JUNE 1996 VOLUME 9, NUMBER 4 © Copyright 1996 by the American Chemical Society
Communications Prostaglandin H Synthase-Catalyzed Oxidation of all-trans- and 13-cis-Retinoic Acid to Carbon-Centered and Peroxyl Radical Intermediates Mary Ann Freyaldenhoven,† Roger V. Lloyd,‡ and Victor M. Samokyszyn*,† Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, and Department of Chemistry, University of Memphis, Memphis, Tennessee 38152 Received February 19, 1996X
Due to the importance of all-trans-retinoic acid (RA) in the treatment of various dermatological conditions and the wide distribution of prostaglandin H synthase (PGHS) in tissues, we have further examined the mechanisms involved in the hydroperoxide-dependent cooxidation of RA and its isomer, 13-cis-retinoic acid ((13Z)-RA), by PGHS. Hydroperoxide-dependent, PGHS-catalyzed oxidation of RA and (13Z)-RA was shown to form free radical adducts, using electron spin resonance (ESR) spin trapping techniques and 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-OOH-18:2) as hydroperoxide substrates. Utilization of the spin trap R-phenyl-N-tert-butylnitrone (PBN) resulted in the detection of (13Z)-RA-PBN and RA-PBN adducts whose spectra were characterized by hyperfine coupling constants of aH ) 4.16/aN ) 15.69 and aH ) 3.01/aN ) 15.92, respectively. Identical experiments under anaerobic conditions were carried out using the spin trap 2-methyl2-nitrosopropane (NtB) which yielded nitroxide adducts whose spectra were characterized by a triplet of doublets with values of aH ) 3.49/aN ) 15.84 for the (13Z)-RA adduct and aH ) 3.49/aN ) 15.88 for the RA adduct. These results are indicative of secondary carbon-centered radical formation. We also used (+)-benzo[a]pyrene 7(S),8(S)-dihydrodiol ((+)-BP-7,8-diol) as a peroxyl radical probe. The results demonstrated the formation of (+)-BP-7,8-diol-derived tetrols, with the trans-anti tetrol representing the major oxidation product in systems undergoing PPHP-dependent, PGHS-catalyzed oxidation of (13Z)-RA or RA. These results are consistent with the formation of peroxyl radicals in these systems. In all experiments, the (13Z)-RA isomer appeared to be a better substrate for the enzyme compared to the alltrans isomer. Collectively, these results provide further evidence to support the previously proposed mechanism for retinoid oxidation by PGHS involving the intermediacy of C4 carboncentered radicals which subsequently react with dioxygen, yielding retinoid-derived peroxyl radicals.
Introduction all-trans-Retinoic acid (RA)1 and 13-cis-retinoic acid ((13Z)-RA) (Figure 1) represent major in vivo and in vitro * Address correspondence to this author at the Division of Toxicology (Mail Slot 638), University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. Tel: 501-686-5766; FAX: 501-6868970; E-Mail:
[email protected].
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metabolites of retinol (vitamin A) (1). In addition, RA is used therapeutically as a topical agent in the treatment of acne vulgaris and photodamaged skin (2, 3), where it undergoes extensive photoisomerization to the 13Z iso† ‡ X
Division of Toxicology, University of Arkansas for Medical Sciences. Department of Chemistry, University of Memphis. Abstract published in Advance ACS Abstracts, May 15, 1996.
© 1996 American Chemical Society
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results demonstrate retinoid-derived peroxyl radical formation as evidenced by hydroperoxide- and retinoiddependent, PGHS-catalyzed epoxidation of (+)-benzo[a]pyrene-7(S),8(S)-dihydrodiol ((+)-BP-7,8-diol).
Materials and Methods
Figure 1. Structures of RA and (13Z)-RA.
