Comparative metabolism of N-tert-butyl-N-[1-(1-oxy-pyridin-4-yl)-ethyl]- and N-tert-butyl-N-(1-phenyl-ethyl)-nitroxide by the cytochrome P450 monooxygenase system

Article abstract of DOI:10.1021/tx025510g

The use of spin-trapping agents for a direct ESR detection of ·OH in biological systems is limited by the low stability of the hydroxyl radical-derived nitroxides. Among the various probes used for trapping of ·OH, DMSO has proven to be highly efficient.

Full text of DOI:10.1021/tx025510g

Chem. Res. Toxicol. 2002, 15, 749-753
749
Com p a r a tive Meta bolism of
N-ter t-Bu tyl-N-[1-(1-oxy-p yr id in -4-yl)-eth yl]- a n d
N-ter t-Bu tyl-N-(1-p h en yl-eth yl)-n itr oxid e by th e
Cytoch r om e P 450 Mon ooxygen a se System
Christo P. Novakov and Detcho A. Stoyanovsky*
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
Received J anuary 14, 2002
The use of spin-trapping agents for a direct ESR detection of ‚OH in biological systems is
limited by the low stability of the hydroxyl radical-derived nitroxides. Among the various probes
used for trapping of ‚OH, DMSO has proven to be highly efficient. The reaction between ‚OH
and DMSO yields methyl radical (CH3‚), which can react with N-tert-butyl-R-phenylnitrone
(PBN) and R-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) to form stable, ESR-detectable
nitroxides. The latter approach has been successfully used in in vivo experiments for analysis
of ‚OH; in these experiments, POBN/‚CH3 and PBN/‚CH3 were detected in the bile duct and
the urine of the treated animals. However, the sites of generation of ‚OH produced in vivo are
unknown. Currently, no ESR data is available for the formation of ‚OH in liver of animals
subjected to oxidative stress. Since nitroxides containing aromatic rings are likely to be
substrates of cytochrome P450, experiments were carried out for assessing the ability of the
cytochrome P450 monooxygenase system to metabolize PBN/‚CH3 and POBN/‚CH3, respectively.
In the presence of NADPH, rat liver microsomes catalyzed the reduction of POBN/‚CH3 to the
corresponding hydroxylamine (POBN/CH3), while PBN/‚CH3 was metabolized without ac-
cumulation of its hydroxylamine form (PBN/CH3). The metabolism of PBN/‚CH3 was inhibited
by 4-methylpyrazole and ketoconasole, suggesting that cytochrome P450-catalysis was required
for the consumption of this nitroxide. Under anaerobic conditions, both the nitroxide and
hydroxylamine forms of PBN/CH3 were metabolized, implying that these adducts may undergo
reductive cytochrome P450-catalyzed biotransformation. On the basis of the susceptibility of
PBN/‚CH3 to undergo irreversible metabolic transformation, it is discussed that POBN may
prove to be a more efficient spin-trapping agent for the in vivo detection of ‚OH.
hydroxyl radical-derived nitroxides (6, 7). Among the
various scavengers used for trapping of ‚OH, dimethyl
sulfoxide (DMSO) has proven to be highly efficient.
DMSO can be tolerated by living systems in up to 1 M
concentrations (8) and has an appreciable rate of interac-
tion with hydroxyl radical [k ) 7 × 109 M-1 s-1 (9)]. The
reaction between ‚OH and DMSO yields methyl radical,
CH3‚ (9), which can react with PBN and POBN to form
stable, ESR detectable nitroxides (PBN/‚CH3 and POBN/
‚CH3, respectively; Scheme 1).
Recently, Burkitt and Mason reported that the bile
duct of rats treated with iron, DMSO and PBN contained
PBN/‚CH3 (10, 11). This observation suggests that the
hepatic metabolism of iron could be paralleled by the
generation of ‚OH. However, direct ESR evidence for the
formation of PBN/‚CH3 in the liver of animals subjected
to iron overload has not been presented yet. This may
be due to the reduction of the corresponding spin adducts
to ESR-silent hydroxylamines (Scheme 1) and/or their
biotransformation to secondary metabolites. Hence, ex-
periments were carried out to evaluate the NADPH-
dependent rat liver microsomal metabolism of POBN/‚
In tr od u ction
Superoxide ion (O2•-) is an abundant metabolic product
that can cause substantial cellular damage. Although
O2 is a rather inert radical [i.e., a poor H-abstractor
•-
(1)], its reaction with transition metal complexes, or its
self-termination to H2O2 followed by iron-catalyzed gen-
eration of hydroxyl radical [‚OH; Fenton reaction (2)] can
potentiate its toxicological impact. The Fenton reaction
has been implied in the pathogenesis of numerous disease
states (3). Direct detection of ‚OH has proven to be
difficult as this species is highly reactive; ‚OH interacts
indiscriminately with low molecular weight compounds,
lipids, and proteins present in cells in a diffusion-
controlled manner. The methods for detection of ‚OH in
biological systems are based on the introduction of a
molecular probe that successfully competes with the
cellular molecules for ‚OH, forms stable hydroxyl radical-
derived analytes, and is relatively nontoxic (reviewed in
refs 4 and 5).
