Xenobiotica, June 2007; 37(6): 604617

Study on the cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool: Indication of biological epoxidation

R. J. W. MEESTERS1, M. DUISKEN1,2, & J. HOLLENDER1,3

Institute of Hygiene and Environmental Medicine, RWTH Aachen University, Aachen, Germany, nchengladbach, Germany, and 3Swiss Federal Institute of Aquatic LECO Instrumente GmbH, Mo bendorf, Switzerland Science and Technology, Eawag, Du
2 1

(Received 1 March 2007; revised 23 March 2007; accepted 11 April 2007)

Abstract The cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool was studied in vitro by enzymatic assays using recombinant human cytochrome P450 enzymes. Three different enzymatic products of allylic hydroxylation and epoxidation were identified by gas chromatography-mass spectrometry. Identified enzymatic products were 8-hydroxylinalool ((R/S)-3,7-dimethyl-1,6-octadiene-3,8-diol) and the cyclic ethers pyranoid-linalool oxide ((R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol) and furanoid-linalool oxide (R/S)-2-(1,1dimethylethyl)-5-methyl-5-vinyltetrahydrofuran. The cyclic ethers result most likely from the epoxidation of the 6,7-carbon double carbon bond of (R/S)-linalool, followed by the intramolecular rearrangement of the 6,7-epoxy-linalool. Allylic-hydroxylation of the 8-methyl group of linalool was catalyzed by CYP2C19 and CYP2D6 while the enzymatic epoxidation of linalool was only observed with CYP2D6. The results indicate that the electrophilic oxidation products of linalool such as 6,7-epoxy-linalool which may cause sensitization and irritational skin reactions are not only produced by auto-oxidation reactions in the presence of air-oxygen as published in the past, but also by P450-mediated oxidative biological transformation. Keywords: Linalool, cytochromes P450 (CYP), epoxidation, pyranoid-linalool oxide, furanoid-linalool oxide, 8-hydroxylinalool

berlandstr. 133, CH-8600 Correspondence: J. Hollender, Swiss Federal Institute of Aquatic Science and Technology, Eawag, U Du bendorf, Switzerland. Tel: 41-44-823-5493. Fax: 41-44-823-5893. E-mail: juliane.hollender@eawag.ch ISSN 0049-8254 print/ISSN 1366-5928 online 2007 Informa UK Ltd. DOI: 10.1080/00498250701393191

