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

Article abstract of DOI:10.1021/tx970044u

(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 α-(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-epoxy-RA, 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.

Full text of DOI:10.1021/tx970044u

102
Chem. Res. Toxicol. 1998, 11, 102-110
Retin oic Acid -Dep en d en t Stim u la tion of
2,2′-Azobis(2-a m id in op r op a n e)-In itia ted Au toxid a tion of
Lin oleic Acid in Sod iu m Dod ecyl Su lfa te Micelles: A
Novel P r ooxid a n t Effect of Retin oic 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 carbon-
centered 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-epoxy-
RA, 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.
LH f L•
(1)
(2)
In tr od u ction
It is generally accepted that lipid peroxidation is
associated with the toxicities of many xenobiotics (e.g.,
paraquat, adriamycin, CCl4) as well as various patholo-
gies (e.g., atherogenesis, carcinogenesis, arthritis). A
simplified version of the major mechanisms involved in
lipid peroxidation, also termed autoxidation of polyun-
saturated fatty acids, is represented by eqs 1-6, involving
initiation (eq 1), propagation (eqs 2 and 3), and termina-
tion (eqs 4-6) reactions. LH represents a polyunsatu-
rated fatty acid, L• represents a polyunsaturated fatty
acid-derived carbon-centered radical, and LOO• repre-
sents 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•
kp
9
LOO• + LH
8 LOOH + L•
(3)
(4)
2L• f nonradical products
L• + LOO• f nonradical products
2LOO• f nonradical products
(5)
(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 reac-
tions 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
* Address correspondence to this author at the Division of Toxicol-
ogy, 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: victors@biomed.uams.edu.
§ Department of Pharmacology & Toxicology (UAMS).
‡ Department of Dermatology (UAMS).
† University of Memphis.
S0893-228x(97)00044-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

Prooxidant Effects of Retinoic Acid
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-undec-
anone, butylated hydroxytoluene (BHT), R-(4-pyridyl 1-oxide)-
N-tert-butylnitrone (4-POBN), sodium borohydride, 1-methyl-
3-nitro-1-nitrosoguanidine, and Diazald (N-methyl-N-nitroso-
p-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 es-
sentially as described by Wertz et al. (12): EI-MS m/z 330 (M•+),
mechanism of action of antioxidants is to scavenge
peroxyl radicals, resulting in the termination of propaga-
tion reactions and thereby inhibiting hydrocarbon au-
toxidation.
Retinoids, metabolic and synthetic derivatives of vita-
min A, have been shown to function as effective antioxi-
dants 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 peroxi-
dation in vitro in rat brain mitochondria (7), and Tesori-
ere 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 doxorubicin-
induced peroxidation of heart and brain tissue mem-
branes (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 character-
izing 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.
1
315 (M•+ - CH3), 299 (M•+ - OCH3), 271 (M•+ - CO2CH3); H
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 Up ta k e Kin etics. 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 autoxi-
dation. 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.
Styr en e Ep oxid a tion Assa y. 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.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. (E)-Retinoic acid (all-trans) (RA) and 13-cis-
retinoic acid [(13Z)-RA] were purchased from ACROS Organics
(Pittsburgh, PA), and (E)-[11,12-3H]retinoic acid ([3H]RA), spe-
cific 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 per-
formed 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 com-
pounds were carried out in the dark or under yellow light. 2,2′-
Azobis(2-amidinopropane) dihydrochloride (AAPH) was pur-
chased from Monomer-Polymer Laboratories (Windham, NH)
Rela tive Qu a n tita tion of 18:2 Au toxid a tion . 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 acid-
derived alcohols, 13-HODE and 9-HODE, relative to an internal
standard. All glassware used for these experiments was tri-
methylsilylated 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
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-di-
tert-butyl-4-methylphenol); 4-POBN, R-(4-pyridyl 1-oxide)-N-tert-bu-
tylnitrone; AIBN, azobis(isobutyronitrile); 9-HODE, 9-hydroxy-(10E,-
12Z)-octadecadienoic acid; 13-HODE, 13-hydroxy-(9Z,11E)-octadeca-
dienoic acid; PGB1, prostaglandin B1; EI, electron impact; PCI, positive
chemical ionization.

