Photodecomposition of Retinyl Palmitate in Ethanol by UVA Light

Chem. Res. Toxicol. 2005, 18, 129-138

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Photodecomposition of Retinyl Palmitate in Ethanol by UVA LightsFormation of Photodecomposition Products, Reactive Oxygen Species, and Lipid Peroxides† Shu-Hui Cherng,‡,§ Qingsu Xia,‡ Lonnie R. Blankenship,‡ James P. Freeman,‡ Wayne G. Wamer,⊥ Paul C. Howard,‡ and Peter P. Fu*,‡ National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079, and Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740 Received July 20, 2004

Photodecomposition of retinyl palmitate (RP), an ester and the storage form of vitamin A (retinol), in ethanol under UVA light irradiation was studied. The resulting photodecomposition products were separated by reversed-phase HPLC and identified by spectral analysis and comparison with the chromatographic and spectral properties of synthetically prepared standards. The identified products include 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxyRP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), palmitic acid, ethyl palmitate, and four tentatively assigned cis and trans isomeric 15-ethoxy-ARs. AR was formed as a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Z-cis-AR with all-trans-AR predominating. 5,6-EpoxyRP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP were also formed from reaction of RP with alkylperoxy radicals generated by thermal decomposition of 2,2′-azobis(2,4-dimethylvaleronitrile). Formation of these photodecomposition products was inhibited in the presence of sodium azide (NaN3), a free radical inhibitor. These results suggest that formation of 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxyRP from photoirradiation of RP is mediated by a light-initiated free radical chain reaction. AR and the isomeric 11-ethoxy-ARs were not formed from reaction of RP with alkylperoxy radicals generated from 2,2′-azobis(2,4-dimethylvaleronitrile), and their formation was not inhibited when NaN3 was present during the photoirradiation of RP. We propose that these products were formed through an ionic photodissociation mechanism, which is similar to the reported formation of AR through ionic photodissociation of retinyl acetate. RP and all its identified photodecomposition products described above (i) were not mutagenic in Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 in the presence and absence of S9 activation enzymes, (ii) were not photomutagenic in Salmonella typhimurium TA102 upon UVA irradiation, and (iii) did not bind with calf thymus DNA in the presence of microsomal metabolizing enzymes. These results suggest that RP and its decomposition products are not genotoxic; however, photoirradiation of RP, 5,6-epoxy-RP, and AR with UVA light in the presence of methyl linoleate resulted in lipid peroxide (methyl linoleate hydroperoxides) formation. The lipid peroxide formation was inhibited by dithiothreitol (DTT) (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), and superoxide dismutase (SOD) (superoxide scavenger) but was enhanced by the presence of deuterium oxide (D2O) (enhancement of singlet oxygen lifetime). These results suggest that photoirradiation of RP, 5,6-epoxyRP, and AR by UVA light generated reactive oxygen species resulting in lipid (methyl linoleate) peroxidation.

Introduction Sunlight is a complete carcinogen and has been determined to be responsible for the induction of skin cancer in humans (1-4). Sunlight consists of a continuum of frequencies of light that contain infrared (above 800 nm), † This article is not an official guidance or policy statement of U. S. Food and Drug Administration (FDA) or National Toxicology Program (NTP). No official support or endorsement by the U. S. FDA and NTP is intended or should be inferred. * Corresponding author. Tel: 870-543-7207. Fax: 870-543-7136. E-mail: [email protected]. ‡ National Center for Toxicological Research. § Current address: Department of Food Science and Nutrition, Hung Kuang University, Sha-lu, Taichung, Taiwan, ROC. ⊥ Center for Food Safety and Applied Nutrition.

visible (between 400 and 800 nm), UVA (between 315 and 400 nm), UVB (between 280 and 315 nm), and UVC (between 200 and 280 nm) regions. Stratospheric ozone absorbs light of wavelengths below 295 nm, preventing these wavelengths from reaching Earth. Skin is the largest body organ and is constantly exposed to sunlight, environmental toxic chemicals, cosmetics, and body-care products (1, 2). The effects of photoirradiation of skin are dependent on the quantity and wavelength of the radiation (1), UV light being the critical component of sunlight that causes human skin toxicity, including skin cancer formation (1, 2). Thus, understanding the mechanisms by which UV irradiation causes human skin cancer is important in development

10.1021/tx049807l CCC: $30.25 © 2005 American Chemical Society Published on Web 01/04/2005