Scheme 1
mer (4). Previous studies have shown that these retinoids and others inhibit phorbol ester-dependent tumor promotion in the mouse skin two-stage carcinogenesis assay (5). However, other investigators have shown that retinoids, under certain conditions, will actually promote carcinogenesis (6, 7). Neither the mechanisms for tumor enhancement nor tumor inhibition have been elucidated. Our laboratory has demonstrated that RA and (13Z)-RA undergo hydroperoxide-dependent cooxidation by prostaglandin H synthase (PGHS) (8-10). The major products of these oxidations are 4-hydroxy-RA and 4-hydroxy(13Z)-RA, indicating that oxidation occurred at the C4 position of the retinoids. In addition, several lines of evidence suggest that the oxidations proceed by free radical mechanisms. For example, hydroperoxide-dependent, PGHS-catalyzed oxidation of RA and (13Z)-RA was characterized by uptake of dioxygen which is first order with respect to enzyme concentration, and the 13Z isomer was more reactive than RA as evidenced by ∼2.6 times higher rates of dioxygen uptake in microsomal and purified enzymatic systems. In addition, dioxygen uptake in these systems was inhibited by the spin trap nitrosobenzene, and kinetic analysis of the reaction was consistent with the competition of nitrosobenzene with dioxygen for reaction with retinoid carbon-centered radicals. Finally, the reactions demonstrated the formation of ESR-detectable nitroxides when R-phenyl-N-tert-butylnitrone (PBN) was included in the reaction mixtures. Scheme 1 represents the proposed mechanism for PGHScatalyzed oxidation of RA and (13Z)-RA involving H-atom abstraction at C4 by PGHS peroxidase, yielding an endocyclic, secondary, and highly resonance-stabilized carbon-centered radical. The radical diffuses from the active site and couples with dioxygen, yielding a retinoidderived peroxyl radical. In the present study, we have demonstrated quantitative differences in PBN-derived nitroxide formation in systems undergoing hydroperoxide-dependent, PGHS-catalyzed oxidation of (13Z)-RA and RA. We also report evidence for the formation of retinoid C4 carbon-centered radicals using ESR spin trapping techniques and 2-methyl-2-nitrosopropane (NtB) as the spin trap under anaerobic conditions. Finally, our 1Abbreviations: RA, all-trans (all-E)-retinoic acid; (13Z)-RA, 13-cisretinoic acid; PGHS, prostaglandin H synthase; RSVM, ram seminal vesicle microsomes; NtB, 2-methyl-2-nitrosopropane; PBN, R-phenylN-tert-butylnitrone; PPHP, 5-phenyl-4-penten-1-yl hydroperoxide; 13OOH-18:2, 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid; (+)-BP7,8-diol, (+)-benzo[a]pyrene 7(S),8(S)-dihydrodiol.
Materials. (13Z)-RA and RA were purchased from ACROS Organics (Pittsburgh, PA) and stored at -20 °C under argon. Retinoid stock solutions were prepared fresh in Me2SO (Aldrich, Milwaukee, WI), and all procedures were carried out under yellow light or in the dark. R-Phenyl-N-tert-butylnitrone (PBN) and 2-methyl-2-nitrosopropane (NtB) were purchased from Aldrich, 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) was purchased from Cayman Chemical Co. (Ann Arbor, MI), and (+)-benzo[a]pyrene 7(S),8(S)-dihydrodiol ((+)-BP-7,8-diol) was purchased from the NCI Chemical Carcinogen Repository, Midwest Research Institute. Caution: BP-7,8-diol is classified as a known human carcinogen and may be hazardous; use extreme caution when handling this material, such as properly operating ventilation hoods, gloves, safety goggles, and protective clothing. 7,8,9,10-trans-anti, trans-syn, cis-anti, and cis-syn tetrols of benzo[a]pyrene were obtained from the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention (Bethesda, MD). 