P450-mediated oxidative metabolism of linalool Introduction

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Linalool ((R/S)-3,7-dimethyl-1,6-octadiene-3-ol)) is a naturally occurring unsaturated racemic monoterpene alcohol. Because of its pleasant scent it is used frequently as a fragrance ingredient in many consumer products, such as cosmetics, fine fragrance formulations, shampoos, soaps and other toiletries, as well as in cleaners and detergents (Skold et al. 2004). The worldwide usage of linalool is approximately 41000 metric tons per year (Letizia et al. 2003). In personal care products, it was detected in approximately 97% of examined deodorants that are sold on the European consumer market and in 61% of the domestic and occupational products (Rastogi et al. 2001). Linalool is associated with causing fragrance allergies and eczema (de Groot and Frosch, 1997; Rastogi et al. 2001). Results of skin irritation studies in humans reported a mild irritational skin reaction with linalool concentrations ranging from 0.4% to 32% (in petrolatum), while other skin sensitization studies that used the maximization test reported concentrations between 8 and 20% (Letizia et al. 2003). Skin sensitization by fragrances that result in fragrance contact allergies are a significant clinical problem and is probably the most common cause of allergic contact dermatitis due to cosmetics ( Jappe et al. 2005; Larsen et al. 1996, 2001). Fragrance compounds such as monoterpenes, which are highly susceptible to air oxidation (autooxidation) owing to their unsaturated character, promote skin sensitization (Christensson et al. 2006; Karlberg et al. 1992, 1994, 1999; Matura et al. 2005; Skold et al. 2002). The auto-oxidized products often have electrophilic properties and react easily, therefore, with nucleophilic sites of skin proteins (Bezard et al. 1997; Nilsson et al. 2005). The unsaturated carbon double bonds (-carbon bonds) in the linalool molecule are susceptible to oxidation by air oxygen, which results in the formation of (reactive) oxidation products. Oxidation products with electrophilic properties such as hydroperoxides (7-hydroperoxy-3,7-dimethyloxta-1,5-diene-3-ol) as well as other oxidized linalool products such as alcohols, ethers and aldehydes have been identified in air-exposed linalool (Skold et al. 2002, 2006). Some of them, including the aforementioned hydroperoxide, act as weak skin sensitizer agents causing allergic skin reactions with individuals who are or were in the past exposed to linalool-containing products (Basketter et al. 2002; Matura et al. 2005; Skold et al. 2002, 2004). The allergenic potential of the linalool oxidation products was previously confirmed by a study which showed that after removal of the auto-oxidation products from oxidized linalool the allergy-causing potency of the remaining linalool decreased fairly (Basketter et al. 2002). Personal care products are mainly applied onto the skin so that ingredients can penetrate the skin and may be adsorbed by the body. Elimination of ingredients such as linalool from the body occurs by metabolism involving cytochrome P450 enzymes (CYP; Baron and Merk, 2001). Oxidation by CYP sometimes results in more reactive products such as epoxides (Guengerich, 2003). Epoxides are able to react with nucleophilic sites of skin proteins to form haptens, which may result in allergic reactions (Bergstrom et al. 2006). We reported recently, that the enzymatic product 3-carene-epoxide was the result of such a P450-mediated bio-activation reaction of the monoterpene 3-carene (Duisken et al. 2005a). Bergstrom et al. (2006) showed for conjugated dienes that their sensitizing capacity was caused by the metabolically formed biologically active allylic epoxides. Linalool oxidation products with electrophilic properties such as epoxides orginating from P450-mediated oxidative metabolism may be an important indication that skin sensitization reactions are caused not only by auto-oxidation products but also by the metabolism of linalool in skin tissue. Therefore, the aim of our study was to obtain an answer to the hypothesis that oxidative CYP-mediated metabolism of linalool results also in the formation

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of electrophilic compounds similar to the ones identified previously as auto-oxidation products. The CYP-mediated oxidation by CYP2D6- and CYP2C19 enzymes, which next to other CYP are expressed in skin (Ahmad et al. 1996; Yengi et al. 2003) and belong to the five CYP enzymes responsible for approximately 99% of P450-mediated drug metabolism (Bertz and Granneman, 1997), were used for the study.

Materials and methods Chemicals and enzymes All chemicals used in this study were of analytical grade quality or for biochemical use, unless specified otherwise. Ethanol (100%), linalool (97%, (R/S)-3,7-dimethyl-1, 6-octadien-3-ol; Figure 1 (1a, b)), disodium hydrogen phosphate dihydrate (Na2HPO4 2H2O) and potassium dihydrogen phosphate trihydrate (KH2PO4 3H2O) were purchased from Sigma Aldrich (Taufkirchen, Germany). Glucose-6-phosphate potassium salt (G6P), the enzyme glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and nicotinamide adenine dinucleotide phosphate sodium salt (NADP, 98%) were purchased from Roche Diagnostics (Basel, Switzerland); ethyl acetate (EtOAc) was of residue analysis quality and was purchased from LGC Promochem (Wesel, Germany). Purified water produced by a Milli-Q water purification system (Millipore, Eschborn, Germany) was used for the preparation of phosphate buffers in enzymatic assays. The reference substance furanoid-linalool oxide (97%, (R/S)-2-(1,1-dimethylethyl)-5-methyl-5vinyltetrahydrofuran; Figure 1 (2ad)) was purchased from Fluka (Buchs, Switzerland) and

OH

OH

1a (S)

1b (R)

2a (2R,5R)

2b (2S,5R)

2c (2R,5S)

2d (2S,5R )

OH

OH

3a (S)
HO O HO

3b (R)
HO O O HO O

4a (3R,6R)
OH OH OH

4b (3S,6R)

4c (3R,6S)

4d (3S,6S)

OH

OH HO

OH HO

5a (trans)

5b (trans)

5c (cis)

5d (cis)

Figure 1. Structural formula of (R/S)-linalool (1a, b), (R/S)-furanoid-linalool oxide (2ad), (R/S)-dihydrolinalool (3a, b) and (R/S)-pyranoid-linalool oxide (4ad) and (cis/trans-8-hydroxylinalool (5ad).

