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What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization Amar G Chittiboyina, Cristina Avonto, and Ikhlas A Khan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00157 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 15, 2016

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Chemical Research in Toxicology

What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization. Amar G. Chittiboyina,§,* Cristina Avonto,§ and Ikhlas A. Khan.§,¤ §

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National Center for Natural Products Research; Division of Pharmacognosy, Department of BioMolecular Sciences; School of Pharmacy, University of Mississippi, University, MS 38677, USA. Ascaridole, radical intermediates, dansylcysteamine, highthroughput screening, skin sensitization. ABSTRACT: To replace animal testing and improve the prediction of skin sensitization, significant attention has been directed to the use of alternative methods. The direct peptide reactivity assay (DPRA), the regulatory agencies’ approved alternative in chemico method, has been applied for understanding the sensitization capacity of activated ascaridole. Ascaridole, the oxidative metabolite of α-terpinene, is considered to be one of the components responsible for contact allergy associated with essential oils derived from Chenopodium and Melaleuca species. The recently developed highthroughput screening based on dansyl dansyl cysteamine (HTS-DCYA) method was applied to understand the reported enhanced reactivity of activated ascaridole and possibly to identify the resulting elusive, radical or other reactive species. For the first time, a substituted cyclohexenone was identified as potential electrophilic intermediate resulting in higher depletion of nucleophilic DCYA, along with several non-reactive byproducts of ascaridole via radical degradation mechanism. Along with commonly assumed allyl-epoxides, allyl-hydroperoxides, formation of electrophilic species via radical degradation is one of the possible pathways associated with reactivity of peptides in aged tea tree oil or oils rich in terpinenes.

Both freshly distilled and air-exposed tea tree oils (TTO) have been shown to be sensitizing in animal models, with a 3fold increase in the sensitization potency for the aged TTO.1 Ascaridole, the oxidative metabolite of α-terpinene is considered to be one of the components responsible for contact allergy to TTO2 The transformation of α-terpinene to ascaridole has been utilized for the detection of singlet oxygen in gas streams.3, 4 Ascaridole is a natural small endoperoxide classified as a bicyclic monoterpene containing an unusual bridging peroxide functional group.5 The compound has been reported to be a major constituent of the oil of Dysphania ambrosioides (syn. Chenopodium ambrosioides), commonly known as wormseed or Mexican Tea. Due to its unique biological properties, it is a component of herbal medicines, tonic drinks, and food flavoring especially in Latin American cuisine.6 In vitro cytotoxicity has been reported against several tumor cell lines (CCRF-CEM, HL60, MDA-MB-231),7, 8 and it is considered as an indicative marker of aged tea tree oils (TTO) originated from Melaleuca species (Myrtaceae), commonly known as paperbarks, honey-myrtles, or tea-trees. Numerous case reports of contact allergy or photosensitivity due to topical application of essential oils containing ascaridole have appeared in the medical literature during recent decades.9-11 Nevertheless, the cause of allergies is still controversial, and no single culprit has been identified. In addition to positive reactions in patch tests, enhanced irritancy with increased concentrations of ascaridole was observed by Krutz et al.12 The authors concluded that activation of ascaridole modulated its sensitizing capacity and proposed that a free radical mechanism might be involved in protein-

binding or other steps of the sensitization process. Treatment of the fairly stable endoperoxide ascaridole with iron as a radical inducer resulted in the induction of the Nrf2 target gene heme oxygenase 1 and upregulation of CD86 and CD54 on THP-1 cells. Interestingly, in the regulatory agencies’ approved in chemico method, direct peptide reactivity assay (DPRA), 58% more depletion of cysteine peptide was observed than that of ascaridole itself. However, the intriguing question remains of how the activated ascaridole was responsible for the observed12 “exceeding reactivity” towards cysteine- but not lysine-containing peptide. Further information on the characterization of radical species or other reactive species could possibly reveal critical information on how the aging process of TTO has a direct impact on the skin sensitization potential of ascaridole and ascaridole-containing essential oils. The applicability of in chemico methods for the identification of hazardous components in complex mixtures is the missing piece in the battery of regulatory agencies’ recommended methods in risk assessment of potential skin sensitizers. The aim of the current study was to i) understand the differences of ascaridole’s role in the depletion of thiol nucleophiles with and without activation by applying a recently developed13 ‘HTS-DCYA screening method’; ii) identify and characterize the reactive, often elusive, intermediates of the activated ascaridole by trapping them with fluorescent dansyl cysteamine; and iii) assess the implications of such intermediates present in complex mixtures, such as TTO, on skin sensitization potential.

