Initiated Autoxidation of Linoleic Acid in Sodium Dodecyl - American

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Chem. Res. Toxicol. 1998, 11, 102-110

Retinoic Acid-Dependent Stimulation of 2,2′-Azobis(2-amidinopropane)-Initiated Autoxidation of Linoleic Acid in Sodium Dodecyl Sulfate Micelles: A Novel Prooxidant Effect of Retinoic Acid Mary Ann Freyaldenhoven,§ Paul A. Lehman,‡ Thomas J. Franz,‡ Roger V. Lloyd,† and Victor M. Samokyszyn*,§ Departments of Pharmacology & Toxicology (Division of Toxicology) and Dermatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, and Department of Chemistry, University of Memphis, Memphis, Tennessee 38152 Received March 20, 1997

(E)-Retinoic acid (RA) was shown to stimulate the rate of 2,2′-azobis(2-amidinopropane) (AAPH)-initiated autoxidation of linoleic acid (18:2) in sodium dodecyl sulfate (SDS) micelles. RA-dependent stimulation of 18:2 autoxidation was characterized by enhanced rates of dioxygen uptake which were linear with retinoid concentration. In contrast, 5,6-epoxy-RA, a major oxidation product of RA, failed to affect the rate of dioxygen consumption at all concentrations tested. RA was also shown to stimulate peroxyl radical-dependent oxidation of styrene to the corresponding oxirane when styrene was included in the micellar system as a molecular probe. Furthermore, unequivocal evidence of RA-dependent stimulation of 18:2 autoxidation was obtained by relative quantitation of 13-hydroxy-(9Z,11E)-octadecadienoic acid (13-HODE) plus 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-HODE) production. In addition, enhanced carboncentered radical formation was demonstrated in the presence of RA by EPR spectroscopy using R-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN) as a spin trap. Analysis and quantitation of RA oxidation products indicated that RA was oxidized to one primary product, 5,6-epoxyRA, which was identified on the basis of cochromatography with synthetic standard (in a reverse-phase HPLC system), electronic absorption spectroscopy, and positive chemical ionization mass spectrometry of the corresponding methyl ester. Other minor oxidation products were also detected but not characterized. In contrast, reaction mixtures devoid of 18:2 failed to demonstrate significant retinoid oxidation. Mechanisms are proposed to account for the prooxidant effects of RA in this system.

Introduction

LH f L•

(1)

It is generally accepted that lipid peroxidation is associated with the toxicities of many xenobiotics (e.g., paraquat, adriamycin, CCl4) as well as various pathologies (e.g., atherogenesis, carcinogenesis, arthritis). A simplified version of the major mechanisms involved in lipid peroxidation, also termed autoxidation of polyunsaturated fatty acids, is represented by eqs 1-6, involving initiation (eq 1), propagation (eqs 2 and 3), and termination (eqs 4-6) reactions. LH represents a polyunsaturated fatty acid, L• represents a polyunsaturated fatty acid-derived carbon-centered radical, and LOO• represents a polyunsaturated fatty acid-derived peroxyl radical (see refs 1 and 2 for reviews of mechanisms involved in the autoxidation of polyunsaturated fatty acids).

L• + O2 f LOO•

(2)

* Address correspondence to this author at the Division of Toxicology, Mail Slot 638, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. Tel: (501) 686-5810, -5811, or -5766. Fax: (501) 686-8970. E-mail: [email protected]. § Department of Pharmacology & Toxicology (UAMS). ‡ Department of Dermatology (UAMS). † University of Memphis.

kp

LOO• + LH 98 LOOH + L•

(3)

2L• f nonradical products

(4)

L• + LOO• f nonradical products

(5)

2LOO• f nonradical products

(6)

At ambient dioxygen tension, eq 2 is diffusion-limited [k ∼ 109 M-1 s-1 (3)]. Thus, the rate-limiting step in propagation involves the bimolecular reaction of peroxyl radicals with bis(allylic) C-H bonds by a H atom abstraction mechanism (eq 3) which is characterized by the bimolecular rate constant kp (kp ) 62 M-1 s-1 for linoleic acid) (reviewed in ref 4). The propagation reactions establish a cycle that will continue until all of the substrate (LH) is depleted and/or when the rate of initiation is less than the rate of termination. The

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Prooxidant Effects of Retinoic Acid

mechanism of action of antioxidants is to scavenge peroxyl radicals, resulting in the termination of propagation reactions and thereby inhibiting hydrocarbon autoxidation. Retinoids, metabolic and synthetic derivatives of vitamin A, have been shown to function as effective antioxidants and to inhibit the peroxidation of polyunsaturated fatty acids in lipid bilayers. For example, we have shown that (13Z)-retinoic acid [(13Z)-RA1] effectively inhibits ascorbate-dependent, iron-catalyzed lipid peroxidation in rat liver microsomes (5, 6). Similarly, (E)-retinoic acid (RA), retinol, and other retinoids have been shown to inhibit ascorbate-dependent, iron-catalyzed lipid peroxidation in vitro in rat brain mitochondria (7), and Tesoriere et al. demonstrated the antioxidant effects of retinol in phosphatidylcholine liposomes (8). Recently, retinol palmitate was shown to function as an antioxidant in vivo in rat tissues and to effectively inhibit doxorubicininduced peroxidation of heart and brain tissue membranes (9). In contrast, under certain conditions retinoids have been shown to exhibit the opposite effect and function as prooxidants, particularly in homogeneous liquid-phase systems. For example, RA and its methyl ester were shown to undergo azobis(isobutyronitrile) (AIBN)-initiated autoxidation in chlorobenzene by a mechanism involving free radical propagation reactions (10). Although the investigators published a thorough kinetic analysis, the actual mechanisms involved in AIBN-initiated retinoid autoxidation are unclear because the authors failed to identify the major retinoid oxidation product(s). The mechanisms involved in the antioxidant or the prooxidant chemistry of retinoids have not been elucidated. Therefore, our laboratory performed further investigations into the antioxidant versus prooxidant effects of retinoids utilizing a classic assay for characterizing antioxidant kinetics (4, 11). Surprisingly, we observed a prooxidant effect of RA in this system, which consisted of a mixture of 2,2′-azobis(2-amidinopropane) (AAPH), as the initiator, and 18:2 in SDS micelles at T ) 313 K (40 °C). A mechanism is proposed to account for this prooxidant activity.