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pyranoid-linalool oxide (97%, (R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol; Figure 1 (4ad)) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).

Recombinant human cytochrome P450 enzymes and NADPH generating system Recombinant human CYP (EC 1.14.14.1), CYP2D6 and CYP2C19 (amount 1 nmol) with co-expressed CYP-reductase in Escherichia coli (bactosomes) were obtained from Cypex (Dundee, Scotland) and stored at a temperature of 80 C until usage. The combination of the enzyme G6PDH (1.5 U ml1), NADP (0.5 mM) and G6P (4.7 mM) dissolved in phosphate buffer ( pH 7.2) functioned as NADPH-generation system in all enzymatic assays. The NADPH-generation system solution was always freshly prepared.

Identification of enzymatic products by GC/MS analysis A Hewlett-Packard gas chromatograph (GC) model 5860 Series II (Waldbronn, Germany) equipped with a programmable temperature vaporizer (PTV ) and an MPS large volume sampler (CIS 3) all from Gerstel (Mu hlheim a. d. Ruhr, Germany) were directly connected by a heated transfer line to a Hewlett-Packard 5972 mass spectrometer (MS). Samples were separated on an RTX-5SIL MS (Restek, Bad Homburg, Germany) fused silica capillary column (0.28 mm 30 m, 0.25 mm film thickness) using helium (linear gas velocity of 0.7 ml/min) as the carrier gas. The GC temperature program conditions were: initial oven temperature 37 C, heating to 200 C by a temperature ramp of 6 C min1 followed by another temperature ramp of 15 C min1 heating up to a temperature of 330 C. From the sample extracts 40 ml was injected (injection speed: 29 ml min1) into the liner (93 1 mm I.D., Gerstel, Mu hlheim a. d. Ruhr, Germany) of the PTV stuffed with deactivated silanized glass wool. The PTV operated in the solvent vent stop flow mode using a vent flow of 200 ml min1 helium gas. Purging of the samples organic solvent started directly after sample injection (injector temperature of 20 C) and lasted 0.5 min while the GC temperature program was running. The PTV injector temperature was held for 2 min at a temperature of 20 C, followed by an increase of the injector temperature (split less mode) up to 300 C by a temperature ramp of 600 C min1. The GC/MS transfer line was set at a temperature of 300 C, this resulted in an ion source and quadrupole temperature of 180 C. The electron impact (EI) ionization voltage was set to 70 eV and positive charged ions were analyzed in full scan mode applying a scan range of m/z 30300. Quantification of metabolite concentrations was performed by external calibration for the metabolites furanoid-and-pyranoid-linalool oxide. 8-Hydroxylinalool was quantified using linalool as surrogate reference substance.

P450-mediated enzymatic assay Phosphate buffer ( pH 7.2) was prepared by mixing 62.1 ml of a Na2HPO4 (0.05 M) solution with 39.8 ml of a KH2PO4 (0.02 M) solution. CYP-mediated enzymatic assays of the substrate linalool using CYP2D6 and CYP2C19 enzymes were carried out as follows. A substrate stock solution was prepared by dissolving 10 ml of linalool with phosphate buffer containing 10% ethanol (100%). The enzymatic biotransformation assays were carried out in 1.5-ml safe-lock micro-tubes from Eppendorf (Hamburg, Germany). The CYP