The ESR spin-trapping technique is the most direct
method for analysis of radical intermediates. While many
spin-trapping agents (e.g., POBN and PBN)1 are rela-
tively nontoxic, their use for a direct detection of ‚OH in
biological systems is limited by the low stability of the
1
Abbreviations: PBN, N-tert-butyl-R-phenylnitrone; POBN, R-(4-
pyridyl-1-oxide)-N-tert-butylnitrone; POBN/‚CH3, N-tert-butyl-N-[1-(1-
oxy-pyridin-4-yl)-ethyl] nitroxide; PBN/‚CH3, N-tert-butyl-N-(1-phenyl-
ethyl) nitroxide; POBN/CH3, N-tert-butyl-N-[1-(1-oxy-pyridin-4-yl)-
ethyl] hydroxylamine; PBN/CH3, N-tert-butyl-N-(1-phenyl-ethyl)
hydroxylamine; ESR, electron spin resonance; EC, electrochemical.
* To whom correspondence should be addressed. Phone: (412) 647-
6087. Fax: (412) 647-5959. E-mail: stoyanovsky@hotmail.com.
10.1021/tx025510g CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/23/2002

750 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Novakov and Stoyanovsky
Sch em e 1
obtained via direct coupling of PBN (0.08 M) with CH3MgBr
(0.3 M) in helium-deaerated, dry diethyl ether (10 mL). After
an incubation of 10 min (20 °C), the excess of the Grignard
reagent was carefully decomposed with 50% ethanol (v/v; 2 mL),
the organic layer was separated, dried over Na2SO4, and rotor-
evaporated (25 °C). The dry residue containing both the nitrox-
ide and hydroxylamine forms of the methylated PBN was
redissolved in methanol and subjected to HPLC purification.
CH3 and PBN/‚CH3, respectively. These experiments
aimed to optimize spin-trapping methodology for the
detection of ‚OH in biological systems.
Ma ter ia ls a n d Meth od s
Rea gen ts. All reagents used were purchased from Sigma
Chem. Co. (St. Louis, MO). The solutions used in the experi-
ments were prepared in deionized and Chelex-100 treated water
or potassium phosphate buffer. Standard rat liver microsomes,
as well as microsomes enriched with cytochrome P450 1A and
3A were purchased from InVitro Technologies, Inc. (Baltimore,
MD). Human P450 reductase microsomes that do not contain
cytochrome P450 and microsomes enriched with cytochrome
P450 2E1 were purchased from Gentest Corporation (Woburn,
MA). Product characterization tables for the microsomes used
could be accessed at http://www.invitrotech.com and http://
www.gentest.com, respectively.
ESR Mea su r em en ts. ESR measurements were performed
on a Bruker ECS106 spectrometer with 50 kHz magnetic field
modulation at room temperature (25 °C). ESR spectrometer
settings were modulation amplitude 0.7 G, scan time 40 s, time
constant 0.64 s, microwave power 20 mW, and receiver gain 1
× 105.
HP LC An a lysis. HPLC was performed with a Waters liquid
chromatograph (Milford, MA). Separation was achieved with a
C-18 reverse phase column (Microsorb, 4.6 mm × 25 cm, 5 µm,
100 A Rainin Instrument Company, Inc., Emeryville, CA). The
mobile phase was saturated with helium and contained 10 mM
lithium perchlorate and either water with 30% (v/v) methanol
for analysis of POBN/CH3, or 70% methanol for analysis of PBN/
CH3. All HPLC analyses were conducted at a flow rate of 1 mL/
min. Electrochemical detection of the POBN/CH3 and PBN/CH3
was carried out at +0.8 V with a LC-4C/CC5 amperometric
system (Bioanalytical Systems, West Lafayette, IN) equipped
with glassy carbon electrode and a Ag/AgCl reference electrode
(12).