13-Hydroperoxy-9-cis-11-transoctadecadienoic acid (13-OOH-18:2) was synthesized as described by Funk, et al. (11). Ram seminal vesicle glands were purchased from Oxford Biomedical Research Inc. (Oxford, MI), and ram seminal vesicle microsomes (RSVM) were prepared according to the procedure of Marnett and Wilcox (12). All reactions were carried out in 0.1 M phosphate buffer (pH 7.8) which had been treated with Chelex 100 resin (Bio-Rad, Hercules, CA). Heat-denatured RSVM were prepared by subjecting to a boiling water bath for approximately 3 min. All other reagents were obtained through commercial sources. ESR Spin Trapping Experiments. Reaction mixtures involving spin trapping with PBN consisted of 2 mg of RSVM protein/mL or heat-denatured RSVM, 600 µM (13Z)-RA or RA, and 10 mM PBN dissolved in buffer, followed by the addition of 800 µM 13-OOH-18:2. Controls consisted of the complete system minus either retinoid or hydroperoxide or substitution of viable RSVM with heat-denatured microsomes. Experiments involving NtB consisted of the same controls and components except that 1.25 mM NtB was dissolved in buffer instead of PBN and 800 µM PPHP was used as the hydroperoxide instead of 13-OOH-18:2. In addition, NtB reaction mixtures were rendered hypoxic by bubbling nitrogen through the NtB/buffer solution and the retinoid stock solution. Depletion of dioxygen was necessary to eliminate or minimize competing reaction of retinoid-derived carbon-centered radicals with dioxygen. ESR spectra were obtained using a Varian E-104 spectrometer custom-interfaced with an IBM-compatible computer for data acquisition and analysis. All spectra were stored on the computer for later analysis, and signal intensities were measured from the stored spectra using software written by Duling (NIEHS) (13). All reactions were carried out at room temperature, and solutions were aspirated into a 10.5-mm Wilmad flat cell centered in the TM110 microwave cavity immediately after preparation. Polyethylene tubing rather than stainless steel was used for the aspiration to avoid the possibility of transition metal contamination. Utilization of (+)-BP-7,8-diol as a Peroxyl Radical Probe during Retinoid Oxidation by RSVM. Reactions were carried out at 37 °C and consisted of 0.5 mg of RSVM protein/mL or heat-denatured RSVM, 150 µM (13Z)-RA or RA, and 200 µM (+)-BP-7,8-diol, followed by the addition of 200 µM PPHP. After 5 min, the solutions were acidified by the addition of HCl and extracted with anhydrous diethyl ether. The ether was evaporated using an argon stream, and the residue was dissolved in HPLC-grade methanol, filtered through 0.45 µm nylon filters (Scientific Resources inc., Eatontown, NJ), and analyzed by reverse-phase HPLC. HPLC analyses were carried
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out using a Waters 600E controller system and a Waters 994 programmable photodiode array detector run on Waters Millennium software (version 2.1) using a Gateway 2000 P5-90 computer. The system was fitted with a Waters µBondapak C18 125 Å 10 µm column, the flow rate was 1.5 mL/min, and eluents were detected by absorbance at 344 nm. The composition of solvent A was 45:55 methanol/water, and solvent B was 100% methanol. The solvent system consisted of 100% solvent A for 20 min, a linear gradient to 80% solvent A over the next 20 min, followed by a linear gradient to 100% solvent B over the next 10 min, and ending with isocratic conditions of 100% solvent B for the final 10 min. Identification of eluting diastereomeric tetrols was made on the basis of comparison of retention times with synthetic standards (14).