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of preventive strategies for minimizing damage induced by sunlight (2, 4-7). Vitamin A (retinol), a natural retinoid, is required for a vast number of biological processes, including regulation of skin epidermal cell growth, normal cell differentiation, and cell maintenance (8, 9). Retinyl palmitate (RP), an ester, is the storage form of vitamin A (retinol). RP is thermally more stable than retinol and frequently used in cosmetic products (10). The number of cosmetic retail products containing RP has increased rapidly in the last 2 decades, with more than 660 RPcontaining cosmetic products on the U. S. market in 2000 (11). Retail products containing RP include moisturizing preparations, skin care preparations, night skin care preparations, lipsticks, suntan gels and preparations, makeup preparations, and bath soaps and detergents (12). While people using these cosmetic products are unavoidably exposed to sunlight, it is not known whether use of RP-containing cosmetics with concomitant exposure to sunlight results in any deleterious effects (13). RP appreciably penetrates the skin in vitro and in vivo (14, 15). Boehnlein et al. reported that after RP was topically applied in acetone to human skin in vitro, about 18% of RP penetrated the skin in 30 h (14). It should be noted, however, that at the end of the study only 0.2% had penetrated through the skin and would be systemically available. As a result, the topical application of RP to the skin of humans would result in significant levels of RP both inside and on top of the skin that would be available for exposure to sunlight. Although it has been shown that RP is more photochemically labile than the parent retinol (16), the photodecomposition products of RP following irradiation with UVA, UVB, or solar light have not yet been clearly identified. We hypothesize that since RP has a maximum UV-visible absorption at 326 nm (9), it would absorb UVA light and form photodecomposition products. Also, RP, in its excited state, may transfer energy to molecular oxygen, leading to the formation of reactive oxygen species (ROS). In this paper, we address these hypotheses describing the photodecomposition of RP by UVA light, identifying photodecomposition products, and determining the mechanisms of photodecomposition.

Materials and Methods Materials. RP, retinyl acetate, retinol, retinal, palmitic acid, ethyl palmitate, m-chloroperbenzoic acid, MnO2, 1,1′-azobis(cyclohexane carbonitrile), sodium azide (NaN3), DTT, SOD, and 8-methoxypsoralen (8-MOP) were purchased from the Sigma Chemical Co. (St. Louis, MO). Cloned T4 polynucleotide kinase (PNK) was obtained from U. S. Biochemical Corp. (Cleveland, OH). [γ-32P]Adenosine 5′-triphosphate ([γ-32P]ATP) (sp. act. > 7000 Ci/mmol) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). All other reagents were obtained through commercial sources and were the highest quality available. All solvents used were HPLC grade. Anhydroretinol, a mixture of trans and cis isomers, was synthesized by reaction of all-trans-retinol in ethanol with anhydrous HCl according to the procedure published by Derguini et al. (17). 5,6-Epoxy-RP and 4-keto-RP were prepared by oxidation of RP in benzene with m-chloroperbenzoic acid at ambient temperature for 1 h (18).

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Figure 1. Reversed-phased HPLC profile of the photodecomposition products of RP (0.5% in ethanol) after irradiation with 14 J/cm2 of UVA light. HPLC analysis was conducted on a Prodigy 5 µm ODS column (4.6 mm × 250 mm) eluted isocratically with methylene chloride in methanol (1/9, v/v) at 1 mL/ min.

Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 were kindly provided by Dr. Bruce Ames from the University of California (Berkeley, CA). Light Source. The UVA light box was custom-made with a four-lamp unit using UVA lamps (National Biologics). The irradiance of light was determined using an Optronics OL754 Spectroradiometer (Optronics Laboratories, Orlando, FL), and the light dose was routinely measured using a Solar Light PMA-2110 UVA detector (Solar Light Inc., Philadelphia, PA). The maximum emission of the UVA is between 340 and 355 nm. The light intensities at wavelengths below 320 nm (UVB light) and above 400 nm (visible light) are about 2 orders of magnitude lower than the maximum at 340-355 nm. Photoirradiation of RP. A solution (2-3 mL) of 0.5% RP dissolved in ethanol was placed in a UV-transparent cuvette and photoirradiated under UVA light for a period of time to receive a light dose of 14 J/cm2. The reaction mixture was then concentrated to about 200 µL under reduced pressure. Reversed-phase HPLC separation of the resulting photodecomposition products was accomplished using a Prodigy 5 µm ODS column (4.6 mm × 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 10% methylene chloride in methanol (v/v) at 1 mL/ min. Inhibition of Photodecomposition of RP by Sodium Azide (NaN3). To determine whether any of the photodecomposition products are formed through a free radical mechanism, 0.5% RP in ethanol was photoirradiated with UVA light in the presence of 1 mM NaN3, a free radical scavenger (19, 20). Irradiation conditions were similar to those described above. The resulting photodecomposition products were analyzed by reversedphased HPLC as described above. Free Radical Reaction of RP. Following the procedure of Yamauchi et al. for the reaction of the peroxylradical with retinyl acetate (21), a free radical reaction with RP (50 mg, 95 µmol) in 10 mL ethanol was initiated by generation of an alkylperoxyl radical through thermal decomposition of 1,1′-azobis(cyclohexane carbonitrile) (100 mg, 410 µmol). After reaction in the dark at 37 °C for 6 h, the resulting reaction products were analyzed by reversed HPLC. 32 P-Postlabeling/HPLC and 32P-Postlabeling/TLC Analyses. Binding of RP and its photodecomposition

Photodecomposition of Retinyl Palmitate in Ethanol

products to DNA, leading to DNA adduct formation, was investigated. RP or its photodecomposition products were incubated with calf thymus DNA. DNA was then isolated, and DNA adducts were analyzed by 32P-postlabeling/ HPLC and 32P-postlabeling/TLC following previously published procedures (22-24). Mutagenicity Assays. Mutagenicity assays of RP and its photodecomposition products were conducted in the presence and absence of S9 enzyme using S. typhimurium TA98, TA100, TA102, and TA104 as described by Maron and Ames (25). Triplicate plates were used for each test. Photomutagenicity of RP, 5,6-Epoxy-RP, and AR in Salmonella typhimurium TA102. The assays were conducted in Salmonella typhimurium histidine auxotrophic strain TA102 concomitant with irradiation by light in the presence of RP, 5,6-epoxy-RP, or AR following the method of Wang et al. (26). The dose of UV light was restricted to the level that induced mutations that were less than 2-fold of the control and that maintained the bacterial viability higher than 50% in the absence of any test compound as suggested by Utesch and Splittgerber (27). In our case, a UVA dose of 170 mJ/cm2 was used. 8-MOP (10 µg/plate) irradiated with UVA at a dose of 170 mJ/cm2 was used as a positive control, and the number of revertant bacteria colonies for TA102 increased steadily as the light dose increased before reaching a maximum number of about 6 times the revertant colonies of the spontaneous mutation. This result is comparable to literature values (28-30). The variability in assays conducted in triplicate was generally less than (20%. A negative solvent control and an 8-MOP positive control were used in all experiments. The average numbers for revertant colonies of TA102 per plate were 371 ( 19 for solvent control (no light) and 529 ( 49 (with light). Peroxidation of Methyl Linoleate Initiated by Photoirradiation of RP, 5,6-Epoxy-RP, and AR. Experiments were conducted with a solution of 100 mM methyl linoleate and either 1.0 mM RP, 5,6-epoxy-RP, or AR in ethanol. Samples were placed in a UV-transparent cuvette and irradiated with 0, 7, 14, 21, 28, 35, 56, or 70 J/cm2 of UVA light. After irradiation, the levels of lipid peroxidation were measured either by calculation of the amount of methyl linoleate hydroperoxides from the HPLC peak area by monitoring the elution at 235 nm (31) or by colorimetric determination of the conversion of 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP) into methylene blue in the presence of hemoglobin (Lipid Peroxides kit, Kamiya Biomedical Company, Thousand Oaks, CA). Methyl linoleate hydroperoxides were separated by HPLC using a Prodigy 5 µm ODS column (4.6 mm × 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 10% water in methanol (v/v) at 1 mL/min. Effect of NaN3, DTT, SOD, and D2O on Peroxidation of Methyl Linoleate Initiated by Photoirradiation of RP. The effect on the levels of lipid peroxide formation induced from photoirradiation of RP with UVA light in the presence of DTT (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), SOD (superoxide scavenger), and deuterium oxide (D2O) (enhancement of singlet oxygen lifetime) was studied with experimental conditions similarly as described above. The concentration of NaN3, DTT, and SOD was 50 mM, and

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Figure 2. UV-visible absorption spectra of RP (- - -), 5,6epoxy-RP (‚‚‚), and 4-keto-RP (s).