Materials and Methods Materials. (E)-Retinoic acid (all-trans) (RA) and 13-cisretinoic acid [(13Z)-RA] were purchased from ACROS Organics (Pittsburgh, PA), and (E)-[11,12-3H]retinoic acid ([3H]RA), specific activity 52 Ci/mmol, was purchased from DuPont Co., NEN Research Products (Boston, MA); all retinoids were stored at -20 °C under argon. In addition, [3H]RA was purified by reverse-phase HPLC on the same day experiments were performed in order to ensure purity and to remove the antioxidant which is present in the NEN preparation. Stock solutions of the retinoids were prepared fresh in Me2SO (Aldrich Chemical Co., Milwaukee, WI) and all procedures involving these compounds were carried out in the dark or under yellow light. 2,2′Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Monomer-Polymer Laboratories (Windham, NH) 1 Abbreviations: RA, (E)-all-trans-retinoic acid [3,7-dimethyl-9(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid]; [3H]RA, (E)-[11,12-3H]retinoic acid; (13Z)-RA, (13Z)-retinoic acid; AAPH, 2,2′azobis(2-amidinopropane) dihydrochloride; 18:2, linoleic acid; SDS, dodecyl sulfate, sodium salt; BHT, butylated hydroxytoluene (2,6-ditert-butyl-4-methylphenol); 4-POBN, R-(4-pyridyl 1-oxide)-N-tert-butylnitrone; AIBN, azobis(isobutyronitrile); 9-HODE, 9-hydroxy-(10E,12Z)-octadecadienoic acid; 13-HODE, 13-hydroxy-(9Z,11E)-octadecadienoic acid; PGB1, prostaglandin B1; EI, electron impact; PCI, positive chemical ionization.

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 103 and linoleic acid (18:2) from Sigma Chemical Co. (St. Louis, MO). Sodium dodecyl sulfate (SDS), styrene, styrene oxide, 2-undecanone, butylated hydroxytoluene (BHT), R-(4-pyridyl 1-oxide)N-tert-butylnitrone (4-POBN), sodium borohydride, 1-methyl3-nitro-1-nitrosoguanidine, and Diazald (N-methyl-N-nitrosop-toluenesulfonamide) were purchased from Aldrich. 9-Hydroxy(10E,12Z)-octadecadienoic acid (9-HODE), 13-hydroxy-(9Z,11E)octadecadienoic acid (13-HODE), and prostaglandin B1 (PGB1) were purchased from Cayman Chemical Co. (Ann Arbor, MI). 5,8-Oxy-RA was a generous gift of Dr. Larry J. Marnett (Vanderbilt University, Nashville, TN). 5,6-Epoxy-RA was synthesized by epoxidation of RA with perphthalic acid essentially as described by Wertz et al. (12): EI-MS m/z 330 (M•+), 315 (M•+ - CH3), 299 (M•+ - OCH3), 271 (M•+ - CO2CH3); 1H NMR (300 MHz, Me2SO-d6) δ 6.97 (dd, J ) 15.0, 15.3 Hz, 1H), 6.42 (d, J ) 15.1 Hz, 1H), 6.25 (d, J ) 6.5, 11.1 Hz, 2H). 6.04 (d, J ) 15.7 Hz, 1H), 5.78 (s, 1H), 2.26 (s, 3H), 1.95 (s, 3H), 1.73 (brs, 2H), 1.37 (brs, 2H), 1.09 (s, 3H), 1.08 (s, 3H), 0.85 (s, 3H). All other reagents were of the highest quality available and were obtained through commercial sources. Dioxygen Uptake Kinetics. Reactions were carried out at 40 °C in 0.05 M phosphate buffer (pH 7.0) containing 1.5 × 10-2 M SDS and 3 × 10-3 M 18:2, in the presence or absence of RA or 5,6-epoxy-RA (at concentrations indicated in figure legends). Hydrocarbon autoxidation was initiated by addition of AAPH (3 × 10-3 M). Reactions were also carried out in the absence of 18:2 to investigate the rate of AAPH-initiated retinoid autoxidation. Dioxygen uptake was measured polarographically with a Clark electrode (Yellow Springs Instrument Co., Yellow Springs, OH). The linear portions of the curves were used to determine rates of oxygen consumption. Styrene Epoxidation Assay. Styrene (1 × 10-3 M) was incubated at 40 °C in 0.05 M phosphate buffer (pH 7.0) containing 1.5 × 10-2 M SDS, 3 × 10-3 M 18:2, and either RA (2 × 10-4 M) or vehicle (0.2%, v/v, Me2SO) followed by the addition of AAPH (3 × 10-3 M) in a total volume of 10.0 mL. After 10 min, the reactions were inhibited with BHT (4 × 10-5 M). 2-Undecanone was added as an internal standard (final concentration ) 1 × 10-4 M), and the mixtures were extracted with 4.0 mL of HPLC-grade ethyl acetate (containing 1 × 10-5 M BHT). The solvent was dried over Na2SO4, and 1.0 µL was split-injected (1:25) onto a Hewlett-Packard 5890 GC, connected to a DB-5 phenylmethyl capillary column (J & W Scientific Co.; 30 m × 0.25 mm i.d.), interfaced to a Hewlett-Packard 5988A mass spectrometer. Initial column temperature was set at 50 °C and maintained for 2 min after injection. Subsequently, the oven temperature was ramped to 250 °C at 20 °C/min. Injector, MS interface, and MS source were maintained at 250 °C. The MS was operated in the 70 eV EI mode for spectral analysis. Once full spectra had been obtained to confirm retention time and identification of the test compounds, the MS was switched to SIM mode for improved sensitivity with reduced background noise. Eleven key ions, ranging from m/z 43 to 170, were selected which were representative of significant or unique ions present in each test compound. Relative Quantitation of 18:2 Autoxidation. The extent of 18:2 autoxidation was determined by indirectly quantitating the formation of 18:2-derived fatty acid-derived hydroperoxides. The assay measured the amount of the corresponding fatty acidderived alcohols, 13-HODE and 9-HODE, relative to an internal standard. All glassware used for these experiments was trimethylsilylated using trimethylchlorosilane (Aldrich). Reactions were carried out at 40 °C in 0.05 M phosphate buffer (pH 7.0) containing 1.5 × 10-2 M SDS, 18:2 (3 × 10-3 M), and retinoid (5.0 × 10-5 M) or vehicle (0.2%, v/v, Me2SO) followed by the addition of AAPH (3 × 10-3 M). After 30 min, the reactions were inhibited with BHT (2.0 × 10-5 M), and PGB1 (1.1 × 10-4 M) was added as an internal standard. The mixtures were acidified to pH 4 and extracted with ethyl acetate (containing 1 × 10-5 M BHT). After the solvent was removed under an argon stream, the residues were dissolved in methanol and the hydroperoxides were reduced by the addition of NaBH4. The