Results and Discussion We have carried out ESR spin trapping experiments as well as studies involving (+)-BP-7,8-diol as a peroxyl radical probe in order to demonstrate the formation of retinoid-derived carbon-centered and peroxyl radical intermediates generated during the hydroperoxide-dependent cooxidation of (13Z)-RA and RA by PGHS. Specifically, we have attempted to demonstrate the formation of retinoid-derived carbon-centered radicals at the endocyclic secondary allylic position of the β-ionone ring (C4), to demonstrate subsequent peroxyl radical formation, and to compare the reactivities of these geometric isomers. Experiments utilizing ESR and PBN as a spin trap demonstrated the formation of nitroxide adducts during the hydroperoxide-dependent oxidation of (13Z)-RA and RA by PGHS. Reaction mixtures contained (13Z)-RA or RA, RSVM, 13-OOH-18:2, and PBN, and ESR spectra were obtained as described in the Materials and Methods section. The spectra of the PBN adducts consisted of a triplet of doublets which were characterized by hyperfine coupling constants of aH ) 4.16/aN ) 15.69 for the (13Z)-RA adduct and aH ) 3.01/ aN ) 15.92 for the RA adduct (not shown). In addition, the signal amplitude for the (13Z)-isomer nitroxide adduct was much greater compared with the RA adduct (ratio (13Z)-RA/RA ∼3). These results are consistent with our demonstration that the rates of dioxygen uptake associated with the PGHS-catalyzed cooxidation of the (13Z) isomer were also ∼2.6 times greater compared with the all-trans isomer. Unfortunately, with PBN as the spin trap, it is difficult to distinguish between peroxyl, alkoxyl, and/or carbon-centered radical adducts. Thus, we subsequently replaced PBN with NtB as the spin trap in an attempt to identify the C-H bond in (13Z)-RA or RA at which peroxidase-catalyzed H-atom abstraction occurs. We have previously identified 4-hydroxy-(13Z)-RA and 4-hydroxy-RA as major oxidation products generated during the hydroperoxide- or arachidonic acid-dependent cooxidation of (13Z)-RA and RA, respectively, by microsomal or purified PGHS (8-10). We proposed that PGHScatalyzed oxidation of the retinoids involves H-atom abstraction at the C4 position, by compounds I and/or II of the peroxidase, yielding the corresponding highly resonance-stabilized, carbon-centered radical. This radical couples with dioxygen yielding the corresponding peroxyl radical (Scheme 1) which ultimately yields alcohols by nonenzymatic mechanisms. This would account for the observed uptake of dioxygen associated with hydroperoxide-dependent, PGHS-catalyzed oxidation of the retinoids and inhibition of dioxygen uptake by the spin trap nitrosobenzene. The C4 C-H bond is predicted
Figure 2. ESR spectra of NtB-derived nitroxide adducts generated during hydroperoxide-dependent, PGHS-catalyzed oxidation of (13Z)-RA. (A) Complete system (2 mg of RSVM/ mL, 600 µM (13Z)-RA, 1.25 mM NtB, and 800 µM PPHP at ambient temperature in 0.1 M phosphate buffer (pH 7.8)); (B) complete system minus PPHP; (C) complete system using heatdenatured microsomes; and (D) complete system minus retinoid. ESR instrumental parameters: gain ) 3.2 × 104, modulation amplitude ) 1.0 G, microwave power ) 20 mW, scan time ) 4 min, sweep ) 80 G, and time constant ) 0.5 s.
Figure 3. ESR spectra of NtB-derived nitroxide adducts generated during hydroperoxide-dependent, PGHS-catalyzed oxidation of RA. (A) Complete system (2 mg of RSVM/mL, 600 µM RA, 1.25 mM NtB, and 800 µM PPHP at ambient temperature in 0.1 M phosphate buffer (pH 7.8)); (B) complete system minus PPHP; (C) complete system using heat-denatured microsomes; and (D) complete system minus retinoid. ESR instrumental parameters were identical to those described in Figure 2, except for using a gain of 1.6 × 105.
