15 major marker compounds (Table S1). As anticipated, only four oils from Melaleuca (sample T1, T4, T5 and T7) met all of the ISO standard requirements and were classified as authentic TTOs. Three of the authentic samples originated from M. alternifolia, while sample T5 was obtained from M. linariifolia. Samples T2, T3 and T8 originated from Melaleuca species other than M. alternifolia or linariifolia. These oils contained over 47% (area percent) of 1,8-cineole (Table S2 and Fig. S5) and the overall composition of the oil didn’t meet the ISO requirements of Australian TTO; thus T2, T3 and T8 were classified as non-authentic TTOs. In samples T6, T9 and T10, the elevated content in sesquiterpenoids was remarkably different from authentic TTOs. Authentic TTO samples contained pcymene in the range between 3.44 to 5.03 %, while nonauthentic TTOs contained variable amounts from 0.07 to 6.54%.

Changes in the essential oil composition after aging. The ten TTO samples were stored at room temperature for 18 months in their original bottles and re-analyzed by GC-MS to evaluate the variation in the chemical composition (Table S3). All the aged samples showed an increased amount of pcymene along with limonene loss, which was below the area percent limit in any of the aged samples regardless of the original botanical source. The increase of p-cymene content with aging in authentic tea tree oil samples was in the range 2.2 to 8.9% (Fig. 1), with the exception of sample T7, which contained over 6 times the amount of p-cymene as compared to before aging. The increase of p-cymene content in nonauthentic TTO was minimal in T6 and T8, whereas a maximum increase of 7.7% in sample T9 (kanuka oil) was observed. In samples T2 and T3, a greater increase in 1,8-cineole rather than p-cymene was noticed. The peroxide content was also analyzed as an indicative marker for aging. Eight samples contained concentrations of peroxide below 100 mg/mL (Table 2 and Fig. S6). The oils with minimum amount of peroxides detectable were all nonAustralian TTOs. One oil (T7) from M. alternifolia presented advanced sign of aging, with peroxides content over a hundred times more than the average along with at least four times higher content of p-cymene than the initial amount. Based on the p-cymene content, oils T1, T4 and T5 were classified as moderately oxidized, while T7 was considered to be a severely oxidized sample. Sensitization potential of aged TTOs. Several TTO constituents including terpinolene, α-, ɣ-terpinene and limonene are known to undergo rapid oxidation leading to the formation of electrophilic compounds29-31. Such degradation products could potentially bind to nucleophilic amino acids on proteins thus eliciting an immune-response.32 The reactive potential of the oils has been evaluated using the HTS-DCYA method4 as a tool to determine the total content of reactive compounds contained in aged oils. The amount of reacting species is reported as Reactive Index (RI) which correlates to the amount of DCYA-adducts found after removal of the unreacted DCYA. The use of a dansylated thiol as nucleophilic agent enabled the trapping of electrophilic and other thio-reacting species as stable fluorescent derivatives. Quantification of the total fluorescent emission can in theory be used to estimate the total formation of DCYA-reacted species adducts regardless of the nature and concentrations of such reactive molecules. The strong electrophile massoia lactone (ML) and the moderate sensitizer cinnamaldehyde (CINA) were used as positive controls.

Table 1. Name and origin of TTO samples. SAMPLE

NAME

PLANT SOURCE

T1

Tea Tree oil*

Melaleuca alternifolia

T2

Cajuput EO*

Melaleuca cajeputi

T3

Cajuput Oil*

Melaleuca cajeputi

T4

Tea tree oil*

Melaleuca

T5

Tea tree oil*

Melaleuca linariifolia

T6

#

Manuka oil

Leptospermum scoparium

T7

Melaleuca alternifolia*

Melaleuca alternifolia

T8

Niaouli - Esoteric oils*

Melaleuca viridiflora

T9

#

Kanuka oil

Kunzea ericoides

T10

Lema® Oil*

Leptospermum +

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Melaleuca alternifolia #

*Commercial sample Non-commercial sample

4


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20

15

10

Variation (%)