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suspension was divided by diluting 100 ml of the homogenized enzyme suspension with 1000 ml of the phosphate buffer and stored at a temperature of 80 C until use, for every enzymatic assay one tube with CYP was used. The metabolism and formation of enzymatic products of linalool by selected CYP was studied using time-, enzyme- and substrate-dependent enzymatic assays. The time-dependent formation of the enzymatic products was studied by mixing 200 ml of the phosphate buffer solution with 200 ml of the NADP/G6P solution and 200 ml of the G6PDH solution. Thereafter 20 ml of the linalool stock solution (0.215 mmol ml1) were added and 100 mL of the recombinant human CYP2D6 (74 pmol ml1) or CYP2C19 (110 pmol ml1), respectively. The safe-lock micro-tubes were vortexed and incubated and vigorously shaken at a temperature of 37 C using an Eppendorf Thermomixer (Hamburg, Germany). This procedure was carried out with various incubation times (e.g. 5, 10, 15, 20, 30 and 40 min), each in a separate safe-lock micro-tube. Formation of enzymatic products depending on CYP enzyme concentrations was identically carried out with two different CYP concentrations, namely CYP2D6, 74 and 148 pmol ml1 and CYP2C19, 110 and 220 pmol ml1, respectively. Substrate dependency of the formation of the enzymatic products was studied with enzyme concentrations as applied in the time-dependent enzymatic assay with 4, 6, 8, 10, 12, 14 and 20 ml of a diluted linalool stock solution (0.0215 mmol ml1) as substrate. Incubation time of the substrate-dependent enzymatic assay was 40 min. With each type of enzymatic assay two separate control incubations were carried out: one control consisted of incubation with CYP in combination with the NADPH regeneration system but without substrate addition; the second control consisted of incubation with substrate in combination with the NADPH regeneration system but without addition of CYP. Enzymatic reactions were stopped by denaturation of the CYP by the addition of 1 ml of EtOAc to the enzymatic assay and mixing vigorously for 1 min using a vortex mixer. The metabolites were extracted by the previously added EtOAc by mixing the safe-lock micro-tubes for an additional 30 min. The EtOAc layer was separated by centrifugation of the safe-lock micro tubes 4000 rpm for 15 min (Eppendorf centrifuge, Hamburg, Germany). The EtOAc with extracted enzymatic products was transferred into brown-colored glass GC septum vials and stored at 4 C until GC/MS analysis.

Results Identification of enzymatic products of linalool catalyzed by P450s A typical enzymatic conversion of the substrate linalool (4.3 nmol) by one of the selected enzymes (CYP2D6, 74 pmol ml1) is illustrated by the GC-MS chromatogram in Figure 2. GC-MS analysis of the two different control incubations showed that no additional substances such as auto-oxidation products of linalool or impurities of the enzyme suspension were formed during incubation experiments. Identification of the enzymatic products was carried out in two different ways: (i) comparison of mass spectra and retention times of the enzymatic products with reference substances and, if reference substances were not available, (ii) the use of mass spectral match factors (MFs) automatically calculated by the used library software (NIST 98 library; Hennig et al. 1994), as well as comparison of Kovacs indices. A structural identification of the enzymatic product was considered to be accurate if the probability (MF) of the unknown enzymatic product as being the proposed identified component by the library database

P450-mediated oxidative metabolism of linalool

(a) 750 650 550 450 350 250 150 50 0 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Retention time [min] (b) 750 650 550 450 14.00 350 250 150 50 0 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Retention time [min] 16.00 3 4 { 2 { 3 5 TIC * 10E3 1 TIC *10E3 1

609

Figure 2. (a) GC/MS total ion current (TIC) chromatogram of the substrate (R/S)-linalool after incubation for 40 min without CYP, (R/S)-linalool (1), (R/S)-dihydro-linalool (3); (b) GC/MS total ion current (TIC) chromatogram of the substrate (R/S)-linalool after incubation for 40 min with CYP2D6 (74 pmol ml1), (R/S)-linalool (1), (R/S)-furanoid-linalool oxide (2), (R/S)-dihydro-linalool (3), (R/S)-pyranoid-linalool oxide (4) and (cis/trans)-8-hydroxylinalool (5); GC/MS conditions see Materials and methods.

was 490% (Table I), main fragments could be explained and the calculated Kovacs indices showed good agreement with literature values. The enzymatic conversion of linalool catalyzed by both CYP resulted in the formation and identification of three different enzymatic products (#2, 4, 5). The substance ( peak #3) was tentatively identified as dihydrolinalool ((R/S)-3, 7-dimethyl-6-octene-3-ol; Figure 1 (3a, b))

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Table I. Calculated match factors (MFs) by the NIST GC/MS library. Match factor (MF) (%) (Enzymatic) product Furanoid-linalool oxidea (2) Dihydrolinalool (3) Pyranoid-linalool oxidea (4) 8-Hydroxylinalool (5)
a

P450 2C19 82.8 79.2 78.7 95.5

P450 2D6 86.8 76.9 77.8 94.1

Confirmed also by reference substance.