the amount of deuterium oxide and water was 10% in ethanol. Statistical comparison between experimental groups was performed by general linear model (GLM) using the SAS software, version 8.0 (SAS Institute Inc., Cary, NC). P e 0.05 was considered to indicate a statistically significant difference. Instrumentation. A Waters HPLC system consisting of a model 600 controller, a model 996 photodiode array detector, and a pump was used for the separation and purification of photodecomposition products of RP. Direct exposure probe (DEP) mass spectrometry (MS) was performed on a ThermoFinnigan TSQ 700 triple quadrupole mass spectrometer operated in the electron ionization (EI) mode. The DEP current was ramped to 800 mA at 5 mA/s. Gas chromatography/mass spectrometry (GC/ MS) was performed on the same TSQ 700 triple quadrupole mass spectrometer operated in the electron ionization (EI) mode. Separation was achieved in a J&W DB5ms capillary column (30 m × 0.25 mm i.d. × 0.25 µm). HPLC/electrospray ionization-mass spectrometry (LC/ ESI-MS) was performed on a ThermoFinnigan TSQ 7000 triple quadrupole mass spectrometer operated in the negative ion and positive ion modes. Separation was achieved on a MetaChem Polaris C18 column (250 mm × 2.0 mm i.d. × 5 µm). The mobile phase was acetonitrile/ water with 0.1% formic acid. The acetonitrile gradient was increased from 5% to 95% over 30 min. The 1H nuclear magnetic resonance (NMR) experiments were carried out on a Bruker AM 400 MHz spectrometer (Bruker Instruments, Billerica, MA).

Results Photoirradiation of RP. Photoirradiation of RP in ethanol by UVA light at a level of 14 J/cm2 was conducted, and the reaction mixture was fractionated by reversed-phase HPLC (Figure 1). Based on comparison of the HPLC retention time, UV-absorption spectrum, and mass spectrum with those of RP, the material contained in the chromatographic peak that eluted at 28.5 min was identified as the recovered substrate, RP. The material in the chromatographic peak eluting at 16.4 min (Figure 1) had a UV-visible absorption spectrum with strong absorptions at 295, 310, and 324 nm (Figure 2). Mass spectral analysis of this photodecomposition product indicated that it has a molecular ion M+ at m/z 540 and fragment ions at m/z 302, 284, 241, 199, and 171, suggesting that this is a product with an oxygen atom added to the substrate RP (Figure 3A). The structure was confirmed as 5,6-epoxy-RP by analysis of its

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Figure 3. Mass spectra of the RP photodecomposition products eluted at 16.4 and 12.7 min (Figure 1) identified as (A) 5,6-epoxyRP and (B) 4-keto-RP, respectively.

proton NMR. The proton NMR assignments are as follows: 5,6-epoxy-RP (CD3CN) 0.90 (t, 3H, CH3 of the palmitate), 0.91 (s, 3H, CH3 at C5), 1.10 and 1.12 (2s, 6H, CH3 at C1), 1.30-1.65 (m, 2H, CH2 at C4), 1.391.46 (m, 2H, H2 at C2), 1.57-1.60 (t, 2H, CH2 at C3), 1.90 (s, 3H, CH3 at C9), 1.95-1.98 (m, 28H, CH2 of the palmitate), 2.17 (s, 3H, CH3 at C13), 2.30 (t, 3H, CH3 of the palmitate), 4.71 (t, 2H, H15), 5.64 (t, 1H, H14), 6.01 (d, 1H, H8), 6.17 (d, 1H, H10), 6.24 (d, 1H, H7), 6.35 (d, 1H, H12), and 6.71 ppm (d, 1H, H11); J7,8 ) 15.6, J10,11 ) 11.3, J11,12 ) 15.2, and J14,15 ) 7.2 Hz. The chemical shift of the methyl group at C5 (0.91 ppm) is different from that of the substrate RP (1.70 ppm, data not shown), which clearly indicates that the epoxy group is located at the C5 and C6 positions. Furthermore, the HPLC retention time, UV-visible absorption, mass, and 1H NMR spectra of the product are identical to those of the authentic 5,6-epoxy-RP prepared from epoxidation of RP with m-chloroperbenzoic acid (data not shown). Thus, the structure of this compound identified as 5,6-epoxy-RP was further confirmed. The material in the chromatographic peak eluted at 12.7 min in Figure 1 has a molecular ion at m/z 540 (Figure 3B). Its UV-visible absorption spectrum (Figure 2) had maximal absorptions at 335, 349, and 369 nm, respectively. The absorption at 369 nm is 42 nm hyperchromic compared with that of RP (at 327 nm) (Figure 2) suggesting that this photodecomposition product has a π-conjugate system more extensive than RP. This product has the HPLC retention time (Figure 1), UVvisible absorption spectrum (Figure 2), and mass (Figure 3B) spectrum identical to those of 4-keto-RP synthetically prepared by oxidation of RP with m-chloroperbenzoic acid. Thus, the compound eluted at 12.7 min was identified as 4-keto-RP. Mass spectral analysis of the chromatographic peaks eluted at 10.9 and 11.8 min (Figure 1) indicated that each of these peaks contained more than one compound. Further separation of the fraction eluting between 10.7 and 12.0 min by HPLC employing a different column and solvent system resulted in separation of five chromatographic peaks (data not shown). Three peaks had molecular ions at m/z 586 and characteristic fragment ions at m/z 571 (loss of a methyl group), m/z 540 (loss of a molecule of ethanol), and m/z 287 and 247 (Figure 4). The molecular ions at m/z 586 are 62 Da (an oxygen atom