to be more reactive compared with other allylic C-H bonds because it involves a secondary rather than a primary allylic center. Furthermore, the C4 carbon is endocyclic, and thus a radical at this position would exhibit optimal extents of p-orbital overlap. We have therefore carried out ESR spin trapping experiments under anaerobic conditions using NtB as a spin trap to confirm the intermediacy of carbon-centered radicals at C4. The ESR profiles for the complete systems and the appropriate controls are shown in Figures 2 and 3. The ESR spectrum of adducts detected using (13Z)-RA, Figure 2(A), consisted of a triplet of doublets having hyperfine coupling constants of aH ) 3.49 and aN ) 15.84, which are characteristic of a secondary carbon-centered radical
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adduct as a consequence of nitrogen hyperfine splitting by a single proton. Thus, a primary allylic nitroxide adduct is expected to yield a spectrum characterized by a triplet of triplets. No signal was observed in the absence of PPHP or (13Z)-RA, or when viable microsomes were substituted with heat-denatured preparations. In reaction mixtures containing RA, the spectrum (Figure 3(A)) was also consistent with secondary carbon-centered radical formation. The spectrum was characterized by hyperfine coupling constants of aH ) 3.49 and aN ) 15.88, and again, the controls failed to exhibit any signal. These results demonstrate that retinoid-derived carbon-centered radicals are generated and are consistent with H-atom abstraction occurring at the C4 position. As noted previously, the (13Z)-isomer exhibited greater ESR signal intensity compared with the all-trans isomer,2 which again suggests that the cis isomer may serve as a better substrate for the enzyme. Additional experiments were performed using (+)-BP7,8-diol as a molecular probe to confirm peroxyl radical formation as has been proposed in Scheme 1. (+)-BP7,8-diol oxidation by peroxyl radicals yields diol epoxides (Scheme 2). The reaction is stereoselective, giving primarily the anti-diol epoxide because of H-bonding between the 8-hydroxyl group and the attacking peroxyl radical, and this stereochemistry is opposite to that observed with cytochrome P450-catalyzed oxidations (14). The oxiranes undergo hydrolysis, and the major peroxyl radical product is the trans-anti tetrol arising by trans addition of water to the anti diol epoxide. Reactions were carried out with RSVM, retinoid, and PPHP in the presence of (+)-BP-7,8-diol and subsequently analyzed for tetrol formation by reverse-phase HPLC as described in the experimental section. The HPLC profiles for both isomers are presented in Figure 4. When retinoid was reacted with intact RSVM and PPHP in the presence of (+)-BP-7,8-diol, there was significant formation of tetrols which was not observed in reaction mixtures containing heat-denatured enzyme. Similarly, significant tetrol formation was not observed in the absence of retinoid or PPHP. Our demonstration of tetrol formation is consistent with the formation of retinoid-derived peroxyl radicals during hydroperoxide-dependent, PGHS-catalyzed oxidation. The ESR spin trapping experiments as well as the results obtained using (+)-BP-7,8-diol as a peroxyl radical probe provide further evidence supporting the proposed mechanism for PGHS-catalyzed oxidation of RA and (13Z)-RA. Our results suggest that compounds I and/or II of the peroxidase abstract an H-atom from (13Z)-RA or RA at the C4 position generating retinoid carboncentered radical intermediates. These radicals diffuse from the active site and react with dioxygen to produce peroxyl radicals as illustrated by the mechanism in Scheme 1. Our results also suggest that (13Z)-RA functions as a more efficient substrate, compared with RA, for PGHS peroxidase. These results are consistent with our demonstration that the rates of dioxygen uptake associated with hydroperoxide-dependent, PGHS-catalyzed oxidation of these retinoids were greater for the 13Z isomer. We have recently carried out molecular mechanics and molecular orbital calculations on RA, (13Z)-RA, and their corresponding C4 radicals.3 Our 2ESR spectra for both the 13Z and the all-trans isomers were obtained 13 min after initiation of the reaction. 3Unpublished results.
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Figure 4. HPLC profiles of (+)-BP-7,8-diol products generated during hydroperoxide-dependent, PGHS-catalyzed oxidation of (13Z)-RA (left) and RA (right). Reactions conditions, procedures for product isolation, and HPLC methods are described in the Materials and Methods section. The eluting (+)-BP-7,8-diolderived tetrols are represented by peaks A-D and correspond to the diastereomers in Scheme 2.
Scheme 2
results indicated that the values for enthalpies of formation of C4 carbon-centered radicals from the parent geometric isomers are virtually equivalent. Thus, the differences in reactivities of these isomers probably reflect steric and/or hydrophobic effects which influence binding and interaction at the active site surface. Retinoic acid oxidation by PGHS, analogous to the mechanism proposed by Reed et al. for PGHS-catalyzed oxidation of phenylbutazone (15), involves a hybrid of peroxidase and peroxyl radical chemistry. However, PGHS-catalyzed oxidation of phenylbutazone involves H-atom abstraction from an O-H bond, whereas retinoid oxidation involves the homolysis of C-H bonds. Our results represent the first reports ever demonstrating C-H bond homolysis by a peroxidase, which makes RA and (13Z)-RA novel peroxidase reducing substrates.