by the GC/MS mass spectra library with a low reliability (Table I). The substance was also detected in the linalool control incubations and could be thus excluded as being a P450-mediated enzymatic product of linalool, but was an impurity of the linalool. The two small chromatographic peaks (#2) were identified by the GC/MS library as (R/S)-furanoidlinalool oxide with an MP of 470%, whereby the first peak (Rt 11.5 min) of the couple was identified as cis-isomers (Figure 1 (2a, b)) and the second peak (Rt 12.0 min) as being the trans-isomers (Figure 1 (2c, d)). A commercially available (R/S)-furanoid-linalool oxide reference substance confirmed the identity of both these enzymatic products by retention times. According to this, the two peaks (#4) were identified by the GC-MS library comparison also with a high MF (Table I) and with the reference substance as (R/S)-pyranoid-linalool oxide. The first peak (Rt 14.8 min) of the couple was most likely the cis-isomer (Figure 1 (4a, b)) while the trans-isomer (Figure 1 (4c, d)) had a retention time of 15.0 min. The enzymatic products ( peak #5) were identified as (cis/trans)-8-hydroxylinalool by the GC/MS library with match factors of 94 or 95%, respectively. Owing to the fact that a (cis/trans)-8-hydroxylinalool-reference substance was not available, further confirmation was carried out by calculation of Kovacs indices (KI) and interpretation of the EI fragmentation pattern. Calculated Kovacs index of (cis)-8-hydroxylinalool (Figure 1 (5c, d), Rt 18.5 min) was KI 1302 and of the isomer (trans)-8-hydroxylinalool (Figure 1 (5a, b), Rt 19.0 min) was KI 1322. Both calculated RI indices varied only approximately 2.5% from the KI values reported by Chassagne et al. (1999), indicating high consistence. The EI-mass spectrum of (cis/trans)-8-hydroxylinalool is presented in Figure 3; and typical fragment ions resulting from the EI-ionization process and their elucidation and fragmentation reactions are presented in Table II. The intensity of the molecular peak at m/z 170 (M) was very low (abundance 2%), which is very common for secondary and primary alcohols. Some of the fragment ions are very common for EI-induced fragmentation of alcohols. Loss of H2O (dehydration) is an example of such a typical fragmentation. Dehydration of (cis/trans)-8-hydroxylinalool and of other main fragments was observed in the mass spectrum several times. Accordingly, the enzymatic conversion of (R/S)-linalool by CYP2C19 was studied (chromatograms not shown). Only 8-hydroxylinalool was detected as metabolites, both cyclic ethers were not found.

P450-mediated enzymatic assays The relationship between enzymatic product formations catalyzed by both CYP was studied as described before on incubation time, enzyme concentration and substrate

P450-mediated oxidative metabolism of linalool

100

611

90 71 80 m/z=43 70 H 60 Abundance [10E3] m/z=18 50 m/z=71 40 m/z=18 HO OH H H

30 53 20

84 119 110 137

97

10 152 40 60 80 100 120 140 160 180 m/z 200

Figure 3. EI-mass spectrum and proposed fragmentation of (cis/trans)-8-hydroxylinalool.

Table II.

Fragment ions used for identification of the enzymatic product (cis/trans)-8-hydroxylinalool. Description Neutral loss Loss of m/z 15 from m/z 152 Neutral loss of m/z 18 from m/z 137 Dissociation of fragment ion m/z 137 Dissociation of alkyl group Neutral loss of m/z 18 from m/z 71 Postulated dissociation reaction/fragment ion MH2O CH 3 H2O C4 H 5 MC6H11O H2O

Fragment ion m/z 152 137 119 84 71 53

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concentration dependency. The time dependency of the enzymatic product formation by enzymatic reactions catalyzed by both CYP was linear for all identified enzymatic products. Using incubation times from 0 to 40 min, calculated regression coefficients showed a mean r2 of 0.95 with SD 0.04. Increase of enzyme concentrations by a factor of 2 resulting in CYP concentrations of 148 pmol ml1 for CYP2D6 and 220 pmol ml1 for CYP2C19, respectively, led also to an increase of the enzymatic activity by a factor of 2 (mean r2 0.992, SD 0.01). Linalool concentration dependency of the enzymatic products formation was studied by the use of LineweaverBurk analysis and was linear for all identified enzymatic products (mean r2 0.92, SD 0.09). Calculated specific enzymatic properties of the CYP enzymes are presented in Table III.