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5

0

-5

-10

-15

-20

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

Figure 1. Variation of the composition of main TTO constituents

The samples were tested in triplicate and the average score is reported in Table 2. Six oils out of ten scored RI < 15 and were considered to be non- or weakly reactive. One cajuput oil (T2) and manuka oil (T6) exhibited weak sign of aging and low electrophilic potential. Whereas, for all authentic TTOs, a trend in increased reactivity with increased amounts of pcymene was observed, although p-cymene was found nonreactive in the DCYA assay. The sample T7 with higher amounts of peroxide content, resulted in the highest RI, almost four times greater than the other authentic TTOs. The second most reactive sample was cajeput oil T3, which contained elevated amounts of limonene and α-pinene (7.7 and 16.2%, respectively). After 18 month storage, a total loss of limonene and 50% decrease in α-pinene content was observed in T3 along with 5% increase in p-cymene and 15% in 1,8-cineole content. As a general trend, oxidized oils also contained elevated amounts of peroxides. Along with the formation of organic peroxides, hydrogen peroxide can also be formed upon aging.31 Hydrogen peroxide is a strong oxidizer which could potentially react with the fluorescent thiol to yield nonscavengeable dimer (DCYA)2 with higher fluorescence response, RI. To estimate the contribution of H2O2 to the observed RI of these oils, different concentrations of H2O2 from 0.1 to 100 mM were prepared and tested in the HTS-DCYA assay. An increased fluorescence response was obtained at all the concentration tested, with a strong response for H2O2 concentrations above 10 mM. The potential H2O2 content in these oils was thus estimated using a conservative approach. For all the oils with the exception of T7, the total peroxide content was estimated to be below 0.1 mM at the concentration tested in the fluorescence assay, thus the contribution of H2O2 to the fluorescence response is expected to be minimal. The fact that samples with comparable amount of peroxides resulted in different reactivity (RI) also reinforced the hypothesis that com-

pounds other than H2O2 may be responsible for the differences in chemical reactivity of these oils. In the case of sample T7, the elevated concentration of peroxides may substantially contribute to the reactivity. The sample was thus deoxygenated with polymer-bound triphenylphosphine (P-TPP) to reduce the potential peroxides to unreactive alcohols/ethers.33 The P-TPPtreated sample was compared to the untreated one in the HTSDCYA assay. As a result, the fluorescence response of the deoxygenated oil (RI = 28.6) dropped compared to the untreated sample but still remained significantly higher than the average authentic TTOs response (Fig. 2). Table 2. Quantified content of p-cymene, peroxide content and RI after 18 months, tabulated based on RI value. Sample

p-Cymene (%)

Peroxide (mg/L)

content

T6

0.04

0

-14.4

T10

5.1

0

-1.6

T2

2.1

0

6.6

T5

7.4

15

9.8

T1

2.2

3

11.2

T9

7.7

30

12

T8

1.4

15

17.2

T4

8.9

200

19.9

T3

5.2

30

36.8

RI

T7

19.5

5000

53.2

CINA*

-

-

14.9

ML*

-

-

89.2

*Positive controls in HTS-DCYA assay: CINA = cinnamaldehyde; ML =

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Chemical Research in Toxicology massoia lactone