plus a molecule of ethanol) higher than the substrate (RP) suggesting that these three photodecomposition products are formed from epoxidation of RP followed by addition of one molecule of ethanol (the solvent used for photoirradiation of RP). The structures of these photodecomposition products were elucidated by analysis of their mass fragment ions and comparison with those reported by Yamauchi et al. (21). Yamauchi et al. (21) reported that free radical reaction of retinyl acetate in methanol resulted in the formation of cis- and trans-9-methoxy-10-hydroxyretinyl acetate, cis- and trans-11-methoxy-12-hydroxyretinyl acetate, and cis- and trans-13-methoxy-14-hydroxyretinyl acetate. These isomeric methoxy-hydroxyretinyl acetates all show a characteristic mass fragment formed from R-cleavage of the methoxy group. 9-Methoxy-10-hydroxyretinyl acetate cleavage at the C9-C10 double bond results in the formation of characteristic fragment ions at m/z 207 (Figure 5). Similarly, characteristic fragment ions at m/z 233 and m/z 273 are formed from 11-methoxy-12-hydroxyretinyl acetate and 13-methoxy-14-hydroxyretinyl acetate through the cleavage of C11-C12 double bond and C13-C14 double bond, respectively (Figure 5). Mass spectral analysis of the photodecomposition products of RP eluted between 10.1 and 11.8 min (Figure 1) indicated that similar characteristic fragment ions were formed. They are the mass fragment ions at m/z 247 and m/z 287, which were formed from cleavage the C11-C12 and C13C14 double bonds of 11-ethoxy-12-hydroxy-RP and 13ethoxy-14-hydroxy-RP, respectively (Figure 4). Thus, we conclude that the chromatographic peaks eluted between 10.1 and 11.8 min (Figure 1) contain both 11-ethoxy-12hydroxy-RP and 13-ethoxy-14-hydroxy-RP and each consists of the trans and cis isomers. The eluents in this region (between 10.7 and 11.7 min) contained a component with its molecular ion at m/z 256. Since the mass spectral profile (data not shown) and the molecular ions are identical to those of palmitic acid, this photodecomposition product was identified as palmitic acid (Figure 1). The material contained in chromatographic peak eluted at 7.8 min (Figure 1) had a molecular ion at m/z 268 (data not shown). The mass spectral profile, UV-visible absorption spectrum, and HPLC retention time are identical to those of the synthetic standard anhydroretinol (AR) prepared from reaction of RP with hydrochloric



Figure 4. Mass spectra of the RP photodecomposition products eluted between 10.6 and 11.7 min of Figure 1 identified as transand cis-11-ethoxy-12-hydroxy-RP and trans- and cis-13-ethoxy-14-hydroxy-RP.

Figure 5. Structures of 9-methoxy-10-hydroxyretinyl acetate (top), 11-methoxy-12-hydroxyretinyl acetate, 13-methoxy-14hydroxyretinyl acetate, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14hydroxy-RP (bottom), and their characteristic mass spectral fragments.