Discussion The knowledge that CYP-mediated metabolism occurs in skin tissue resulted in the hypothesis that reactive oxidation products that cause skin sensitization may not only result from air auto-oxidation but also from enzymatic origin. To prove this hypothesis we studied the CYP-mediated oxidative metabolism of linalool and tried to identify biological reactive enzymatic products equal or different to reactive auto-oxidation products as reported in the past. Results from enzymatic assays showed that enzymatic reactions catalyzed by CYP2C19 and CYP2D6, two CYP identified in skin tissue (Ahmad et al. 1996; Yengi et al. 2003), resulted in the formation of several different enzymatic products. The enzymatic product, cis- and trans-8-hydroxylinalool, could be identified by the GC-MS library and fragmentation pattern and was confirmed by comparison of calculated Kovacs index with literature data. The enzymatic products are probably the result of the allylic-hydroxylation substitution reaction of one of the methyl group situated at the 8-carbon atom of the linalool molecule. Similar to our results Letizia et al. (2003) identified 8-hydroxylinalool as an enzymatic product of linalool using mammalian CYP prepared from rat livers and lung. Comparing the hydroxylation activity of both CYP, CYP2C19 had a higher enzymatic affinity to linalool than CYP2D6, but the catalytic efficiency (Kcat/Km) was 25% less than for CYP2D6 (Table III). Allylic-hydroxylation reaction of substrates containing (conjugated)--bonded carbon atoms resulting in allylic alcohols has been reported as a common P450-mediated catalyzed enzymatic reaction (Bylund et al. 1998; Wrighton et al. 1990). Accordingly, allylic alcohols have been identified as enzymatic products from other monoterpenes such as limonene (Miyazawa et al. 1998), 1,8-cineole (Duisken et al. 2005b; Miyazawa and Shindo, 2001; Miyazawa et al. 2001) and 3-carene (Duisken et al. 2005a). Catalyzed hydroxylation activities for the enzymatic conversion of limonene by CYP2C19 for the enzymatic product carveol (Km 0.46 mM) and perillyl alcohol (Km 0.26 mM) were in approximately the same range as for the catalyzed 80 -allylic hydroxylation reaction of linalool (Table III). The hydroxylation of 1,8-cineol by other CYP such as CYP3A4 and CYP3A5 showed significantly lower Km values (between 19 mM and 141 mM; Duisken et al. 2005b; Miyazawa and Shindo, 2001). The other enzymatic products of CYP2D6 were identified by the use of reference substances as (R/S)-furanoid-linalool oxide and (R/S)-pyranoid-linalool oxide. Both were reported previously in a study concerning the auto-oxidation of linalool and the allergic

Table III. Michaelis-Menten kinetic values of the 6,70 -epoxidation and 80 allylic-hydroxylation shown in Figure 4 (F furanoid, P pyranoid enzymatic product). max mmol min1 mmol1 CYP Epox. Hydrox. Epox. Kcat s1 Hydrox Kcat/Km s1 mmol1 Epox. Hydrox

Km mM

P450

Epox.

Hydrox.

P450-mediated oxidative metabolism of linalool

2C19 2D6

6,70 -F 3.9

6,70 -P 11.7

80 0.14 1.3

6,70 -F 0.13

6,70 -P 0.35

80 11.7 144.9

6,70 -F 5.6 101

6,70 -P 1.5 102

80 5.1 103 6.3 104

6,70 -F 1.4 101

6,70 -P 1.3 101

80 3.6 104 4.8 104

613

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OH

OH

(cis/trans)- 8-Hydroxylinalool

8-Allylic-hydroxylation (P450 2C19 and P450 2D6)

OH

(R/S)-Linalool

6,7-Epoxidation (P450 2D6)

O OH

6,7-Epoxy-linalool

HO O

HO O

(R/S)-pyranoid-linalooloxide

(R/S)-furanoid-linalooloxide

Figure 4. Scheme of postulated enzymatic reactions of the substrate (R/S)-linalool by CYP2C19 and CYP2D6 enzymes followed by intramolecular rearrangement.