Purification of the POBN/CH3 and PBN/CH3 adducts was
carried out with a semipreparative column (C18; 10 mm × 25
cm; Waters, Milford, MA) at a flow rate of 3 mL/min. The mobile
phase was the same as this used for the analytical separations,
except that it did not contain lithium perchlorate. UV detection
was carried out within the wavelength range 200-400 nm using
a SPD-M10VP Shimadzu diode array detector (Kyoto, J apan).
Injection loops of 0.02 mL and 0.2 mL were used for the
analytical and preparative separation, respectively. The metha-
nol from the fractions containing the individual POBN and PBN
adducts was rotor-evaporated at room temperature. The result-
ing water solutions of the adducts were diluted with equal
volume of 0.2 M phosphate buffer (pH 7.4) and used within 1-2
h.
P r epar ation of P OBN/CH3 an d P BN/CH3 Addu cts. POBN/
‚CH3 and PBN/‚CH3 were prepared via ‚OH-dependent oxidation
of DMSO to ‚CH3 in the presence of POBN and PBN, respec-
tively (12). The nitroxide formed after 5 consecutive additions
of 0.02 mL of Fe(NH4)(SO4)2 (0.05 M) into a 1 mL solution of a
spin-trapping reagent (0.02 M), H2O2 (2 mM), EDTA (5 mM),
and DMSO (2%) in 0.1 M phosphate buffer (pH 7.4) was
extracted with 2 × 1 mL of ethyl acetate (POBN/CH3) or 2 × 1
mL of hexane (PBN/CH3). The ethyl acetate (hexane) phase was
separated and the crystals formed after evaporation of the
solvent (stream of nitrogen, 25 °C) were redissolved in 1 mL of
double-distilled water (POBN/CH3) or 1 mL of ethanol. The
solutions of POBN/‚CH3 and PBN/‚CH3 in phosphate buffer (0.1
M, pH 7.4) exhibited ESR spectra with a triplet of doublets. The
hyperfine structure of the ESR spectra was consistent with the
Meta bolism of P OBN/CH3 a n d P BN/CH3 Ad d u cts. Liver
microsomes (2 mg of protein/1 mL of 0.1 M phosphate buffer;
pH 7.4) were incubated at 37° C in a shaking water bath with
NADPH (1 mM) and the corresponding PBN or POBN deriva-
tive. After initiation of the reaction with NADPH, at various
time points a 0.1 mL aliquot of the reaction suspension was
mixed with 0.1 mL of acetonitrile. The resulting suspension was
centrifuged to remove precipitated proteins (5 min × 5000g),
and the supernatant was subjected to HPLC-EC analysis. In
selected experiments, anaerobiosis was achieved via deoxygen-
ation of all reaction solutions with a stream of nitrogen for 15
min at 20 °C. Subsequent additions were made under nitrogen,
the tubes containing the final reaction suspensions were sealed,
and the reactions carried out as described in the Results.
results obtained by Augusto et al. and Saprin and Piette [(aN
)
16.10 G; aH ) 2.77 G) and (aN ) 16.46 G; aH ) 3.36 G,
respectively (13, 14)] and allows the assignment of the nitroxides
as that formed by addition of methyl radical to POBN and PBN,
respectively. Reduction of both POBN/‚CH3 and PBN/‚CH3 to
their hydroxylamine derivatives (POBN/CH3 and PBN/CH3,
respectively) was performed by incubation of the corresponding
nitroxide with ascorbate (5 mM) in 0.1 M phosphate buffer (pH
7.4; 10-40 min of incubation at 25°). Subsequently, the reaction
solution was extracted with ethyl acetate and the crystals
formed after evaporation of the organic solvent (stream of
nitrogen; 25 °C) were redissolved in ethanol. The ethanolic
solutions obtained did not exhibit the typical ESR spectra of
POBN/‚CH3 and PBN/‚CH3, which were observed before the
incubation with ascorbate. The POBN/CH3 and PBN/CH3 ad-
ducts were further purified by semipreparative HPLC and
stored in ethanol at -80° C.