MATERIALS AND METHODS

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Chemicals and Reagents. Ascaridole (CAS# 512-85-6) with >90% purity (Fig. S1-S2 for 1H and 13C NMR spectra) was purchased from City Chemicals LLC. The fluorescent compounds DCYA and DCYA disulfide were synthesized as described by Chittiboyina et al.14 Polymer-supported maleimide (SiliaBond® Maleimide, ≥ 0.81 mmol/g) was purchased from SiliCycle (Quebec City, Quebec, Canada). Standardized buffer solution pH 10 ± 0.02 (cat. # SB116-500), micro centrifuge tubes, polypropylene solvent-resistant 96-well microplates and TLC silica gel supported on aluminum sheets (M1055530001A) were purchased from Fisher Scientific (Suwanee, GA, USA). HTS-DCYA method. The reactivity potential of ascaridole and activated ascaridole was investigated using a modified version of the protocol described by Avonto et al.13 Briefly, 200 µL of a 2.5 mM solution of DCYA was mixed with 200 µL of a solution 5.0 mM of (activated) ascaridole in acetonitrile. For positive/negative controls (PC and NC), 200 µL acetonitrile was added to the DCYA solution in place of the test compound. Such controls (PC and NC) were added to monitor the variation of the fluorescence response (inter- and intraday) of DCYA. The role and experimental details of PC and NC were previously reported.13 Three hundred microliters of the DCYA-sample solution was then split in two micro centrifuge tubes (150 µL each). Thirty microliters of aqueous pH 10 buffer was added to the reaction (R) tubes and negative controls, while 30 µL of acetonitrile was added to the blanks (Bl) and positive controls. The solution was incubated for 20 min at room temperature. Thirty mg of polymer-supported maleimide was then added and 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 HTS assays 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). Fifty readings (high sensitivity) were recorded at emission wavelength 520 nm (excitation 350 nm, cutoff 420 nm) at 23 °C. The Reactivity Index (RI) was calculated based on the average of 9 readings per sample (3 readings/well in triplicate) using the following equation: RI=100*((Bl-R)/(PC-NC)-(PC-Bl)/PC) Activation of Ascaridole and Isolation of ascaridole byproducts. Ascaridole was activated with iron (II) sulphate as previously described.12 Briefly, 200 mM ascaridole (30 mL, 1.01 g) solution was incubated with 40 mM of FeSO4 solution in water and depletion of the ascaridole was monitored by TLC. After 90 min, the mixture was extracted twice with diethyl ether (25 mL), and the combined organic layer was carefully dried over anhydrous MgSO4 and evaporated with stream of nitrogen. A small amount of the resulting mixture was subjected to HTS-DCYA method without any further purification. The remaining crude mixture was loaded on an 80 g silica gel flash cartridge (SiliaPrepTM, SiliCycle) and eluted with hexane/acetone (gradient elution from 5 to 50 % acetone) to yield compounds 2a-e.