104 Chem. Res. Toxicol., Vol. 11, No. 2, 1998 mixtures were again acidified to pH 4 and extracted with ethyl acetate, and the solvent was removed with an argon stream. The final residues were dissolved in 1 mL of ethanol and stored at -80 °C. HPLC analysis was performed using a Waters Novapak C18 (125 Å, 4 µm, 3.9 × 300 mm) HPLC column and a Waters 600E HPLC pumping system with a 994M photodiode array detector. The system was controlled using Waters Millennium software, version 2.1, using a Gateway 2000 P5-90 computer, and the photodiode array detector allowed the acquisition of on-line electronic absorption spectra of eluents. The solvent system consisted of 20% solvent B in solvent A at t ) 0 followed by a linear gradient to 90% solvent B over 30 min followed by isocratic conditions for 25 min. Solvent A consisted of 50:50 methanol/water containing 0.1% trifluoroacetic acid, and solvent B consisted of 90:10 methanol/water containing 0.1% trifluoroacetic acid. Solvents must be prepared fresh daily to avoid the formation of methyl trifluoroacetate. The flow rate was 1.0 mL/min, and eluents were detected by absorbance at 235 nm. Typical retention times for 13-HODE, 9-HODE, and PGB1 in this HPLC system were 35.33, 35.60, and 23.04 min, respectively. The relative quantitative effects of RA on 13- and 9-HODE generation were measured by peak integration relative to the internal standard and are expressed as (13HODE + 9-HODE peak areas)/(PGB1 peak area). EPR Spin-Trapping Experiments. EPR 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 (13). All reactions were carried out at 40 °C, and solutions were aspirated into a 10.5-mm Wilmad flat cell centered in the TM110 microwave cavity after 20-min reaction times. Polyethylene tubing rather than stainless steel was used for the aspiration to avoid the possibility of transition-metal contamination. Composition of reaction mixtures as well as EPR instrumental parameters are described in the figure legend. Analysis and Quantitation of RA Oxidation Products Generated during AAPH-Initiated Autoxidation of 18:2. [3H]RA (2 × 10-4 M, specific activity 4.1 × 10-4 Ci/mmol) was incubated at 40 °C in 0.05 M phosphate buffer (pH 7.0) in the presence or absence of 18:2 (3 × 10-3 M) followed by the addition of AAPH (3 × 10-3 M) in a total volume of 10.0 mL. After all dioxygen was depleted (2.1 × 10-4 M O2), BHT (1.0 × 10-5 M final concentration) was added, the reaction mixtures were acidified to pH 3 with HCl, and they were extracted with 3 × 5 mL of HPLC-grade ethyl acetate. The solvent was removed under an argon stream, and the residue was dissolved in 100 µL of HPLC-grade acetonitrile, filtered through 0.45-µm nylon filters (Scientific Resources Inc., Eatontown, NJ), and analyzed by reverse-phase HPLC. The same analytical column and chromatographic system was used as described for the 13HODE/9-HODE analysis, along with a Packard Radiomatic FloOne\βeta 150TR flow scintillation analyzer using Packard ULTIMA-FLOM LSC-cocktail. DPMs were obtained using a programmed, empirically determined quench curve. The solvent system consisted of 20% solvent B in solvent A at t ) 0 followed by a linear gradient to 90% solvent B over 30 min followed by isocratic conditions for 25 min. Solvent A consisted of 50:50 methanol/water containing 0.01 M ammonium acetate (pH 6.65 ( 0.05), and solvent B consisted of 90:10 methanol/water containing 0.01 M ammonium acetate (pH 6.65 ( 0.05). The flow rate was 1.0 mL/min, and eluents were detected by absorbance at 343 nm. 5,6-Epoxy-RA was identified on the basis of cochromatography with synthetic standard, UV spectroscopy, and mass spectrometry of the corresponding methyl ester. Samples for mass spectrometry were obtained by collecting the HPLC-eluted material from scaled-up reaction mixtures (total volume ) 0.1 or 1 L) using unlabeled RA. The HPLC solvent was removed in vacuo and the corresponding methyl esters were prepared by reaction with ethereal diazomethane generated by reaction