Resu lts
ESR a n d HP LC-EC Mon itor ed Meta bolism of
P OBN/CH3 a n d P BN/CH3 Nitr oxid es. The disappear-
ance of the ESR signal of nitroxides subjected to reduc-
tion appears to be a major obstacle in the bio-application
of the ESR spin-trapping technique. In the presence of
Alternatively, PBN/‚CH3 and PBN/CH3 were prepared as
described in ref 15. Briefly, PBN/‚CH3 and PBN/CH3 were

Metabolism of PBN/ CH3 and POBN/ CH3 Adducts
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 751
F igu r e 2. HPLC-EC-monitored metabolism of PBN/‚CH3 and
POBN/‚CH3 by liver microsomes. All experiments were carried
out for 20 min at 37 °C in 0.1 M phosphate buffer (pH 7.4). (A)
Chromatogram 1, PBN/‚CH3 (3 µM) and microsomes (2 mg of
protein/mL); chromatogram 2, PBN/‚CH3, microsomes, and
NADPH (1 mM); chromatogram 3, PBN/‚CH3, P450 reductase
microsomes lacking cytochrome P450 (2 mg of protein/mL), and
NADPH (1 mM). Peaks I and II reflect the elution of PBN/‚CH3
and PBN/CH3, respectively. (B) Chromatogram 1, POBN/‚CH3
(3.4 µM) and microsomes (2 mg of protein/mL); chromatogram
2, POBN/‚CH3, microsomes, and NADPH (1 mM). Peaks I and
II reflect the elution of POBN/‚CH3 and POBN/CH3, respec-
tively.
F igu r e 1. ESR-monitored metabolism of PBN/‚CH3 and POBN/
‚CH3 by liver microsomes. All reactions were carried out at 25
°C in 0.1 M phosphate buffer (pH ) 7.4) containing rat liver
microsomes (2 mg of protein/mL), NADPH (1 mM) and either
POBN/‚CH3 (0.014 mM; A) or PBN/‚CH3 (0.01 mM; B).
rat liver microsomes, most nitroxides undergo NADH-
and NADPH-dependent reduction to ESR-silent hydroxy-
lamines (R2N-O‚ f R2N-OH). Iannone et al. proposed
that the latter reaction is catalyzed by cytochrome c
reductases without a direct involvement of cytochrome
P450 (16). The corresponding hydroxylamine derivatives
could be reoxidized back to ESR active nitroxides (Scheme
1) with either potassium ferricyanide or O2 in alkaline
solutions (12, 17, 18). However, nitroxides containing
aromatic rings are likely to be substrates of cytochrome
P450. Therefore, experiments were carried out for as-
sessing the ability of the cytochrome P450 monooxyge-
nase system to metabolize PBN/‚CH3 and POBN/‚CH3,
respectively. The biotransformation of these compounds
is of particular interests as they are used as ESR and
HPLC-EC markers of hydroxyl radical.
In the presence of rat liver microsomes and NADPH,
the ESR spectra of both PBN/‚CH3 and POBN/‚CH3
decreased in a time-dependent manner (Figure 1), sug-
gesting the occurrence of reactions with the participation
of the cytochrome P450 monooxygenase system. No
spectral changes were observed in the absence of NADPH
(data not shown). Figure 2A depicts the HPLC-EC profile
of a suspension containing PBN/‚CH3 and rat liver
microsomes (chromatogram 1, peak I); in the presence
of NADPH, PBN/‚CH3 was consumed in a time-dependent
manner without any detectable accumulation of PBN/
CH3 (Figure 2A; chromatogram 2; Figure 3A, open
circles). In the absence of NADPH, PBN/‚CH3 was
relatively stable; no consumption of PBN/‚CH3 was
observed after an incubation of 20 min with rat liver
microsomes at 37 °C (Figure 3A). A cytochrome P450-
dependent consumption of PBN/‚CH3 is suggested by the
formation of PBN/CH3 when the reaction was carried out
with human P450 reductase microsomes that do not
contain cytochrome P450. In these experiments, NADPH
triggered a semiquantitative conversion of PBN/‚CH3 to
the corresponding hydroxylamine (Figure 2A; chromato-
gram 3, peak II). It should be noted that PBN/CH3 is less
efficiently oxidized in the electrochemical cell than PBN/
F igu r e 3. Effects of PBN, 4-methylpyrazole and ketoconasole
on the NADPH-dependent microsomal metabolism of PBN/‚CH3.
All reactions were carried out for 20 min at 37 °C in 0.1 M
phosphate buffer (pH 7.4). The experiments under anaerobic
conditions were performed as outlined in the Materials and
Methods. Quantitation of PBN/‚CH3 was carried out by HPLC-
EC. (A) Consumption of PBN/‚CH3 (4.1 µM) by rat liver
microsomes (2 mg of protein/mL) in the presence of NADPH (1
mM). In these experiments, the metabolism of PBN/‚CH3 was
not paralleled by formation of PBN/CH3 (open circles). (B)
Effects of cytochrome P450 inhibitors on the metabolism of PBN/
‚CH3 (2.6 µM) catalyzed by rat liver microsomes (2 mg of protein/
mL) in the presence of NADPH (1 mM). The data presented are
the mean ( SEM of three independent experiments (n ) 3).