with the fluorescent DCYA offered an expeditious approach for the potential identification of reactive intermediates through characterization of more stable adducts. To illustrate the potential of this approach, the reaction between the fluorescent thiol DCYA and the oxidized oil T7 was conducted and the resulting crude reaction mixture was analyzed by HPLC-DAD-MS. At least 5 major peaks (A-E, Fig. 3) in addition to DCYA2 (F, 23.42 min, MW 618) were observed having a UV profile compatible with dansyl moiety (Fig. 3c). For the peaks A and B at 10.75 and 11.04 min, the experimental m/z 435 was observed in the ESI(-) mode (Fig. 4) after reaction with DCYA. Such value was consistent with potential parent compound(s) derived from the reaction of DCYA (MW 310) with a small molecule having MW 126. In addition to the molecular ion, a characteristic DCYA fragment (m/z 309), and m/z 471 [M + Cl]- fragments were also identified in the mass spectra of these two peaks (Fig. 4). The observed m/z 309 fragment was due to in-source collision-induced dissociation (ISCID) of the parent DCYA-adduct. The ISCID technique was effectively applied for enhanced identification of glutathione-trapped reactive metabolites.35 No fragments corresponding to oxidized DCYA-adducts (expected m/z 325, [M - H + O] or m/z 341, [M - H + O2]) were found. Such oxidized DCYA sulfoxide/sulfone derivatives could be generated in strong oxidative conditions (such as elevated H2O2 concentrations). This information along with reported results for peroxide content (see paragraph above) endorse the conclusion that the observed fluorescence response is not derived from nonspecific oxidized DCYA by-products which could be formed in the presence of potential pro-oxidant compounds.

100 80 60 RI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40 20 0 T7

T7-TPP

ML

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CINA

Figure 2. Electrophilic potential of TTO sample T7 before and after deoxygenation with P-TPP. ML= massoia lactone; CINA = cinnamaldehyde.

Identification of potential electrophilic compounds. As oxidation of essential oils mainly occur through radical mechanisms, potentially reactive radicals can also be formed along with relatively more stable peroxide derivatives. These reactive radicals can potentially contribute to the sensitization potential but their identification may be challenging. On the other hand, their reaction product with a nucleophile may be stable enough to be identified. This hypothesis has been successfully applied by Kao et al.34 to investigate the reactivity of carbon-radical intermediates of allylic hydroperoxides trapped with several nucleophiles including glutathione and a cysteine derivative. In the current investigation, trapping experiments

a)

b)

A B,C

D

c)

E

F

Figure 3. Identification of potential adducts from oil T7 after reaction with DCYA: a) ESI(+) TIC b) ESI(-) TIC and c) UV absorption profile (λ340 nm) of an enriched fraction after fractionation on Sephadex column purification.

A minor peak having ESI(+) m/z 435 (Fig. 5b) was observed at 11.14 min compatible with an unsaturated homolog of compounds A/B. Based on mass fragmentation, these three compounds appears to be structurally inter-related and possibly derived from a C8 scaffold. Interestingly, 6-methylhepta-3,5dien-2-one (C8H12O, MW 124) was reported as a component of aged oil (but not in the fresh tea tree oil) by using compre-

hensive two-dimensional gas chromatography (GCxGC-qMS) by Tranchida et al.36 To validate the hypothesis that such small electrophiles could be forming in aged TTO essential oils, the commercially available 6-methylhepta-3,5-dien-2-one was incubated with DCYA and analyzed by UHPLC-DAD-MS. The resulting DCYA adduct of 6-methylhepta-3,5-dien-2-one was identified at 19.78 min (ESI(+), [M+H] 435) instead of

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10-11 min. Further studies were thus performed to isolate the major fluorescent peaks A/B, which were characterized using NMR and HPLC/MS (Fig. S10 and S11) as two diastereoisomers resulting from the DCYA addition to 4-hydroxy-4methylcyclohex-2-en-1-one. This small electrophilic molecule of MW 126 has been previously reported as thermal byproduct of ascaridole.37 The identification of electrophilic species in aged and commercial TTO using HTS-DCYA method exemplifies the potential advantage of using dansyl cysteamine for the detection of trace-amount(s) of reactive components. Small molecules having MW 126 were not previously reported in aged/commercial TTO, raising the doubt whether such compounds may be present in the oil but elusive to standard analytical methods. At least five additional chromatographic peaks with m/z 479 [M + H + DCYA]+ (Fig. 5a), and two other peaks having m/z 463 [M + H + DCYA] + (Fig. 5d) were also observed in the ESI(+) MS profile, which are compatible with monoterpene derivatives with empirical formula of C10H16O2 and C10H16O (MW 168 and 152, respectively). The presence of such intermediates in tea tree oil is expected due to oxidation and dehydration terpineols, or to oxidation of the corresponding terpinenes to oxygenated monoterpenes. In a separate study (data not shown) several known menthene-diols and mentha-triols were isolated and characterized from the residue obtained after distillation of essential oil of tea tree. Again, the existence of such intermediates in TTO residue validates the supposition of the existence of 1,2and/or 1,4-endoperoxides in tea tree oil. Terpine-4-ol, αterpineol, and terpinenes (α/γ/δ) along with limonene directly/indirectly contribute to the observed increased amounts of cymene in aged TTOs.5 Some plausible examples of oxidized DCYA-reactive intermediates originated from α-terpinene are shown in Fig. 6.