Freyaldenhoven et al. Chart 1. Structures of (E)-Retinoic Acid (RA) and 5,6-Epoxy-RA

of 1-methyl-3-nitro-1-nitrosoguanidine or Diazald with KOH at 4 °C (14). (Caution: It should be noted that diazomethane is carcinogenic and is potentially explosive. Thus, diazomethane generation and handling should be carried out in a fume hood, behind protective shielding, and using gloves and protective clothing. Ground glass and sharp/jagged edges should be avoided.) Mass spectra were obtained using a Hewlett-Packard system consisting of a 5988A mass spectrometer interfaced to a 59980A particle beam interface. Samples were introduced into the interface via a Hewlett-Packard 1090 Series II HPLC. The HPLC was configured without a column (injector output connected directly to the particle beam input), and samples (10 µL in acetonitrile) were introduced in neat acetonitrile at a flow rate of 0.5 mL/min. The particle beam was maintained at 60 °C with 35 psi of helium at the nebulizer. The mass spectrometer was configured in chemical ionization mode using methane as the reagent gas at a source pressure of 1 × 10-4 Torr. Source temperature was maintained at 250 °C, and injections were evaluated in positive chemical ionization (PCI) mode with programmed scanning from m/z 50 to 350.

Results Our laboratory has investigated the effects of RA (Chart 1) on the autoxidation of linoleic acid (18:2) (3 × 10-3 M) in sodium dodecyl sulfate (SDS) micelles (1.5 × 10-2 M SDS), initiated by 2,2′-azobis(2-amidinopropane) (AAPH) (3 × 10-3 M) at 40 °C. AAPH is a water-soluble azo initiator which undergoes thermal homolysis to generate carbon-centered radicals and N2. The carboncentered radicals may either couple (yielding dimers) or diffuse from the solvent cage and react with dioxygen to yield peroxyl radicals which may then function as initiators of polyunsaturated fatty acid autoxidation (Scheme 1) (15). Typical values for the rate of dioxygen uptake associated with AAPH-derived peroxyl radical formation as well as AAPH-initiated autoxidation of 18:2 are shown in Table 1. The rate law characterizing dioxygen uptake associated with hydrocarbon autoxidation is represented by eq 7 (4, 11):

-d[O2]/dt ) [RH](Ri)0.5kp/(2kt)0.5

(7)

where [RH] is the concentration of 18:2, Ri is the rate of initiation, kp is the propagation rate constant, and kt is the termination rate constant [eq 6 is the most prevalent at ambient pO2, which is used in the present study (4, 15)]. Initially, several kinetic parameters characterizing 18:2 autoxidation in this system were determined. The rate of initiation was determined using the inhibitor method (11, 16, 17) and utilizing BHT as the antioxidant. Ri was calculated from eq 8 assuming a molar density for SDS of 0.25 L/mol (17):

Prooxidant Effects of Retinoic Acid

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 105

Scheme 1

Scheme 2

Table 1. Effect of RA on the Rate of Dioxygen Consumption Associated with AAPH-Initiated Autoxidation of 18:2 in SDS Micelles -d[O2]/dt (M s-1 × 10-9) 10-3

AAPH (3 × M) alone (minus 18:2) AAPH + 18:2 (3 × 10-3 M) AAPH/18:2 + RA (2.0 × 10-5 M)

3 ( 0.5a 62 ( 1 150 ( 5

a Reactions were carried out at 40 °C in 0.05 M phosphate buffer (pH 7.0) containing 1.5 × 10-2 M SDS, and dioxygen uptake was measured with a Clark electrode as described in the Materials and Methods section. The values represent the mean ( SD of triplicate measurements.

τ ) n[BHT](Ri)-1

(8)

where τ is the lag period associated with the BHT antioxidant effect and n is the inhibitor number [i.e., mol of peroxyl radical scavenged/mol of antioxidant, n ) 2 for BHT (11, 16)]. The kinetic chain length (λ) (i.e., number of propagation reactions per primordial carboncentered radical generated) was calculated using eq 9 (4):

λ ) (Ri)-1(d[O2]/dt)

(9)