‚CH3. Therefore, the ratio of peaks I and II (Figure 2A,
chromatogram 3) does not reflect the stoichiometry of the
studied analytes (12).
In contrast to PBN/‚CH3, rat liver microsomes plus
NADPH metabolized POBN/‚CH3 (Figure 2B; chromato-
gram 1, peak I) to the corresponding hydroxylamine

752 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Novakov and Stoyanovsky
Ta ble 1. Ra tes of P BN/‚CH3 Meta bolism by Micr osom es
hanced with the advent of ESR spectrometry and the
later development of the spin-trapping technique. The
spin-trapping technique is based on the high rates of
interaction of nitrones or nitroso compounds with radical
species. The paramagnetic nitroxides formed as a result
of the latter interaction exhibit ESR spectra that are
“structural fingerprints” of the parent radical species.
However, direct ESR detection of ‚OH in biological
systems has proven to be difficult as the ‚OH-derived
nitroxides are relatively unstable compounds. The latter
experimental difficulty could be solved with the introduc-
tion of DMSO as a primary molecular probe for the
interception of ‚OH. The reaction between ‚OH and
DMSO yields methanesulfinic acid and CH3‚. Methane-
sulfinic acid can be quantified after derivatization with
diazonium salts (22), while CH3‚ can alkylate PBN and
POBN via 1,3-addition to remarkably stable nitroxides.
The latter approach has been successfully used in in vivo
experiments for detection of cyclosporin A-, iron-, and
copper-dependent generation of ‚OH; in these experi-
ments, POBN/‚CH3 and PBN/‚CH3 were detected in the
bile duct and the urine of the treated animals (11, 23,
24). However, no ESR spin-trapping data is available for
the formation of POBN/‚CH3 and PBN/‚CH3 in liver of
animals subjected to oxidative stress. The reason for this
could be, at least in part, that these nitroxides are
metabolized by the cytochrome P450 monooxygenase
system. To test this hypothesis, we have carried out
experiments for evaluating the potential of liver mi-
crosomes to catalyze the biotransformation of PBN/‚CH3
and POBN/‚CH3, respectively.
En r ich ed w ith Cytoch r om e P 450, Typ es 1A, 3A, a n d 2E1a
rate of PBN/‚CH3 metabolism
[nmol of nitroxide/min/nmol of P450]
cytochrome P450
1A
3A
2E1
0.38 ( 0.04
0.52 ( 0.05
0.29 ( 0.03
a
All reactions were carried out for 20 min at 37 °C in phosphate
buffer (0.1 M; pH 7.4) containing NADPH (1 mM) and liver
microsomes enriched with cytochrome P450. The consumption of
PBN/‚CH3 was monitored by HPLC-EC. The data presented are
the mean ( SEM of three independent experiments (n ) 3).
(POBN/CH3; Figure 2B; chromatogram 2, peak II). In the
absence of NADPH, consumption of POBN/‚CH3 was not
observed. P450 reductase microsomes that do not contain
cytochrome P450 were as efficient in reducing PBN/‚CH3
as the control microsomes. The latter suggests that
POBN/‚CH3 was not metabolized by cytochrome P450 to
any significant extent (data not shown).
Effects of Cytoch r om e P 450 In h ibitor s on th e Ra t
Liver Micr osom a l Meta bolism of P BN/CH3. The
NADPH-dependent rat liver microsomal metabolism of
PBN/‚CH3 was inhibited by 4-methylpyrazole and keto-
conasole (Figure 3B). The latter further supports the
notion that cytochrome P450-catalysis is required for the
metabolism of PBN/‚CH3; at the concentrations used,
both 4-methylpyrazole and ketoconasole are known to
inhibit the activity of most cytochrome P450 isozymes
(19). Recently, Reinke et al. reported that PBN undergoes
a cytochrome P-450 metabolism to 4-hydroxyPBN (20),
suggesting that this nitrone may interfere with the
cytochrome P450-dependent metabolism of PBN/‚CH3.