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Acknowledgment. This work was supported by Grant R29ES06765-02 from the NIH. Dr. Freyaldenhoven is the recipient of the Kappa Epsilon-Nellie Wakeman-American Foundation for Pharmaceutical Education First Year Graduate Scholarship.
References (1) Blaner, W. S., and Olson, J. A. (1994) Retinol and retinoic acid metabolism. In The Retinoids. Biology, Chemistry, and Medicine (Sporn, M. B., Roberts, A. B., and Goodman, D. S., Eds.) 2nd ed., pp 597-630, Raven Press, New York. (2) Kelly, A. P. (1990) Acne and related disorders. In Principles and Practice of Dermatology (Sams, W. M., Jr., and Lynch, R. J., Eds.) pp 781-796, Churchill Livingstone, New York. (3) Weiss, J. S., Ellis, C. N., Headington, J. T., Tincoff, T., Hamilton, T. A., and Voorhees, J. J. (1988) Topical tretinoin improves photoaged skin. A double-blind vehicle-controlled study. J. Am. Med. Assoc. 259, 527-532. (4) Lehman, P. A., and Malany, A. M. (1989) Evidence of percutaneous absorption of isotretinoin from the photo-isomerization of topical tretinoin. J. Invest. Dermatol. 93, 595-599. (5) Moon, R. C., Mehta, R. G., and Rao, K. V. N. (1994) Retinoids and cancer in experimental animals. In The Retinoids. Biology, Chemistry, and Medicine (Sporn, M. B., Roberts, A. B., and Goodman, D. S., Eds.) 2nd ed., pp 573-595, Raven Press, New York. (6) Forbes, P. D., Urbach, F., and Davies, R. E. (1979) Enhancement of experimental photocarcinogenesis by topical retinoic acid. Cancer Lett. 7, 85-90.
(7) Hennings, H., Wenk, M. L., and Donahoe, R., (1982) Retinoic acid promotion of papilloma formation in mouse skin. Cancer Lett. 16, 1-5. (8) Samokyszyn, V. M., Chen, T., Maddipati, K. R., Franz, T. J., Lehman, P. A., and Lloyd, R. V. (1995) Free radical oxidation of (E)-retinoic acid by prostaglandin H synthase. Chem. Res. Toxicol. 8, 807-815. (9) Samokyszyn, V. M., and Marnett, L. J. (1987) Hydroperoxidedependent cooxidation of 13-cis-retinoic acid by prostaglandin H synthase. J. Biol. Chem. 262, 14119-14133. (10) Samokyszyn, V. M., Sloane, B. F., Honn, K. V., and Marnett, L. J. (1984) Cooxidation of 13-cis-retinoic acid by prostaglandin H synthase. Biochem. Biophys. Res. Commun. 124, 430-436. (11) Funk, M. O., Isaac, R., and Porter, N. A. (1976) Preparation and purification of lipid hydroperoxides from arachidonic acid and γ-linolenic acids. Lipids 11, 113-117. (12) Marnett, L. J., and Wilcox, C. L. (1977) Stimulation of prostaglandin biosynthesis by lipoic acid. Biochim. Biophys. Acta 487, 222-230. (13) Simulation of multiple spin-trap EPR spectra. J. Magn. Reson., Ser. B 104, 105-110. (14) Pruess-Schwartz, D., Nimesheim, A., and Marnett, L. J. (1989) Peroxyl radical- and cytochrome P-450-dependent metabolic activation of (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in mouse skin in vitro and in vivo. Cancer Res. 49, 1732-1737. (15) Reed, G. A., Brooks, E. A., and Eling, T. E. (1984) Phenylbutazonedependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene: A new mechanism for prostaglandin H synthasecatalyzed oxidations. J. Biol. Chem. 259, 5591-5595.
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