▬ -ESI TIC ▬ -ESI EIC (249.0) ▬ -ESI EIC (309.0) ▬ -ESI EIC (435.0) ▬ -ESI EIC (471.0)

▬ -ESI TIC ▬ -ESI EIC (477.0) ▬ -ESI EIC (459.0)

Figure 4. ESI(-) EIC profile of selected peaks at 10.66, 10.97 (above) and at 16.2 min (below).

a)

b)

c)

d)

Figure 5. ESI(+) EIC profile of potential DCYA adducts for m/z 479 (a), 435 (b), 437 (c) and 463 (d).

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a)

OH

OH

4-Terpineol

-Terpineol

O

b)

-Terpinene R1

[O]

O

R1

OR1

OOH

R2 a-1

-Terpinene

-Terpinene Terpenolene

R2 a-3

R2 a-2

Cymene

R1= Me, R2= iPr; or R1= iPr, R2= Me [O] -CH(CH3)2

Trapped with DCYA

O

O2S

H N

R1

HO

OH

S R2

N

O2S

O2S

H N

R1 OOH

S R2

N

O

H N

MW 462

MW 478

S HO N A/B MW 436 rt 10.66 and 10.97 min Figure 6 a) Main TTO monoterpenes known to undergo oxidative changes. b) Selected examples of oxygenated species derived from αterpinene degradation and their potential reaction products with DCYA.

levels led to rapid degradation of terpinolene and α-terpinene, with a 50% loss after 2 and 13 days, respectively (Fig. 8). After 28 days, none of the two compounds was present in detectable levels (Fig. S8) but both aged samples scored significantly higher RI values, while no RI increment was found for ɣterpinene, α-terpineol, p-cymene, and sabinene (Fig. 7). In the case of terpinen-4-ol, no byproducts were identified from GCMS data. A possible explanation is that degradation products were unstable or non-volatile enough under the tested GC

Generation of reactive species after aging of pure compounds. In order to identify which TTO components may contribute to the generation of thio-reactive species, the individual major constituents of sample T7 were aged under accelerated conditions. Changes in the chemical composition were monitored by GC-MS (Figs. S7 and S8) and correlated to the RI of the pure components upon aging. All compounds at day 0 scored weak to non-reactive in the HTS-DCYA assay (Fig. 7). Incubation of the compounds under elevated oxygen