Typical kinetic values characterizing AAPH-initiated autoxidation of 18:2 in our system (T ) 313 K) are Ri ) 7.5 × 10-7 M s-1, λ ) 139, and kp/(2kt)0.5 (olefin oxidizability) ) 3.0 × 10-2 M-0.5 s-0.5. Addition of RA (2.0 × 10-5 M) to the above micellar system resulted in significant stimulation in the rate of dioxygen consumption associated with AAPH-initiated autoxidation of 18:2 (Table 1, Figure 1, left). Substitution of RA with vehicle (0.2%, v/v, Me2SO) did not result in this increased rate of dioxygen uptake (not shown). Furthermore, reaction mixtures of SDS, AAPH, and RA (2 × 10-5, 2 × 10-4, or 2 × 10-3 M), in the absence of 18:2, exhibited only very low rates of O2 uptake which were equivalent to those associated with the thermal decomposition of AAPH and subsequent AAPH-derived peroxyl radical formation. Thus, RA-dependent stimulation of dioxygen uptake does not involve a synergistic AAPH-dependent autoxidation of RA. The RA-dependent stimulation of 18:2 autoxidation was concentration-

dependent, exhibiting a linear relationship of -d[O2]/dt with retinoid concentration (Figure 1, right). Interestingly, 5,6-epoxy-RA (Chart 1), which has been identified as the major RA oxidation product in the present study (see below), failed to stimulate dioxygen uptake at all concentrations tested (Figure 1, right). Thus, retinoiddependent stimulation of dioxygen uptake in this system requires an intact double bond within the substituted cyclohexene ring of the retinoid. We also investigated the effects of RA on peroxyl radical formation using styrene as a peroxyl radical probe. As shown in Scheme 2, peroxyl radicals oxidize styrene to the corresponding oxirane (18). Thus, we compared the extents of styrene epoxidation during AAPH-initiated autoxidation of 18:2 in the presence versus absence of RA. GC/MS analysis of reaction mixtures demonstrated that RA stimulates peroxyl radical generation in our system as evidenced by enhanced peroxyl radical-dependent epoxidation of styrene to styrene oxide, relative to an internal standard (Figure 2). The stimulation in peroxyl radical formation in systems containing RA was approximately 5-fold over the systems devoid of RA. In an attempt to confirm and quantitate the RAdependent stimulation of 18:2 autoxidation, we developed an assay to indirectly measure fatty acid-derived hydroperoxide formation by detecting the fatty acid-derived alcohols, 9-HODE and 13-HODE. As seen in eq 3, autoxidation of 18:2 results in generation of fatty acidderived hydroperoxides (LOOH) which can be reduced to the corresponding alcohols using sodium borohydride. By comparison with synthetic standards and using reverse-phase HPLC analysis, we identified these alcohols from reaction mixtures involving AAPH-initiated autoxidation of 18:2 in SDS micelles. PGB1 was used as the internal standard, the detection wavelength was 235 nm, and the relative amounts of the two alcohols were obtained by calculating the ratios of HPLC peak integration values of (13-HODE + 9-HODE)/PGB1. The presence of RA in these reaction mixtures resulted in a 0.40 ( 0.03 ratio, while the ratio was 0.24 ( 0.01 in the absence of RA (mean ( SD of triplicate measurements). This result is a 1.6 times greater production of 9-HODE plus 13-HODE in the presence of RA compared with reaction mixtures devoid of RA. Interestingly, we have also demonstrated that RA stimulates EPR-detectable free radical formation, using 4-POBN as the spin trap, during AAPH-initiated autoxidation of 18:2 in SDS micelles. In the absence of RA, a relatively minor nitroxide signal was detected (Figure 3B). The low amplitude of this signal likely reflects the low rate of 18:2 autoxidation as well as competition of other mechanisms with addition to the nitrone. However, addition of RA to reaction mixtures resulted in an 8.5fold increase in nitroxide adduct formation (trace 3A). The spectrum demonstrated a single adduct which was characterized by hyperfine coupling constants aH and aN of 2.72 and 15.49 G, respectively. These results are similar to published spectra of carbon-centered radical

106 Chem. Res. Toxicol., Vol. 11, No. 2, 1998

Freyaldenhoven et al.

Figure 1. (Left) Effects of RA and 5,6-epoxy-RA on dioxygen consumption associated with AAPH-initiated autoxidation of 18:2 in SDS micelles. The concentrations of 5,6-epoxy-RA and RA were equal (2.0 × 10-5 M), and reaction conditions are as described in Table 1. (Right) Concentration-dependent effects of RA and 5,6-epoxy-RA on the rates of dioxygen uptake associated with AAPHinitiated autoxidation of 18:2 in SDS micelles. The values represent the mean ( SD of triplicate measurements. (Variations in the rate of dioxygen consumption, where no retinoid was present, reflect the fact that the two sets of experiments were conducted on different days. High variations have been noted previously in experiments conducted on different days.)

Figure 2. GC analysis of styrene oxide, generated by peroxyl radical-dependent epoxidation of styrene during AAPH-initiated autoxidation of 18:2 in SDS micelles in the presence (upper) and absence (lower) of RA. IS, internal standard (2-undecanone). The reaction conditions and analytical methods are described in the Materials and Methods section.

adducts of 4-POBN (19-22). In contrast, AAPH alone failed to yield EPR-detectable nitroxide radical formation (trace 3C) indicating that the AAPH-derived carboncentered radicals did not contribute to the observed signals. In addition, reaction mixtures of AAPH plus RA (in the absence of 18:2) failed to yield detectable nitroxide formation (trace 3D). This result is consistent with the dioxygen uptake experiment demonstrating insignificant rates of O2 uptake in the absence of 18:2. Radiolabeled RA ([3H]RA) was employed in order to detect and quantitate RA oxidation products produced during AAPH-initiated autoxidation of 18:2. Radiochemical detection was necessary because the various retinoid products and starting material exhibit differences in UV absorption characteristics (data not shown). The mixtures were extracted with ethyl acetate, and analysis of the ethyl acetate-extractable material was performed using reverse-phase HPLC and scintillation spectrometry. Reaction mixtures of 18:2, [3H]RA, and AAPH in SDS-containing buffer demonstrated the formation of several RA-derived reaction products (Figure 4,