104 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
Freyaldenhoven et al.
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 Mil-
lennium 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% trifluoro-
acetic 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 integra-
tion relative to the internal standard and are expressed as (13-
HODE + 9-HODE peak areas)/(PGB1 peak area).
EP R Sp in -Tr a p p in g Exp er im en ts. 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.
Ch a r t 1. Str u ctu r es of (E)-Retin oic Acid (RA) a n d
5,6-Ep oxy-RA
of 1-methyl-3-nitro-1-nitrosoguanidine or Diazald with KOH at
4 °C (14). (Ca u tion : 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 con-
nected 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 spectrom-
eter 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.
An a lysis a n d Qu a n tita tion of RA Oxid a tion P r od u cts
Gen er a ted d u r in g AAP H-In itia ted Au toxid a tion 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
Resu lts
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 carbon-
centered 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 initia-
tors 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):
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 13-
HODE/9-HODE analysis, along with a Packard Radiomatic Flo-
One\ âeta 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.
-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):
5,6-Epoxy-RA was identified on the basis of cochromatogra-
phy 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

Prooxidant Effects of Retinoic Acid
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 105
Sch em e 1
Sch em e 2
dependent, exhibiting a linear relationship of -d[O2]/dt
with retinoid concentration (Figure 1, right). Interest-
ingly, 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, retinoid-
dependent 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 radi-
cal generation in our system as evidenced by enhanced
peroxyl radical-dependent epoxidation of styrene to sty-
rene 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 RA-
dependent stimulation of 18:2 autoxidation, we developed
an assay to indirectly measure fatty acid-derived hydro-
peroxide 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 acid-
derived 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 alco-
hols 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 integra-
tion values of (13-HODE + 9-HODE)/PGB1. The pres-
ence 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.
Ta ble 1. Effect of RA on th e Ra te of Dioxygen
Con su m p tion Associa ted w ith AAP H-In itia ted
Au toxid a tion of 18:2 in SDS Micelles
-d[O2]/dt (M s-1 × 10-9
)
AAPH (3 × 10-3 M) alone
(minus 18:2)
3 ( 0.5a
AAPH + 18:2 (3 × 10-3 M)
62 ( 1
150 ( 5
AAPH/18:2 + RA (2.0 × 10-5 M)
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 carbon-
centered 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 oxidiz-
ability) ) 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 stimula-
tion of dioxygen uptake does not involve a synergistic
AAPH-dependent autoxidation of RA. The RA-dependent
stimulation of 18:2 autoxidation was concentration-
Interestingly, we have also demonstrated that RA
stimulates EPR-detectable free radical formation, using
4-POBN as the spin trap, during AAPH-initiated autoxi-
dation 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 8.5-
fold 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.
F igu r e 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 AAPH-
initiated 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.)
F igu r e 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 carbon-
centered 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
buffer (pH 7.0) at 40 °C, in the presence and absence of RA (2.0
formation (trace 3D). This result is consistent with the
dioxygen uptake experiment demonstrating insignificant
rates of O2 uptake in the absence of 18:2.
F igu r e 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
× 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.
Radiolabeled RA ([3H]RA) was employed in order to
detect and quantitate RA oxidation products produced
during AAPH-initiated autoxidation of 18:2. Radio-
chemical detection was necessary because the various
retinoid products and starting material exhibit differ-
ences 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 forma-
tion of several RA-derived reaction products (Figure 4,
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 dem-
onstrating 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
F igu r e 5. Electronic absorption spectrum (upper) and PCI
mass spectrum (lower) of RA oxidation product that cochro-
matographed with synthetic 5,6-epoxy-RA in Figure 4 (upper)
(tR ) 34.2 min).
methane, exhibited an M + 1 and molecular ion charac-
terized 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 5,6-
epoxyretinoate (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 RA-
dependent stimulation of 18:2-derived hydroperoxide
formation ([RA] ) 5.0 × 10-5 M) because the concentra-
tions of RA were not identical in all experimental
systems.
F igu r e 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-epoxy-
RA, 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-
Discu ssion
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 hydroper-
oxide formation, which was quantitated indirectly by
measuring relative amounts of 9- and 13-HODE forma-
tion. 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
Freyaldenhoven et al.
Sch em e 3
oxidation product was identified as 5,6-epoxy-RA on the
basis of HPLC analysis along with radiochemical detec-
tion, 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 limita-
tions 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 gener-
ated 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 mecha-
nisms, involving the intermediacy of 18:2-derived alkoxyl
radicals, as discussed below.
We have recently demonstrated that chemically gener-
ated peroxyl radicals react regiospecifically with RA by
addition to C5, yielding almost exclusively the 5,6-epoxide
(24). These results, taken together with the demonstra-
tion that the oxirane represents the major RA-derived
oxidation product in the current system, are consistent
with the mechanisms depicted in Scheme 3. The 18:2-
derived peroxyl radicals, generated during AAPH-initi-
ated 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 â-scission of the peroxide moiety (26,
27). The rate law characterizing hydrocarbon autoxida-
tion can be expressed by eq 7, where kp e 102 M-1 s-1
(4). From eq 7 and applying a steady-state approxima-
tion, 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 sys-
tem is defined by eq 10 (4):