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Accelerated aging of Ascaridole. One milliliter of ascaridole 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 a magnetic stirrer for 28 days. Thin Layer Chromatography. A ten microliters aliquot of each sample was loaded on a silica gel plate and eluted with 40% ethyl acetate in hexane. The TLC plates were examined at λ366 for the presence of the yellow fluorescent spots corresponding to DCYA (Rf 0.67), its dimer (Rf 0.57) or reaction products (Rf 0.25 and 0.14 respectively in Fig. S8, supporting information). Isolation and Characterization of DCYA-adducts. To a solution of activated ascaridole crude mixture (300 mg) in acetonitrile (4 mL), an equal volume of DCYA (50 mM in acetonitrile) was added, followed by the addition of 0.3 mL of pH 10 buffer. After 4 h, the reaction mixture was extracted repeatedly with hexane (total volume about 50 mL). The aqueousacetonitrile layer was concentrated under vacuum and the resulting mixture was then loaded onto a 25 g silica gel flash cartridge (SiliCycle), eluted with hexane/EtOAc (gradient elution from 10 to 70% EtOAc), and resulting fractions were dried and analyzed by NMR and UHPLC-DAD-MS. UHPLC-DAD-MS Analyses. Characterization of potential DCYA adducts was performed using a Waters Acquity UPLCTM system including binary solvent manager, sampler manager, column compartment and PDA connected to a Waters Empower 2 data station and a UHPLC BEH Shield RP18 column (1.7 µm 2.1x 100 mm, Waters Corporation, Milford, MA, USA). The column and sample temperature were maintained at 35 and 15 oC, respectively. The column was equipped with an LC-18 guard column (Vanguard 2.1 x 5 mm, Waters Corp., 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: 35 % to 100% B (0.25 ml/min) in 9 min and then hold for 2 min with 100% B. UV absorption was monitored at 254 and 330 nm. Positive and negative ESI scans were recorded in the range m/z 100-1000.

RESULTS & DISCUSSION Several non-animal alternative tests and integrated approaches including in chemico methods are used for the risk assessment of potential skin sensitizers. In chemico assays are extremely useful to determine earlier haptenation events by mimicking the covalent binding of candidate sensitizers to the biological target.15 The only currently validated in chemico testing method, DPRA, is designed to mimic the covalent binding of electrophilic chemicals to nucleophilic centers in skin proteins by quantifying the reactivity of chemicals towards the model synthetic peptides containing cysteine and lysine. Recently,12 the DPRA method was used to show that activated ascaridole selectively depleted cysteine- but not lysine-containing peptide. Experimentally observed induction of Nrf2 target genes and upregulated protein expression in THP-1 cells implied that activation of ascaridole modulates its sensitizing capacity via radical mechanism. Interest in the enhanced reactivity (depletion) of the peptide together with the lack of structural information associated with activated ascaridole prompted us to explore the applicability of


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the newly developed HTS-DCYA method13 as a tool to determine the total content of reactive compounds. In the HTSDCYA method, the reactive potential is calculated as a Reactive Index (RI) which correlates with the amount of DCYAadducts found after removal of the unreacted DCYA, autooxidized dimer, and test article’s interference. Quantification of the total fluorescence emission can, in theory, be used to estimate the total formation of DCYA-adducts regardless of the nature and concentration of such reactive molecules in the test article. 110 90 70

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Asc

Asc 2 Asc 3

2e

2b

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Figure 1.HTS-DCYA results of pure ascaridole (Asc), aged ascaridole (Asc 2), after activation with FeSO4 (Asc 3) and it’s degradation products (2e, 2b, 2d). CINA = Cinnamaldehyde as a positive control.

In the current study, both ascaridole and Fe-activated ascaridole were tested in duplicate along with the moderate sensitizer cinnamaldehyde as the positive control. The average scores are reported in Figure 1. In the HTS-DCYA assay, ascaridole (Asc) showed very low RI values similar to weakto-non sensitizers. The lack of reactivity was further confirmed with an NMR-DCYA method,14 wherein no depletion of electrophile/DCYA signals for 120 min was observed (Figs. S3 and S4). Even aging the ascaridole (Asc 2) for 28 days under an oxygen atmosphere did not produce any reactivity in the HTS-DCYA assay. Treatment of ascaridole with FeSO4 for 1 h as reported by Krutz et al.12 resulted in small changes in the ascaridole concentration along with several minor, polar compounds observed (monitored by TLC). However, an increased concentration of the FeSO4 and prolonged reaction times up to 4 h led to higher conversion of ascaridole to several polar compounds. The activated ascaridole mixture was indeed more reactive than ascaridole. Activation of ascaridole with FeSO4 for 90 min and after aqueous workup (free from iron and other inorganic impurities) sample (Asc 3) resulted in 17.3 RI compared to inactivated ascaridole (7.5) and aged ascaridole (7.8). (Fig-