properties of identified auto-oxidation products (Skold et al. 2004). In this study the formation of (R/S)-furanoid-linalool oxide and (R/S)-pyranoid-linalool was explained by the formation of a tertiary hydroperoxide (7-hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol), which can rearrange to produce an epoxide as a secondary oxidation product. This epoxide is then readily attacked by the hydroxyl group of linalool, on either one of the two epoxidecarbons (60 and 70 carbon), which then results in the formation of two different cyclic ethers. Both cyclic ethers were also identified in our study, but as the result of an enzymatic reaction catalyzed by CYP2D6. Therefore, we postulate that the CYP2D6 first catalyzes the enzymatic epoxidation of the 6,70 -carbon double bond in the linalool molecule, which is then followed by the intramolecular rearrangement leading to the two cyclic ethers (Figure 4). We speculate that the rearrangement reaction is not a CYP2D6-catalyzed reaction since, like Skold et al. (2004), we found a higher amount for the furanoid derivative (0.3 nmol nmol-1 CYP2D6) than for the pyranoid derivative (0.04 nmol nmol1 CYP2D6). The precursor 6,7-epoxylinalool and the furanoid and pyranoid ethers were detected as natural products in fruits by Winterhalter et al. (1986). Interestingly, biodegradation of linalool by different Aspergillus niger fungus strains yielded also the furanoid and pyranoid linalool oxides (Demyttenaere et al. 2001) showing that this biological transformation of linalool seems to be general in different species.

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Enzymatic epoxidation reactions of substrates catalyzed by CYP are common for substrates with -bonded carbons in the molecule, for example aromatic terpenes and olefins (Martinez and Stewart 2000). Recently, Nilsson et al. (2005) described the formation of epoxides from the diene 5-isopropenyl-2-methyl-1-methylene-2-cyclohexene by human liver microsomes; and Duisken et al. (2005a) identified 3-carene-epoxide as an enzymatic product of 3-carene by CYP1A2. Epoxidation of linalool by recombinant human CYP has, to our knowledge, not been reported until now. Kinetic analysis of the ether formation needs to be carefully interpreted because Km and max values include the epoxidation as well as the following cyclization reactions. The kinetic values characterize the biological epoxidation only if epoxidation is the rate-limiting reaction step. The enzymatic affinity of the CYP2D6 for the enzymatic epoxidation of the 6,70 -carbon double bound of linalool was approximately a factor 310 lower then the enzymatic affinity for the catalyzed 80 -carbon allylic hydroxylation (Table III). The catalytic efficiencies for the formation of the furanoid and pyranoid enzymatic products were approximatley in the same range but about a factor 30004000 lower than for the 80 -allylic hydroxylation. Results from the enzymatic assays indicate that there is no mechanism-based inactivation of the CYP by the one of the enzymatic products as reported for other olefins by Murray and Reidy (1990). Regression analysis between enzyme product concentration in time and enzyme concentration and substrate concentrations obtained for both CYP regression lines with regression coefficients r240.90.

Conclusion The results of our study confirm our hypothesis that reactive oxidation products of linalool such as epoxides may also be formed by CYP catalyzed biological transformation. This is in accordance with other recently published studies (Bergstrom et al. 2006; Duisken et al. 2005a; Nilsson et al. 2005). The formation of the identified cyclic ethers seems to be possible by two different reactions: (i) an enzymatic conversion by oxidative CYP enzymes followed by a rearrangement; and (ii) an auto-oxidation in the presence of air forming a hydroperoxide, followed by the formation of an epoxide as a secondary oxidation product, which is again rearranged to the cyclic ethers. The intermediary formed electrophilic epoxide may cause sensitization and irritational skin reactions by linalool-containing consumer products similar to other previously described auto-oxidation products such as hydroperoxides (Skold et al. 2004). As a result, the use of preservatives against oxidation of the cosmetic ingredients may prohibit air oxidation to a certain extent, but the formation of epoxides by CYP located in skin is still possible. Although in this in vitro system the biological oxidation mainly proceeds via the non-epoxide route, a more pronounced 6,7-epoxide route in vivo cannot be excluded.

Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We thank our project partner Brunhilde Blo meke of the University of Trier for helpful discussions as well as Wolfgang Dott of the RWTH Aachen for his continuous support.

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R. J. W . Meesters et al.

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