Prooxidant Effects of Retinoic Acid
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 109
secondary alkoxyl radical (L′O•) and 5,6-epoxy-RA. Un-
like 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 bimolecu-
larly with 18:2 by H atom abstraction/addition mecha-
nisms or to undergo intramolecular â-scission reactions
(2). Alternatively, this secondary alkoxyl radical may
react bimolecularly with RA by addition (or H atom
abstraction) mechanisms yielding RA-derived carbon-
centered radicals [L′O-(RA)•] and ultimately RA-derived
peroxyl radicals [L′O-(RA)-OO•]. These oxidation prod-
ucts may ultimately represent the unidentified RA
products detected by radiochemical HPLC analysis (Fig-
ure 4). The alkoxyl radical addition and subsequent
dioxygen coupling mechanism are represented by eqs 12
and 13, respectively:
[LOO•] ) (Ri/2kt)0.5
(10)
If we assume that peroxyl radical addition to the RA
olefinic site is rate-limiting during peroxyl radical-
dependent 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 18:2-
derived 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 com-
pared with peroxyl radicals. For example, the homoge-
neous liquid-phase reaction of tert-butoxyl with olefins
containing allylic centers occurs by both H atom abstrac-
tion 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 investiga-
tors 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 pri-
mary 18:2-derived alkoxyl radicals (LO•) because of
possible unique solvent cage effects imposed in our
micellar system. The generation of epoxyallylic carbon-
centered radicals may, in part, account for the RA-
dependent stimulation of EPR-detectable, 4-POBN-
derived 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.
L′O• + RA f L′O-(RA)•
(12)
(13)
L′O-(RA)• + O2 f L′O-(RA)-OO•
Analogous to our system, Liebler and McClure have
demonstrated, employing LC/MS/MS analysis, the gen-
eration of â-carotene-alkoxyl radical addition products
during the azobis(2,4-dimethylvaleronitrile)-dependent
autoxidation of the carotenoid in a homogeneous liquid-
phase 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 character-
izing 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 ab-
straction from 18:2 bis(allylic) centers. This would
mechanistically account for our observed RA-dependent
increase in the kinetic chain length as well as RA-
dependent stimulation of peroxyl radical-dependent oxi-
dation 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-termina-
tion reactions (eqs 14-16),
L(O)O• + RA f L(O)O-(RA)•
L(O)O-(RA)• + LOO• f L(O)O-(RA)-OOL (15)
L(O)O-(RA)• + L• f L(O)O-(RA)-L
(14)
(16)
where L(O)O• represents a polyunsaturated fatty acid-
derived 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 mi-
cellar system has been shown to demonstrate significant
intermicellar diffusion of the 18:2-derived peroxyl radi-
cals (11). Support for the possibility that retinoids
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