Figure 6. Reversed-phased HPLC profile and UV-visible absorption spectra of RP photodecomposition products identified as all-trans-AR (AR) and cis-ARs (6Z-, 8Z-, and 12Z-AR). HPLC analysis was conducted on a Vydac C18 column (4.6 mm × 250 mm) eluted isocratically with water in methanol (14/86, v/v) at 1 mL/min.

acid (17). Upon further separation by HPLC using a Vydac C18 column, this AR was determined to be a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Zcis-AR with the all-trans-AR isomer as the predominant product (Figure 6). By comparison of their HPLC retention times, UV-visible absorption spectra (Figure 6 inserted), and mass spectra (data not shown) with those of the synthetic standards, the materials contained in chromatographic peaks eluted at 28.1, 33.2, 34.1, and 39.5 min of Figure 6 were identified as 8Z-AR, 12Z-AR, 6Z-AR, and all-trans-AR, respectively. The material contained in the chromatographic peak eluted at 6.8 min in Figure 1 had a mass spectrum with a molecular ion at m/z 314 (data not shown). Further HPLC separation of the collected material employing a Vydac C18 column eluted isocratically with 14% of water in methanol identified multiple components (data not shown). There are four major chromatographic peaks that

had nearly identical UV-visible absorption spectra (data not shown) and identical molecular ions at m/z 314 (Figure 7). These results suggest that these compounds are formed from addition of one molecule of ethanol to AR. Reddy and Rao (32) reported that direct excitation of retinyl acetate in methanol generated AR and retinyl methyl ether (15-methoxy-AR), both containing a trans and several cis isomers. Based on the similarity between this study and our photoirradiation of RP, we propose that the products contained in these four chromatographic peaks are isomeric 15-ethoxy-ARs with each tentatively assigned as 8Z-15-ethoxy-AR, 12Z-15-ethoxyAR, 6Z-15-ethoxy-AR, and all-trans-15-ethoxy-AR. Based on comparison of mass spectra (data not shown) with that of the commercially available ethyl palmitate, the material eluted at 6.85 min was identified as ethyl palmitate (Figure 1).

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Figure 7. Mass spectra of RP photodecomposition products identified as all-trans-15-ethoxy-AR and cis-15-ethoxy-ARs. The numbering assignments for 6Z-, 8Z-, and 12Z-ARs are arbitrary.

Photoirradiation of RP, 5,6-epoxy-RP, and AR in the Presence of Methyl Linoleate. Photoirradiation of RP with UVA light in the presence of methyl linoleate was studied to determine whether photoirradiation of RP can initiate lipid peroxidation. Photoirradiation of RP, methyl linoleate, and a mixture of methyl linoleate and RP with 0, 7, 14, 21, 28, 35, and 70 J/cm2 of UVA light was conducted in parallel. The extent of lipid peroxide formation was measured following irradiation both by colorimetric determination of the conversion of MCDP into methylene blue in the presence of hemoglobin (Figure 8A) or by calculation of the amount of methyl linoleate hydroperoxides based on the HPLC peak area detected at 235 nm (Figure 8B) (31). With light dose at 35 J/cm2 or lower, photoirradiation of methyl linoleate did not generate peroxidation in significant levels, and significant peroxidation occurred until a light dose of 70 J/cm2 was used (P e 0.05). However, peroxidation of methyl linoleate catalyzed by photoirradiation of RP was present even when the light dose was 7 J/cm2. Lipid peroxidation increased in a dose response manner (Figure 8A,B). As expected, in the absence of methyl linoleate, photoirradiation of RP did not produce any methyl linoleate hydroperoxides (Figure 8). Photoirradiation of AR and 5,6-epoxy-RP by UVA in the presence of methyl linoleate was similarly conducted (Figure 9). Photoirradiation of AR and 5,6-epoxy-RP in the presence of methyl linoleate generated methyl linoleate hydroperoxides at all the light doses, and the extent of peroxidation increased in a dose (light)-dependent manner. Photoirradition of both AR and 5,6-epoxyRP at 70 J/cm2 generated methyl linoleate hydroperoxides at a quantity higher than that for RP (Figure 9) (P e 0.05). These results indicate that RP and its photodecomposition products, AR and 5,6-epoxy-RP, all can generate lipid peroxidation (methyl linoleate hydroperoxides) upon photoirradiation with UVA light. Mechanistic Study on Peroxidation of Methyl Linoleate Initiated by Photoirradiation of RP, AR, and 5, 6-Epoxy-RP. The involvement of free radical

Figure 8. Lipid peroxidation induced by photoirradiation of RP with UVA light. The levels of peroxidation were measured by (A) colorimetric determination and (B) HPLC analysis monitoring the elution at 235 nm.