8


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conditions. As in the case of the oils, compounds which scored higher in the HTS-DCYA assay contained an increased amount of total peroxides content (Fig. S9), although this correlation alone is not sufficient to explain the increased reactivity of the aged compounds. It is worth recalling that the presence of oxygen is only one of the factors involved in aging. Light irradiation, heat and humidity can also accelerate the aging process. It is thus possible that compounds stable under these conditions may be unstable if different aging conditions are applied. Such comprehensive analysis was outside of the scope of this work and will be object of further investigations. In conclusion, the results obtained herein are in line with previously reported data on TTO which associate increased sensitization potential with aging processes.5 This case study demonstrates how both crude oils and pure components might undergo chemical changes upon aging, but such changes may not unequivocally correlate to increased accumulation of reactive species. Compounds such as α-terpinene and terpinolene have been found to rapidly age under oxygen with generation of thio-reactive species and potential reaction adducts have been found by UHPLC-DAD-MS. Such results endorse previous authors’ hypothesis on the potential of cyclic dienes to act as pre-/pro-haptens through generation of reactive species upon chemical or metabolic activation.38 The use of DCYA trapping experiments served as an expeditious approach to identify reactive intermediates in aged tea tree oil without any activation (viz., Fenton reaction, or FeSO4 to form reactive radicals), supporting the possibility that electrophilic species may also be formed upon aging and thus be responsible for the generation of such adducts, as confirmed by the identification of a DCYA adducts A/B. The applicability of in chemico methods to rapidly identify potential hazardous compounds in complex mixtures is most needed to complement the battery of approaches used in skin sensitization risk assessment. A rational use of chemical

methods can be extremely useful to assist in prioritizing risk analyses as required by international regulatory agencies. 60

Day 0

Day 28

50

RI

40

30

20

10

0

Figure 7. HTS-DCYA results for major TTO components before and after aging

This work exemplifies how the HTS-DCYA assay can be used as an expeditious approach to analyze complex mixtures for the presence of stable or unstable reactive compounds as a proof-of-concept. The method could thus serve as useful strategy to the rapid identification of potential hazardous compounds in known or unknown mixtures, filling the gap of current ex vivo approaches.

120

100

80 Area %

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60

40

20

0 0

5

10

15

20

25

30

Day

α-Terpinene p-Cymene Figure

8.

Stability

Terpinolene Sabinene

ɣ-Terpinene Terpinen-4-ol of

major

TTO

components

analyzed

9


α-Terpineol by

GC-MS

upon

aging.


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phytes and other filamentous fungi. J. Antimicrob. Chemother. 50, 195-199. (8) Hammer, K. A., Carson, C. F., and Riley, T. V. (2003) Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J. Appl. Microbiol. 95, 853-860. (9) Carson, C., Hammer, K., and Riley, T. (2006) Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev. 19, 50-62. (10) Standardisation, I. O. f. (2004) ISO 4730:2004, oil of Melaleuca, terpinen-4-ol type (tea tree oil). In International Organisation for Standardisation, Geneva, Switzerland. (11) Rubel, D. M., Freeman, S., and Southwell, I. A. (1998) Tea tree oil allergy: what is the offending agent? Report of three cases of tea tree oil allergy and review of the literature. Australas. J. Dermatol. 39, 244-247. (12) Wang, M., Zhao, J., Avula, B., Wang, Y.-H., Chittiboyina, A. G., Parcher, J. F., and Khan, I. A. (2015) Quality Evaluation of Terpinen-4-ol-Type Australian Tea Tree Oils and Commercial Products: An Integrated Approach Using Conventional and Chiral GC/MS Combined with Chemometrics. J. Agric. Food Chem. 63, 2674-2682. (13) Brophy, J. J., Davies, N. W., Southwell, I. A., Stiff, I. A., and Williams, L. R. (1989) Gas chromatographic quality control for oil of Melaleuca terpinen-4-ol type (Australian tea tree). J. Agric. Food Chem. 37, 1330-1335. (14) Porter, N. G., and Wilkins, A. L. (1999) Chemical, physical and antimicrobial properties of essential oils of Leptospermum scoparium and Kunzea ericoides. Phytochemistry 50, 407-415. (15) a) Monti, D., Chetoni, P., Burgalassi, S., Najarro, M., Saettone, M. F., and Boldrini, E. (2002) Effect of different terpenecontaining essential oils on permeation of estradiol through hairless mouse skin. Int. J. Pharm. 237, 209-214. b) https://npgsweb.arsgrin.gov (16) Ireland, B. F., Hibbert, D. B., Goldsack, R. J., Doran, J. C., and Brophy, J. J. (2002) Chemical variation in the leaf essential oil of Melaleuca quinquenervia (Cav.) S.T. Blake. Biochem. Syst. Ecol. 30, 457-470. (17) Maddocks-Jennings, W., Wilkinson, J., Shillington, D., and Cavanagh, H. (2005) A fresh look at manuka and kanuka essential oils from New Zealand. Int. J. Aromather. 15, 141-146. (18) Christoph, F., Kaulfers, P. M., and Stahl-Biskup, E. (2000) A Comparative Study of the in vitro Antimicrobial Activity of Tea Tree Oils s.l. with Special Reference to the Activity of β-Triketones. Planta Med. 66, 556-560. (19) Aptula, A., Patlewicz, G., and Roberts, D. (2005) Skin sensitization: reaction mechanistic applicability domains for structureactivity relationships. Chem. Res. Toxicol. 18, 1420-1426. (20) Karlberg, A. T., Bergstrom, M. A., Borje, A., Luthman, K., and Nilsson, J. L. (2008) Allergic contact dermatitis-formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21, 53-69. (21) Hausen, B. M., Reichling, J., and Harkenthal, M. (1999) Degradation products of monoterpenes are the sensitizing agents in tea tree oil. Dermatitis 10, 68-77. (22) Smith Pease, C. K., Basketter, D. A., and Patlewicz, G. Y. (2003) Contact allergy: the role of skin chemistry and metabolism. Clin. Exp. Dermatol. 28, 177-183. (23) OECD. (2015) Test No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD Publishing. (24) Schwöbel, J., Koleva, Y., Enoch, S., Bajot, F., Hewitt, M., Madden, J., Roberts, D., Schultz, T., and Cronin, M. (2011) Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111, 2562-2596. (25) Andres, E., Sá-Rocha, V., Barrichello, C., Haupt, T., Ellis, G., and Natsch, A. (2013) The sensitivity of the KeratinoSens™ assay to evaluate plant extracts: a pilot study. Toxicol. In Vitro 27, 12201225. (26) Chittiboyina, A. G., Avonto, C., Rua, D., and Khan, I. A. (2015) Alternative Testing Methods for Skin Sensitization: NMR