110 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
Freyaldenhoven et al.
(16) Boozer, C. E., Hammond, G. S., Hamilton, C. E., and Sen, J . N.
(1955) Air oxidation of hydrocarbons. The stoichiometry and fate
of inhibitors in benzene and chlorobenzene. J . Am. Chem. Soc.
77, 3233-3237.
(17) Barclay, L. R. C., Locke, S. J ., and MacNeil, J . M. (1983) The
autoxidation of unsaturated lipids in micelles. Synergism of
inhibitors vitamins C and E. Can. J . Chem. 61, 1288-1290.
(18) Ortiz de Montellano, P. R., and Grab, L. A. (1987) Cooxidation of
styrene by horseradish peroxidase and phenols: A biochemical
model for protein-mediated cooxidation. Biochemistry 26, 5310-
5314.
(19) Makino, K., Mossoba, M. M., and Riesz, P. (1983) Chemical effects
of ultrasound on aqueous solution. Formation of hydroxyl radicals
and hydrogen atoms. J . Phys. Chem. 87, 1369-1377.
(20) Iwahashi, H., Albro, P. W., McGown, S. R., Tomer, K. B., and
Mason, R. P. (1991) Isolation and identification of POBN radical
adducts formed during oxidation of linoleic acid, linolenic acid,
and arachidonic acid by soybean lipoxidase. Arch. Biochem.
Biophys. 285, 172-180.
function as antioxidants in lipid bilayers involving cross-
termination mechanisms will require structural identi-
fication 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
2
(37), and H 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.
(21) Ortiz de Montellano, P. R., Augusto, O., Viola, F., and Kunz, K.
L. (1983) Carbon radicals in the metabolism of alkyl hydrazines.
J . Biol. Chem. 258, 8623-8629.
(22) Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982)
The catalytic mechanism of cytochrome P-450. Spin trapping
evidence for the one-electron substrate oxidation. J . Biol. Chem.
257, 11288-11295.
Ack n ow led gm en t. This study was supported by a
grant from the NIH (5 R29 ES06765-03). Dr. Freyalden-
hoven is the recipient of the Kappa Epsilon-Nellie Wake-
man-American Foundation for Pharmaceutical Educa-
tion First-Year Graduate Scholarship.
(23) 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.
(24) Samokyszyn, V. M., Freyaldenhoven, M. A., Chang, H. C.,
Freeman, J . P., and Compadre, R. L. (1997) Regiospecificity of
peroxyl radical addition to (E)-retinoic acid. Chem. Res. Toxicol.
10, 795-801.
(25) Mayo, F. R., Miller, A. A., and Russell, G. A. (1958) The oxidation
of unsaturated compounds. IX. The effects of structure on the
rates and products of oxidation of unsaturated compounds. J . Am.
Chem. Soc. 80, 2500-2507.
(26) Porter, N. A., and Nixon, J . R. (1978) Stereochemistry of free
radical substitution on the peroxide bond. J . Am. Chem. Soc. 100,
7116-7117.
(27) Dix, T. A., Fontana, R., Panthani, A., and Marnett, L. J . (1985)
Hematin-catalyzed epoxidation of 7,8-dihydroxy-7,8-dihydroben-
zo[a]pyrene by polyunsaturated fatty acid hydroperoxides. J . Biol.
Chem. 260, 5358-5365.
(28) Wong, P. C., Griller, D., and Scaiano, J . C. (1982) A kinetic study
of the reactions of tert-butoxyl with alkenes: Hydrogen abstrac-
tion vs addition. J . Am. Chem. Soc. 104, 5106-5108.
(29) Forni, L. G. (1990) Free radical reactions involving fatty acids:
The pulse radiolysis approach. In Membrane Lipid Oxidation,
Volume 1 (Vigo-Pelfrey, C., Ed.) pp 15-32, CRC Press, Boca
Raton, FL.
(30) Dix, T. A., and Marnett, L. J . (1985) Conversion of linoleic acid
hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy
fatty acids by hematin. J . Biol. Chem. 260, 5351-5357.
(31) Dix, T. A., and Marnett, L. J . (1981) Free radical epoxidation of
7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by hematin and poly-
unsaturated fatty acid hydroperoxides. J . Am. Chem. Soc. 103,
6744-6746.
(32) Wilcox, A. L., and Marnett, L. J . (1993) Polyunsaturated fatty
acid alkoxyl radicals exist as carbon-centered epoxyallylic radi-
cals: A key step in hydroperoxide-amplified lipid peroxidation.
Chem. Res. Toxicol. 6, 413-416.
(33) Bors, W., Tait, D., Michel, C., Saran, M., and Erben-Russ, M.
(1984) Reactions of alkoxy radicals in aqueous solutions. Israel
J . Chem. 24, 17-24.