intermediates in the peroxidation of methyl linoleate initiated by photoirradiation of RP was examined. The free radical scavengers DTT and NaN3 (19, 20, 33) and the superoxide free radical scavenger SOD (19, 34) were employed for study. NaN3 is also an effective singlet oxygen (1O2) (20) and hydroxyl radical (•OH) scavenger (20, 34). As a result, NaN3 alone cannot be relied upon to determine whether singlet oxygen is involved in peroxidation of methyl linoleate by photoirradiation of RP. Singlet oxygen has a longer half-life in deuterium water (D2O) (20, 35). Therefore, use of both NaN3 and



Figure 9. Peroxidation of methyl linoleate initiated by RP, AR, and 5,6-epoxy-RP and irradiation with UVA light.

Figure 11. Inhibitory effect of NaN3 on the peroxidation of methyl linoleate initiated by (A) AR and (B) 5,6-epoxy-RP and irradiation with UVA light.

Figure 10. Peroxidation of methyl linoleate initiated by RP under UVA irradiation and the effects of DTT, NaN3, SOD, and D2O on peroxidation. The levels of peroxidation were measured by HPLC analysis monitoring the elution at 235 nm.

D2O should provide a reliable approach for determining whether singlet oxygen is involved in peroxidation. The results are summarized in Figure 10. Lipid peroxidation was inhibited by DTT, NaN3, and superoxide dismutase (SOD) (all with P e 0.05) but was enhanced by the presence of D2O (P e 0.05). These results clearly suggest that peroxidation of methyl linoleate initiated by photoirradiation of RP is mediated by free radicals. The inhibition of peroxidation by NaN3 and enhancement by D2O indicate that singlet oxygen is involved in peroxidation. The involvement of free radicals on peroxidation of methyl linoleate initiated by photoirradiation of AR and 5,6-epoxy-RP was similarly studied using NaN3 as a free radical scavenger. The results are shown in Figure 11A,B

and indicate that NaN3 significantly inhibited peroxidation initiated by AR and 5,6-epoxy-RP. Genotoxicity and Photomutagenicity of RP Photodecomposition Products. 1. Mutagenicity Assays. The mutagenicity of RP, 5,6-epoxy-RP, 4-keto-RP, AR (a mixture of trans and cis isomers), and a mixture containing 11-ethoxy-12-hydroxy-RP and 13-ethoxy-14-hydroxyRP in S. typhimurium TA98, TA100, TA102, and TA104 was assayed in the presence and absence of an exogenous metabolic activation system (i.e., S9) as described by Maron and Ames (25). The quantities of each substrate used for the assay were in the range of 0.1-0.5 mg/plate. Neither RP nor its photodecomposition products were mutagenic under experimental conditions (data not shown). Under experimental conditions employed, these compounds were also not photomutagenic in S. typhimurium TA102. 2. 32P-Postlabeling Analyses. RP and its photodecomposition products described above were each incubated with calf thymus DNA and the resulting incubation products were analyzed by 32P-postlabeling/TLC or 32 P-postlabeling/HPLC. Under our experimental conditions, no DNA adducts were detected from any of the incubations (data not shown).

Discussion We report that photoirradiation of RP in ethanol with UVA light results in the formation of photodecomposition products, including 5,6-epoxy-RP, 4-keto-RP, trans- and cis-AR, trans- and cis-11-ethoxy-12-hydroxy-RP, transand cis-13-ethoxy-14-hydroxy-RP, palmitic acid, and ethyl palmitate. There are four isomeric ethoxy-AR photodecomposition products that are tentatively assigned to be

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Figure 12. Proposed pathways for photodecomposition of RP dissolved in ethanol by UVA light. The photodecomposition products shown are generated through a free radical mechanism.