ASSOCIATED CONTENT Supporting Information. GC-MS chromatograms, essential oils chemical composition, peroxide content results, UHPLC-DADMS chromatograms and NMR spectra are included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Prof. Ikhlas A. Khan, National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA. Tel: +1-662- 915-7821; Fax:+1-662-915 7062. E-mail: [email protected].

Funding Sources This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01FD004246-04, the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6408-1-603-06.

ACKNOWLEDGMENT Would like to thank Prof. Parcher for the editing and proofreading of the current findings.

ABBREVIATIONS ACN, Acetonitrile; CINA, Cinnamaldehyde; DCYA, Dansyl Cysteamine; EO, essential oil, ESI, Electron Spray Ionization; GC, Gas Chromatography; HTS, High Throughput Screening; LLNA, Local Lymph Node Assay; ML, Massoia lactone; NC, Negative Control; PC, Positive Control; P-TPP, Polymer-bound triphenylphosphine; RI, Reactive Index; TTO, Tea Tree Oil; UHPLC-DAD-MS, Ultra High Pressure Liquid Chromatography coupled with Diode Array Detector and Mass Spectrometer

REFERENCES (1) Pauwels, M., and Rogiers, V. (2004) Safety evaluation of cosmetics in the EU. Reality and challenges for the toxicologist. Toxicol. Lett. 151, 7-17. (2) Antignac, E., Nohynek, G. J., Re, T., Clouzeau, J., and Toutain, H. (2011) Safety of botanical ingredients in personal care products/cosmetics. Food Chem. Toxicol. 49, 324-341. (3) Schnuch, A., Lessmann, H., Geier, J., Frosch, P. J., and Uter, W. (2004) Contact allergy to fragrances: frequencies of sensitization from 1996 to 2002. Results of the IVDK*. Contact Dermatitis 50, 65-76. (4) Avonto, C., Chittiboyina, A. G., Rua, D., and Khan, I. A. (2015) A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers. Toxicol. Appl. Pharmacol. 289, 177-184 (5) Anonymous. (2008) Scientific Committee on Consumer Products, Opinion on tea tree oil, SCCP/1155/08. (6) Carson, C. F., and Riley, T. V. (1995) Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. J. Appl. Bacteriol. 78, 264-269. (7) Hammer, K. A., Carson, C. F., and Riley, T. V. (2002) In vitro activity of Melaleuca alternifolia (tea tree) oil against dermato-