(34) Liebler, D. C., and McClure, T. D. (1996) Antioxidant reactions
of â-carotene: Identification of carotenoid-radical adducts. Chem.
Res. Toxicol. 9, 8-11.
(35) Armstrong, R. B., Ashenfelter, K. O., Eckhoff, C., Levin, A. A.,
and Shapiro, S. S. (1994) General and reproductive toxicology of
retinoids. In The Retinoids. Biology, Chemistry, and Medicine, 2nd
ed. (Sporn, M. B., Roberts, A. B., and Goodman, D. S., Eds.) pp
545-572, Raven Press, New York.
(36) Wassall, S. R., Phelps, T. M., Albrecht, M. R., Langsford, C. A.,
Stillwell, W. (1988) Electron spin resonance study of the interac-
tions of retinoids with a phospholipid model membrane. Biochim.
Biophys. Acta 939, 393-402.
(37) Meeks, R. G., Zaharevitz, D., Chen, R. F. (1981) Membrane effects
of retinoids: Possible correlation with toxicity. Arch. Biochem.
Biophys. 207, 141-147.
(38) De Boeck, H., and Zidovetzki, R. (1988) NMR study of the
interaction of retinoids with phospholipid bilayers. Biochim.
Biophys. Acta 946, 244-252.
Refer en ces
(1) Porter, N. A. (1990) Autoxidation of polyunsaturated fatty acids:
Initiation, propagation, and product distribution (basic chemistry).
In Membrane Lipid Oxidation, Volume 1 (Vigo-Pelfrey, C., Ed.)
pp 33-62, CRC Press, Boca Raton, FL.
(2) Gardner, H. W. (1989) Oxygen radical chemistry of polyunsatu-
rated fatty acids. Free Radical Biol. Med. 7, 65-86.
(3) Maillard, B., Ingold, K. U., and Scaiano, J . C. (1983) Rate
constants for the reactions of free radicals with oxygen in solution.
J . Am. Chem. Soc. 105, 5095-5099.
(4) Pryor, W. A. (1984) Free radicals in autoxidation and in aging.
Part I. Kinetics of the autoxidation of linoleic acid in SDS
micelles: Calculations of radical concentrations, kinetic chain
lengths, and the effects of vitamin E. Part II, The role of radicals
in chronic human diseases and in aging. In Free Radicals in
Molecular Biology, Aging and Disease: Aging (Armstrong, D.,
Sohal, R. S., Cutler, R. G., and Slater, R. F., Eds.) pp 1-26, Raven
Press, New York.
(5) Samokyszyn, V. M., and Marnett, L. J . (1990) Inhibition of liver
microsomal lipid peroxidation by 13-cis-retinoic acid. Free Radical
Biol. Med. 8, 491-496.
(6) Samokyszyn, V. M., and Marnett, L. J . (1990) Inhibition of
microsomal lipid peroxidation by 13-cis-retinoic acid. Methods
Enzymol. 190, 281-288.
(7) Das, N. P. (1989) Effects of vitamin A and its analogues on
nonenzymatic lipid peroxidation in rat brain mitochondria. J .
Neurochem. 52, 585-588.
(8) Tesoriere et al. (1993) Antioxidant activity of all-trans-retinol in
homogeneous solution and in phosphatidylcholine liposomes.
Arch. Biochem. Biophys. 307, 217-223.
(9) Ciaccio, M., Valenza, M., Tesoriere, L., Bongiorno, A., Albiero,
R., and Livrea. (1993) Vitamin A inhibits doxorubicin-induced
membrane lipid peroxidation in rat tissues in vivo. Arch. Biochem.
Biophys. 302, 103-108.
(10) Finkelshtein, E. I., Rubashinskaya, Y. F., and Kozlov, Y. I. (1981)
Kinetics and mechanism of the autoxidation of retinoic acid and
its methyl ester. Zh. Org. Khim. 17, 936-943 (in Russian).
(11) Castle, L., and Perkins, M. J . (1986) Inhibition kinetics of chain-
breaking phenolic antioxidants in SDS micelles. Evidence that
intermicellar diffusion rates may be rate-limiting for hydrophobic
inhibitors such as R-tocopherol. J . Am. Chem. Soc. 108, 6381-
6382.
(12) Wertz, P. W., Kensler, T. W., Mueller, G. C., Verma, A. K., and
Boutwell, R. K. (1979) 5,6-Epoxyretinoic acid opposes the effects
of 12-O-tetradecanoylphorbol-13-acetate in bovine lymphocytes.
Nature 277, 227-229.
(13) Duling, D. (1994) Simulation of multiple spin-trap EPR spectra.
J . Magn. Reson., Series B 104, 105-10.
(14) Black, T. H. (1983) The preparation and reactions of diazo-
methane. Aldrichim. Acta 16, 3-10.
(15) Barclay, L. R. C., Locke, S. J ., MacNeil, J . M., VanKessel, J .,
Burton, G. W., and Ingold, K. U. (1984) Autoxidation of micelles
and model membranes. Quantitative kinetic measurements can
be made by using either water-soluble or lipid-soluble initiators
with water-soluble or lipid-soluble chain breaking antioxidants.
J . Am. Chem. Soc. 106, 2479-2481.
TX970044U