cis and trans isomeric 15-ethoxy-ARs. When photoirradiated in the presence of NaN3, the formation of 5,6epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13ethoxy-14-hydroxy-RP was inhibited. These compounds were also formed from a free radical chemical reaction of RP. Thus, these results indicate that photoirradiation of RP with UVA light results in the formation of 5,6epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13ethoxy-14-hydroxy-RP through a free radical mechanism (Figure 12). On the other hand, AR and 15-ethoxy-AR were not formed from a chemical free radical reaction of RP. Also, formation of these compounds from photoirradiation of RP was not inhibited by NaN3. AR and retinyl methyl ether (15-methoxy-AR) were reported as photodecomposition products from photoirradiation of retinyl acetate (32, 36-38). The pentaenylic retinyl cation was detected from photoirradiation of retinol, retinoic acid, and retinyl acetate using laser flash photolysis and pulse radiolysis methodologies (32, 38, 39). Based on our and these published results (32), we propose that the formation of AR and the isomeric 15-ethoxy-ARs reported in our study is mediated by an ionic photodissociation mechanism (Figure 13). We have also determined that RP and its photodecomposition products are not mutagenic in S. typhimurium tester strains TA98, TA100, TA102, and TA104 and are not photomutagenic in S. typhimurium TA102. In addition, these compounds did not bind to calf thymus DNA in the presence of metabolizing enzymes. Taken together, our results suggest that these compounds are not genotoxic. However, we have determined that upon photoirradiation with UVA light in the presence of methyl linoleate, RP, AR, and 5,6-epoxy-RP can initiate lipid peroxidation forming methyl linoleate hydroperoxides. Our mechanistic studies have revealed that lipid peroxidation is mediated by reactive oxygen species. Thus, photoirradiation of RP with UVA light results in the formation of photodecomposition products and ROS through three distinct mechanisms: a UVA-initiated free radical mechanism, an ionic photodissociation mechanism, and RP photosensitization (Figure 14). The results of our study also suggest that photoirradiation of RP generates singlet oxygen. Delmelle (40, 41) has observed that illumination of retinal resulted in generation of singlet oxygen. Singlet oxygen can react with biological

Cherng et al.

Figure 13. Proposed pathways for photodecomposition of RP dissolved in ethanol by UVA light. The photodecomposition products shown are generated through an ionic photodissociation mechanism. The wavelengths given indicate peak absorptions for RP and the retinyl cation.

Figure 14. The three mechanistic pathways initiated by photoirradiation of RP with UVA light leading to generation of RP photodecomposition products and, when photoirradiation occurs in the presence of methyl linoleate, the formation of lipid peroxidation products.

molecules, including amino acid, proteins, lipids, and DNA and, thus, possibly play an important role in damaging skin (42). Thus, since upon photoirradiation, RP, AR, and 5,6-epoxy-RP generate ROS, singlet oxygen, and lipid peroxidation, concomitant exposure to RP and UV light may pose a human health risk. Short-lived ROS have been shown to damage DNA and proteins and lead to aging, inflammation, cardiovascular diseases, and cancer (43-46). As shown from our study, ROS can initiate lipid peroxidation. It is well established that lipid peroxidation produces lipid alkoxy radicals and aldehydes of low molecular weight, such as malondialdehyde, formaldehyde, crotonaldehyde, acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal. All of the low molecular weight aldehydes have been found to bind covalently with cellular DNA, form DNA adducts, and induce tumors in experimental animals (47-52). Humphries and Curley (53) reported that photooxygenation of triplet-sensitized retinyl acetate, methyl retinoate, and methyl 13-cis-retinoate resulted in one major peroxide product. Thus, our results are consistent with this finding that RP can similarly act as a photosensitizer


under employed experimental conditions. Besides the possible adverse health effects exerted by ROS and lipid peroxidation products, photodecomposition products may also exert toxicological and biological effects. It has been reported that AR can prevent mammary cancer induced by N-methyl-N-nitrosourea (54), inhibit cell growth in lymphocytes, and induce cell death by inducing oxidative stress in human B lymphoblastoid 5/2 cells (55). While RP and other retinoids must bind to nuclear receptors to exert their biological effects, AR exerts its biological activities independent of RARs and RXRs. Therefore, this direct biological activity of AR must be considered in any attempts to understand the biological sequence of events initiated by photoirradiation of RP. Our results suggest that the effects of RP on sun-exposed skin may involve complex mechanisms. Additional in vivo studies are needed to explore more fully the phototoxicological properties of RP.

Acknowledgment. We thank Leslie Coop for performing the NMR measurements and Dr. Frederick A. Beland for critical review of this manuscript. This research was supported in part by an Interagency Agreement, No. 2143-0001, between the Food and Drug Administration/National Center for Toxicological Research (FDA/NCTR) and the National Institute for Environmental Health Sciences/National Toxicology Program (NIEHS/ NTP). Through this agreement, this research was supported by an appointment (S.C.) to the Postgraduate Research Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA.


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TX049807L

Photodecomposition of Retinyl Palmitate in Ethanol by UVA Light

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