Figure 3. EPR spectra of nitroxide radical adducts generated during AAPH (3 × 10-3 M)-initiated autoxidation of 18:2 (3 × 10-3 M) in SDS (1.5 × 10-2 M) micelles in 0.05 M phosphate buffer (pH 7.0) at 40 °C, in the presence and absence of RA (2.0 × 10-4 M), using 4-POBN (5 × 10-2 M) as the spin trap. EPR instrumental parameters: field center ) 3350 G, modulation amplitude ) 2.0 G, time constant ) 0.5 s, gain ) 104, and power ) 20 mW. Traces: A, complete system plus RA; B, complete system minus RA; C, AAPH alone in SDS/buffer system; D, AAPH plus RA (minus 18:2) in SDS/buffer system. Spectral acquisitions were carried out at ambient temperature.

upper) which were not significantly detectable in the absence of 18:2 (Figure 4, lower). The latter result is consistent with the dioxygen uptake experiments demonstrating insignificant rates of O2 uptake in the absence of 18:2 as well as the EPR experiment resulting in undetectable nitroxide formation in the absence of 18:2. Not considering the isomerization of RA, the extent of

Prooxidant Effects of Retinoic Acid

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 107

Figure 5. Electronic absorption spectrum (upper) and PCI mass spectrum (lower) of RA oxidation product that cochromatographed with synthetic 5,6-epoxy-RA in Figure 4 (upper) (tR ) 34.2 min).

Figure 4. Reverse-phase HPLC analysis of [3H]RA (2 × 10-4 M)-derived oxidation products generated during AAPH (3 × 10-3 M)-initiated autoxidation of 18:2 (3 × 10-3 M) in SDS (1.5 × 10-2 M) micelles in 0.05 M phosphate buffer (pH 7.0) (upper) as compared with reaction mixtures devoid of 18:2 (i.e., AAPH + [3H]RA in SDS micelles) (lower). The reaction conditions and analytical methods are described in the Materials and Methods section.

RA oxidation in the absence of 18:2 was ∼3.8%, whereas the extent of oxidation in the presence of 18:2 was ∼23%. In the presence of 18:2 (Figure 4, upper), peak B represents the major oxidation product at ∼7.6% of RA oxidation, whereas the two peaks labeled A together comprised ∼10% and all other products totaled less than 5%. The major peak eluting at 50.2 min (Figure 4, peak D) represents unreacted [3H]RA, and the smaller peaks eluting around this time point as well as those at 46.1 min (peak C) represent (13Z)-RA and other geometric isomers of RA (5, 6, 23). Peak B, with a retention time of 34.2 min, was identified as 5,6-epoxy-RA, on the basis of cochromatography with synthetic standard. To better characterize this oxidation product, we performed an unlabeled study under the same conditions, collected the HPLC-eluted material with the identical retention time, and confirmed product identity on the basis of electronic absorption spectroscopy and mass spectrometry. The oxidation product exhibited a UV spectrum identical with that of the synthetic 5,6-epoxyRA, characterized by an absorption maximum at 330 nm (Figure 5, upper). The positive chemical ionization mass spectrum of the corresponding methyl ester, synthesized by reaction of the isolated product with ethereal diazo-

methane, exhibited an M + 1 and molecular ion characterized by m/z 331 and 330, respectively (Figure 5, lower), which correspond to the methyl ester of 5,6-epoxy-RA. Furthermore, the fragmentation pattern is consistent with the mass spectrum characterizing methyl 5,6epoxyretinoate (5, 6, 23). Other minor RA oxidation products were also detected in the presence of 18:2, which were not characterized. It should be noted that the quantitative extent of RA oxirane formation, the effects of RA on 4-POBN-derived nitroxide formation, and the RA-dependent stimulation of styrene oxide formation (where [RA] ) 2.0 × 10-4 M in each system) cannot be directly compared with RA-dependent effects on rates of dioxygen consumption [[RA] ) (0-2.0) × 10-5 M] or RAdependent stimulation of 18:2-derived hydroperoxide formation ([RA] ) 5.0 × 10-5 M) because the concentrations of RA were not identical in all experimental systems.

Discussion We report evidence that RA functions as a prooxidant in a micellar system consisting of 18:2 in SDS micelles and the water-soluble, azo initiator AAPH. RA was shown to stimulate the rate of dioxygen consumption associated with hydrocarbon autoxidation, which was linear with retinoid concentration, whereas the major RA oxidation product, 5,6-epoxy-RA, failed to affect dioxygen uptake at all concentrations tested (Figure 1, Table 1). RA also stimulated peroxyl radical-dependent oxidation of styrene to styrene oxide when styrene was included as a peroxyl radical probe (Figure 2). Evidence that RA actually stimulated 18:2 autoxidation was demonstrated by RA-dependent stimulation of 18:2-derived hydroperoxide formation, which was quantitated indirectly by measuring relative amounts of 9- and 13-HODE formation. In addition, EPR spin-trapping experiments (using 4-POBN as the spin trap) demonstrated RA-dependent stimulation of free radical formation which may represent carbon-centered radicals (Figure 3). The major RA