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Spectroscopy for Probing the Reactivity and Classification of Potential Skin Sensitizers. Chem. Res. Toxicol. 28, 1704-1714. (27) Rudbaeck, J., Ramzy, A., Karlberg, A.-T., and Nilsson, U. (2014) Determination of allergenic hydroperoxides in essential oils using gas chromatography with electron ionization mass spectrometry. J. Sep. Sci. 37, 982-989. (28) Johansson, S. G. H., Emilsson, K., Groetli, M., and Boerje, A. (2010) Structural Influence on Radical Formation and Sensitizing Capacity of Alkylic Limonene Hydroperoxide Analogues in Allergic Contact Dermatitis. Chem. Res. Toxicol. 23, 677-688. (29) Christensson, J. B., Johansson, S., Hagvall, L., Jonsson, C., Boerje, A., and Karlberg, A.-T. (2008) Limonene hydroperoxide analogues differ in allergenic activity. Contact Dermatitis 59, 344352. (30) Karlberg, A.-T., and Dooms-Goossens, A. (1997) Contact allergy to oxidized d-limonene among dermatitis patients. Contact Dermatitis 36, 201-206. (31) Rudbaeck, J., Bergstroem, M. A., Boerje, A., Nilsson, U., and Karlberg, A.-T. (2012) α-Terpinene, an Antioxidant in Tea Tree Oil, Autoxidizes Rapidly to Skin Allergens on Air Exposure. Chem. Res. Toxicol. 25, 713-721. (32) Kao, D., Chaintreau, A., Lepoittevin, J.-P., and GimenezArnau, E. (2011) Synthesis of Allylic Hydroperoxides and EPR SpinTrapping Studies on the Formation of Radicals in Iron Systems as Potential Initiators of the Sensitizing Pathway. J. Org. Chem. 76, 6188-6200.

(33) Balci, M. (1981) Bicyclic endoperoxides and synthetic applications. Chem. Rev. 81, 91-108. (34) Kao, D., Chaintreau, A., Lepoittevin, J.-P., and GiménezArnau, E. (2014) Mechanistic studies on the reactivity of sensitizing allylic hydroperoxides: investigation of the covalent modification of amino acids by carbon-radical intermediates. Toxicol. Res. 3, 278289. (35) Zhu, X., Kalyanaraman, N., and Subramanian, R. (2011) Enhanced screening of glutathione-trapped reactive metabolites by insource collision-induced dissociation and extraction of product ion using UHPLC-high resolution mass spectrometry. Anal. Chem. 83, 9516-9523. (36) Tranchida, P. Q., Shellie, R. A., Purcaro, G., Conte, L. S., Dugo, P., Dugo, G., and Mondello, L. (2010) Analysis of fresh and aged tea tree essential oils by using GC× GC-qMS. J. Chromatogr. Sci. 48, 262-266. (37) Naya, Y., Nagahama, Y., and Kotake, M. (1978) Volatile components of Ledum palustre var. nipponicum et yesoense. Heterocycles 10, 29-36. (38) Bergström, M., Luthman, K., Nilsson, J. and Karlberg, AT. (2006) Conjugated dienes as prohaptens in contact allergy: in vivo and in vitro studies of structure-activity relationships, sensitizing capacity, and metabolic activation. Chem. Res. Toxicol. 19, 760-769.

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In Chemico Evaluation of Tea Tree Essential Oils as Skin Sensitizers

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