ure 1). The enhanced reactivity of activated ascaridole prompted us to investigate further. Mechanistically, an activation of the endoperoxide with Fe(II) would facilitate the cleavage of the O-O bond to form oxygenated radicals as proposed in Scheme 1. The radicals can rearrange, due to the presence of the terpene double bond, into many possible carbon centered radicals. These radical intermediates are suspected to covalently bind to skin proteins through radical mechanisms resulting in elicitation of earlier sensitization events. However, due to their short half-lives, characterization and identification of such radical or other reactive intermediate species can be challenging. In the current approach, trapping experiments with fluorescent DCYA would serve as a novel tool/probe for the potential identification of reactive, electrophilic intermediates via the formation of stable adducts. To test the practicality of such an approach, ascaridole was activated with Fe(II) and the resulting mixture was treated with DCYA in a pH 10 buffer. Thin layer chromatography indicated the formation of at least two fluorescent spots along with unreacted DCYA and its dimer (Fig. S8). Further analysis with UHPLC-DAD-MS (Fig. S9) indicated formation of two major adducts with the same m/z 436 at approximately 4.9 min and a minor adduct with m/z 478 at 6.88 min. The major compounds were isolated by column chromatography and characterized as 2e-DCYA diastereomeric adducts by NMR and HPLC-MS (Table S9, Fig.S9-S13), suggesting that these diastereomers originated from the substituted cyclohexenone, 2e (Path A). The structural characterization of the minor adduct with m/z 478 could not be achieved due to minor amounts, however, based on the mass and fragmentation pattern, the adduct could have originated from isoascaridole via the nucleophilic thiol ring opening of oxirane (Path B).16, 17 Isoascaridole is a known byproduct obtained after the thermal decomposition of ascaridole.18 To understand the formation of such reactive small molecules, gram quantity of ascaridole was subjected to activation with Fe(II) for 4 h, and at least five products 2a-2e (Fig. S5) were isolated by column chromatography. As shown in Scheme 1 Path A, formation of α,β-unsaturated ketone 2e is possible via fragmentation and loss of a stable isopropyl radical upon activation with a radical inducer. Formation of such an intermediate as a minor product was reported with ruthenium (II) catalysis from 1,4-epiperoxides.19 Due to its inherent structural feature, α,β-unsaturation, 2e would be expected to undergo Michael addition with nucleophilic thiols, such as DCYA in this case, to yield the diastereomeric adducts with m/z 436. On the other hand, four closely related compounds 2a-2d were possibly derived from the corresponding syndiepoxide of dihydroascaridole, isoascaridole via Path B of Scheme 1.



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Scheme 1. Possible reaction schema for the production of isolated reaction products (2a-2e) of activated ascaridole and its two major DCYA adducts. Upon activation of ascaridole in the current study, formation of several medium-polarity spots including isoascaridole were observed by TLC. After about 60 min the isoascaridole and other non-polar spots were converted to several new, polar spots on TLC. Compound 2b may be formed via epoxide ring opening of compound 2 and further deprotonation, whereas compounds 2d and 2c are possible via oxirane ring opening and addition of water. It is also possible that 2d can undergo further hydrolysis to yield 2c. Formation of 2a can be envisioned from the hydrolysis of precursor epoxide, 2b. All these products of activated ascaridole were isolated by column chromatography, and the structures were confirmed by comparing NMR and MS with the literature data20, 21 Table S3 and Fig.S6-S7). Several Dysphania (Chenopodium) ambrosioides essential oils have been shown to contain isoascaridole along with ascaridole and our results are in agreement with the results reported by Naya et al.20 on the generation of 2b and 2d as byproducts of (iso)ascaridole after treatment with ferrous sulfate. On the contrary, 2e was found as a byproduct of activation rather than a thermal byproduct of ascaridole under GC conditions as suggested by the same authors. The monoterpene α-terpinene is used as a fragrance compound and is present in various essential oils. It is one of the components responsible for the antioxidative properties of many species originating from the genera Melaleuca and Chenopodium.22, 23 Interestingly, α-terpinene endoperoxide (ascaridole), possibly resulting from photo-oxidation of αterpinene, has also been reported24, 25 from these plants in significant quantities. On the other hand, aged oils of Malaleuca have been linked to potential adverse effects with increased skin sensitization potential due to the presence of reactive intermediates such as allylic-epoxides and –hydroperoxides.26, 27

In order to assess the applicability of the newly developed method in estimating the sensitization potential of complex mixtures, the ‘HTS-DCYA’ method was applied to tea tree oils for the first time in a previous study.28 (Avonto et al.