108 Chem. Res. Toxicol., Vol. 11, No. 2, 1998

oxidation product was identified as 5,6-epoxy-RA on the basis of HPLC analysis along with radiochemical detection, cochromatography with synthetic standard, UV spectroscopy, and PCI mass spectrometric analysis of the corresponding methyl ester (Figures 4 and 5). The observed prooxidant effects of RA in this SDS micellar system may occur by an RA-dependent increase in the rate of initiation, by a decrease in the rate of termination, or by increasing the apparent rate constant of propagation according to eq 7. In a separate study, we measured and compared the effects of RA (2.0 × 10-5 M) on both the rate of dioxygen uptake, associated with AAPH-initiated autoxidation of 18:2, and the rate of initiation using the inhibitor method described above. RA was shown to approximately double the rate of dioxygen uptake which is consistent with the results in Figure 1 and Table 1. However, the measured rates of initiation in the absence versus presence of RA were similar and were characterized by values of (1.7 ( 0.2) × 10-7 and (2.7 ( 0.2) × 10-7 M s-1, respectively. The differences appear to be statistically significant, based on the Student’s t-test (p e 0.05), and may result from limitations of the inhibitor method for calculating Ri in this system; i.e., 18:2-derived peroxyl radicals, arising from the “primordial” 18:2-derived carbon-centered radicals generated during the initiation phase, may compete for reaction with the antioxidant BHT versus RA which may ultimately affect the measured lag phase (τ, eq 8). Thus, use of the inhibitor method may not accurately reflect the true rate of initiation in such a complex system. Regardless of the latter, the ratio of calculated Ri values in the presence versus absence of RA is 1.6 ( 0.4. A doubling of rates of dioxygen uptake would necessarily require a 4-fold increase in the rate of initiation because d[O2]/dt is proportional to the square root of Ri (eq 7). Our results, however, do not reflect a 4-fold increase in Ri (the square root of 1.6 is only 1.26). Collectively, these results suggest that addition of RA to the SDS/18:2/ AAPH system results in the stimulation of the kinetic chain length (i.e., number of propagation cycles per primordial 18:2-derived carbon-centered radicals generated during initiation) according to eq 9. Furthermore, an effect of RA on the rate of termination is unlikely. Thus, the major kinetic contribution of RA, regarding its demonstrated prooxidant effects, probably occurs via an increased apparent rate of propagation. We believe that this occurs as a consequence of chain-transfer mechanisms, involving the intermediacy of 18:2-derived alkoxyl radicals, as discussed below. We have recently demonstrated that chemically generated peroxyl radicals react regiospecifically with RA by addition to C5, yielding almost exclusively the 5,6-epoxide (24). These results, taken together with the demonstration that the oxirane represents the major RA-derived oxidation product in the current system, are consistent with the mechanisms depicted in Scheme 3. The 18:2derived peroxyl radicals, generated during AAPH-initiated autoxidation, react with RA yielding 5,6-epoxy-RA and 18:2-derived alkoxyl radicals (LO•). These results are consistent with the work of Mayo et al. demonstrating preferential reactivity of peroxyl radicals with conjugated allylic centers by addition rather than H atom abstraction mechanisms (25). In addition, the rate-limiting step characterizing peroxyl radical epoxidation of alkenes appears to be represented by the addition reaction rather than subsequent β-scission of the peroxide moiety (26,

Freyaldenhoven et al. Scheme 3

27). The rate law characterizing hydrocarbon autoxidation can be expressed by eq 7, where kp e 102 M-1 s-1 (4). From eq 7 and applying a steady-state approximation, where the rate of initiation must equal the rate of termination, the steady-state concentration of peroxyl radicals generated during 18:2 autoxidation in our system is defined by eq 10 (4):

Prooxidant Effects of Retinoic Acid

[LOO•] ) (Ri/2kt)0.5

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 109

(10)

If we assume that peroxyl radical addition to the RA olefinic site is rate-limiting during peroxyl radicaldependent oxirane formation, then:

d[5,6-epoxide]/dt ) d[LO•]/dt ) ko[RA](Ri/2kt)0.5 (11) where ko is the rate constant characterizing the reaction of LOO• with C5 of RA and LO• represents the 18:2derived alkoxyl radical. The rate constant characterizing this reaction (ko) is estimated to be greater than kp by over 2 orders of magnitude (27). Alkoxyl radicals are considerably more reactive compared with peroxyl radicals. For example, the homogeneous liquid-phase reaction of tert-butoxyl with olefins containing allylic centers occurs by both H atom abstraction and addition reactions which are characterized via bimolecular rate constants which are g106 M-1 s-1 (28). The reaction of tert-butoxyl with 18:2 is characterized by rate constants of 1.6 × 108 and 1.3 × 107 M-1 s-1 for reactions in aqueous solutions and nonpolar solvents, respectively (29). However, the 18:2-derived alkoxyl radical (LO•) in our system probably does not directly participate in propagation reactions because Marnett and co-workers have demonstrated that it rapidly cyclizes yielding epoxyallylic carbon-centered radicals (Scheme 3) (30-32). Although the rate constant for this reaction has not been determined, it is estimated to be considerably higher compared with rate constants characterizing the bimolecular reaction of alkoxyl radicals with olefins because the reaction is intramolecular. In fact, this cyclization reaction is so rapid that the latter investigators were unable to reduce the alkoxyl radical [generated by reaction of 13-hydroperoxy-(9Z,11E)-octadecadienoic acid with hydroxoporphyrinatoiron(III)] and to detect the corresponding alcohol (13-HODE) in reaction systems containing an excess of 2,4,6-tri-tert-butylphenol (32); the rate constant of the latter reaction is estimated to be ∼1010 M-1 s-1 (33). On the other hand, at present we cannot completely rule out the involvement of the primary 18:2-derived alkoxyl radicals (LO•) because of possible unique solvent cage effects imposed in our micellar system. The generation of epoxyallylic carboncentered radicals may, in part, account for the RAdependent stimulation of EPR-detectable, 4-POBNderived nitroxide adducts (Figure 3) which exhibit a hyperfine structure characteristic of carbon-centered radical adducts (19-22). These carbon-centered radicals can diffuse from the solvent cage and react with dioxygen at diffusion-limited rates yielding epoxyallylic peroxyl radicals (L′OO•). Ultimately, the latter reaction may partially account for the RA-dependent stimulation of rates of dioxygen uptake [and would be expected to be first-order with respect to [RA] (Figure 1, right)] because the rate-limiting step is represented by peroxyl radical generation during propagation reactions (eq 3) associated with AAPH-initiated autoxidation of 18:2. However, the latter chemistry cannot account for our demonstration of RA-dependent stimulation of peroxyl radical-dependent oxidation of styrene (Figure 2) and stimulated 18:2 autoxidation because, as shown in Scheme 3, this mechanism does not increase the net stoichiometry of peroxyl radicals. We believe that the RA-dependent prooxidant effect may be explained if L′OO• reacts with another equivalent of RA yielding the corresponding