Indeed, PBN inhibited the metabolism of both PBN/‚CH3
and PBN/CH3 in a concentration-dependent manner
(Figures 3B and 4, respectively). However, this effect was
pronounced at concentrations of PBN that were consider-
ably higher than its LD50 (21). Under anaerobic condi-
tions, both PBN/‚CH3 and PBN/CH3 were metabolized by
rat liver microsomes plus NADPH (Figures 3B and 4,
respectively), suggesting that these adducts may undergo
a reductive cytochrome P450-catalyzed biotransforma-
tion. Cytochrome P450 2E1, 1A, and 3A exhibited similar
activity in metabolizing PBN/‚CH3 (Table 1). The con-
sumption of PBN/‚CH3 by microsomes enriched with
cytochrome P450 2E1, 1A, and 3A was not affected by
superoxide dismutase, catalase, and desferrioxamine,
suggesting that O2-•, H2O2 and metal ions were not
involved in the overall reaction mechanism (data not
shown).
Liver microsomes plus NADPH mediated the conver-
sion of PBN/‚CH3 to ESR-silent products without a
detectable formation of PBN/CH3. However, human P450
reductase microsomes that did not contain cytochrome
P450 reduced PBN/‚CH3 to PBN/CH3, suggesting that
both cytochrome P450 and the cytochrome P450 reduc-
tase can metabolize this nitroxide. The microsomal
metabolism of PBN/‚CH3 was inhibited by 4-methylpyra-
zole and ketoconasole, which further supports the notion
for a preponderant cytochrome P450 catalysis. The
consumption of PBN/‚CH3 by microsomes enriched with
cytochrome P450 2E1, 1A, or 3A was not affected by
superoxide dismutase, catalase, and desferrioxamine,
suggesting that O2-•, H2O2, and ‚OH-like species were
not involved in the overall reaction mechanism (25).
Under anaerobic conditions, NADPH triggered microso-
mal consumption of both PBN/‚CH3 and PBN/CH3, which
implies that these adducts may undergo a reductive
cytochrome P450-catalyzed biotransformation. Since the
metabolism of PBN/‚CH3 was not paralleled by changes
in its ESR spectrum, it could be speculated that cyto-
chrome P450 modified the side chain of the nitroxide
rather than hydroxylated its aromatic ring; mechanisti-
cally, the latter reaction may proceed via a reductive
cleavage of the N-CR function of PBN/‚CH3 (Scheme 2).
Discu ssion
The results of this work provide evidence that the ESR
markers of hydroxyl radical, PBN/‚CH3, and POBN/‚CH3
could be metabolized by the cytochrome P450 monooxy-
genase system. In the presence of NADPH, liver mi-
crosomes reduced POBN/‚CH3 to POBN/CH3, whereas
PBN/‚CH3 was metabolized to ESR-silent product(s)
without a detectable formation of its hydroxylamine form.
Studies of oxidative stress have attracted considerable
interest and have been the focus of much research in
recent years. Cumulative oxidative damage to tissues has
been implicated in a number of disease states, e.g. the
aging process, cancer, and ischemia reperfusion. In the
last two decades, the understanding of the molecular
mechanisms of bio-oxidative damage was strongly en-
Contrary to PBN/‚CH3, liver microsomes plus NADPH
catalyzed the reduction of POBN/‚CH3 to its ESR-silent
hydroxylamine form. This reduction was most likely
mediated by the microsomal cytochrome P450 reductase
and can be reversed; POBN/CH3 could be reoxidized back
to POBN/‚CH3 with potassium ferricyanide, or with O2
in alkaline solutions (Scheme 1). Alternatively, both
POBN/‚CH3 and POBN/CH3 could be directly quantified
by HPLC-UV/EC.

Metabolism of PBN/ CH3 and POBN/ CH3 Adducts
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 753
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These results suggest that the cytochrome P450 mo-
nooxygenase system may interfere with the ESR analysis
of PBN/‚CH3 and POBN/‚CH3, respectively. PBN is often
used for the in vivo detection of ‚OH. However, the ESR
detection of PBN/‚CH3 could be successful if its rate of
generation is higher that that of its metabolism. Hence,
the susceptibility of PBN/‚CH3 to undergo an irreversible
metabolic transformation should be taken in consider-
ation when spin-trapping experiments with PBN are to
be carried out. In contrast, POBN/‚CH3 can undergo a
reductive microsomal metabolism to POBN/CH3, whereas
the structural information of the parent analyte is
preserved. The latter suggests that POBN may prove to
be a more efficient spin-trapping agent than PBN for the
in vivo analysis of ‚OH.
Ack n ow led gm en t. This work was supported by
Grant ES-09648 from the National Institute of Environ-
mental Health Sciences, NIH.
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