Chem Res Tox., 2016, under review) Based on experimentally observed DCYA adducts, several elusive intermediates with postulated m/z 124, 126, 152 and 168 were identified in (aged) tea tree oils. In the current study some of the same adducts with m/z 126 and 168 were also observed after activation of pure ascaridole. We hypothesize that compounds with m/z 126 and 168 in TTO might have originated from α-terpinene and/or its photo-oxidative product, ascaridole, via radical mechanisms such as those proposed in Scheme 1. Several TTOs were screened for the presence of substituted cyclohexenone 2e. It was found that many tea tree oils do contain 2e in trace amounts, suggesting that such radical mechanisms may operate in the essential oils of Malaleuca. In summary, treatment of the fairly stable endoperoxide ascaridole with iron mediated the radical induction and resulted in two-fold more depletion of fluorescent thiol compared to that of ascaridole itself in the ‘HTS-DCYA’ method. For the first time, the major adducts (as a set of diastereomers) with DCYA were isolated and a possible pathway involved in the radical degradation of ascaridole to yield the reactive, electrophilic species was proposed. Taking a cue from this data, the existence of such intermediates in aged TTO was also identified. Until this finding, many researchers believed and assumed that the sensitization potential of aged oils was partly due to formation of various allyl-epoxides and allylhydroperoxides. Our data indicate that formation of electrophilic species via radical degradation is one of the possible mechanistic pathways associated with nucleophilic reactivity (DPRA or HTS-DCYA) in aged tea tree oil or oils rich in terpinenes. The experimental results lead to several hypotheses; first of all the small electrophilic, cyclohexenone may be among the principal components responsible for the observed “exceeding reactivity” towards cysteine- but not lysinecontaining peptide. Secondly, α-terpinene may be one of the crucial components that undergo singlet oxygen addition to give stable endoperoxides andfurther activation with radical initiator would lead to the formation of reactive, elusive spe-


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cies such as the α,β−unsaturated ketone. Presence of such intermediates may be responsible for the observed concomitant irritancy with increased concentrations of ascaridole and ascaridole-containing essential oils.

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ASSOCIATED CONTENT

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Supporting Information NMR data for ascaridole, compounds (2b-2e) and DCYA-adducts of 2e are included. Arrayed spectra for ascaridole with DCYA along with data and data elaboration tables are also included. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION Corresponding Author *Dr. Amar G. Chittiboyina, National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA. Tel: +1-662- 915-1572; Fax:+1-662-915 7989. E-mail: [email protected]..

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Funding Sources This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01FD004246-05, the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6408-1-603-05.

ACKNOWLEDGMENT Would like to thank Prof. Parcher for the editing, proof-reading and Dr. Avula for UHPLC-MS analysis.

ABBREVIATIONS

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REFERENCES

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DCYA, dansyl cysteamine; DPRA, direct peptide reactivity assay; GC, gas chromatography; HTS, highthroughput screening; MS, mass spectroscopy; NMR, nuclear magnetic resonance spectrocopy; TLC, thin layer chromatography;TTO, tea tree oil; UHPLC, ultra high performance liquid chromatography

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and in vitro studies of structure-activity relationships, sensitizing capacity, and metabolic activation. Chem. Res. Toxicol. 19, 760-769. (28) Avonto, C., Chittiboyina, A. G., Wang, M., Vasquez, Y., Rua, D., and Khan, I. A. (2016) In chemico evaluation of tea tree essential oils as skin sensitizers: Impact of the chemical composition on aging

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