secondary alkoxyl radical (L′O•) and 5,6-epoxy-RA. Unlike the dienylalkoxyl radical (LO•), C-O• functions R to a single double bond do not appear to undergo cyclization reactions. Instead, they are expected to react bimolecularly with 18:2 by H atom abstraction/addition mechanisms or to undergo intramolecular β-scission reactions (2). Alternatively, this secondary alkoxyl radical may react bimolecularly with RA by addition (or H atom abstraction) mechanisms yielding RA-derived carboncentered radicals [L′O-(RA)•] and ultimately RA-derived peroxyl radicals [L′O-(RA)-OO•]. These oxidation products may ultimately represent the unidentified RA products detected by radiochemical HPLC analysis (Figure 4). The alkoxyl radical addition and subsequent dioxygen coupling mechanism are represented by eqs 12 and 13, respectively:

L′O• + RA f L′O-(RA)•

(12)

L′O-(RA)• + O2 f L′O-(RA)-OO•

(13)

Analogous to our system, Liebler and McClure have demonstrated, employing LC/MS/MS analysis, the generation of β-carotene-alkoxyl radical addition products during the azobis(2,4-dimethylvaleronitrile)-dependent autoxidation of the carotenoid in a homogeneous liquidphase system (34). Collectively, mechanisms involving the reaction of 18:2-derived alkoxyl radicals with 18:2 and/or RA, whether as LO• or L′O• in Scheme 3, may account for the prooxidant effects of RA exhibited during AAPH-initiated autoxidation of 18:2 in SDS micelles. This is because the bimolecular rate constants characterizing the reaction of alkoxyl radicals by olefinic addition and allylic H atom abstraction mechanisms are over 4 orders of magnitude higher than the rate constant (kp) characterizing peroxyl radical-dependent H atom abstraction from 18:2 bis(allylic) centers. This would mechanistically account for our observed RA-dependent increase in the kinetic chain length as well as RAdependent stimulation of peroxyl radical-dependent oxidation of styrene and enhanced 18:2 autoxidation. In general, this provides a kinetic basis for the observed prooxidant effects of the retinoid in our system. It is interesting that RA functions as a prooxidant in our micellar system whereas RA and other retinoids function as antioxidants in liposomal or microsomal lipid bilayers. Possible explanations for this paradoxical behavior may involve differences in solvent cage effects. For example, lipid bilayers may promote cross-termination reactions (eqs 14-16),

L(O)O• + RA f L(O)O-(RA)•

(14)

L(O)O-(RA)• + LOO• f L(O)O-(RA)-OOL

(15)

L(O)O-(RA)• + L• f L(O)O-(RA)-L

(16)

where L(O)O• represents a polyunsaturated fatty acidderived alkoxyl or peroxyl radical. This mechanism has been proposed by Liebler and McClure to account for the antioxidant effects of carotenoids (34). In addition, lipid bilayer systems do not exhibit significant interliposomal/ microsomal diffusion of phospholipids, whereas our micellar system has been shown to demonstrate significant intermicellar diffusion of the 18:2-derived peroxyl radicals (11). Support for the possibility that retinoids

110 Chem. Res. Toxicol., Vol. 11, No. 2, 1998

function as antioxidants in lipid bilayers involving crosstermination mechanisms will require structural identification of retinoid adducts in liposomal systems. Our demonstration of the prooxidant effects of RA in micellar systems may be toxicologically relevant because high concentrations of retinoids are cytotoxic (35) and have been shown to severely affect membrane structure and fluidity as evidenced on the basis of EPR spin-labeling studies (36), fluorescence spectroscopic investigations (37), and 2H and 31P NMR studies (38) involving model membrane systems. These perturbations may result in the generation of significant micellar structures within the membrane which may in turn result in susceptibility to the demonstrated prooxidant effects of RA.

Acknowledgment. This study was supported by a grant from the NIH (5 R29 ES06765-03). Dr. Freyaldenhoven is the recipient of the Kappa Epsilon-Nellie Wakeman-American Foundation for Pharmaceutical Education First-Year Graduate Scholarship.

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