4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) inhibits peroxynitrite-mediated phenol nitration

Article abstract of DOI:10.1021/tx990159t

Peroxynitrite (PN), a very reactive oxidant formed by the combination of superoxide and nitric oxide, appears to play a role in producing tissue damage in a number of inflammatory conditions. Pharmacological scavenging and decomposition of PN within these areas has therapeutic value in several tissue injury models. Recently, we have been interested in nitroxide free radical-containing compounds as possible scavengers of PN decomposition products. Nitroxides can undergo redox reactions to the corresponding hydroxylamine anion or oxo-ammonium cation in biological systems as shown by its ability to react with superoxide, leading to the formation of hydrogen peroxide and molecular oxygen. We found that 4-hydroxy-2,2,6,6- tetramethylpiperidine-1-oxyl (Tempol) inhibits PN-mediated nitration of phenolic compounds in the presence of a large molar excess of PN, suggesting a catalytic-like mechanism. In these experiments, Tempol inhibited PN- mediated nitration over the pH range of 6.5-8.5. This inhibition was specific for nitration and had no effect on hydroxylation. After the inhibition of PN- mediated nitration, Tempol was recovered from the reaction mixtures unmodified. In addition, Tempol was effective in protecting PC-12 cells from death induced by SIN-1, a PN-generating compound. The exact mechanism of Tempol's interaction with PN is not clear; however, we propose that an intermediate in this reaction may be a nitrogen dioxide radical - Tempol complex. This complex could react with water to form either nitrite or nitrate, or with a phenol radical to produce nitrophenol or nitrosophenol products and regenerate the nitroxide.

Full text of DOI:10.1021/tx990159t

294
Chem. Res. Toxicol. 2000, 13, 294-300
4-Hyd r oxy-2,2,6,6-tetr a m eth ylp ip er id in e-1-oxyl (Tem p ol)
In h ibits P er oxyn itr ite-Med ia ted P h en ol Nitr a tion
Richard T. Carroll,*,† Paul Galatsis,‡ Susan Borosky,† Karla K. Kopec,†
Vikram Kumar,† J ohn S. Althaus,† and Edward D. Hall†
Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert
Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105, and Neuroscience Chemistry,
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company,
2800 Plymouth Road, Ann Arbor, Michigan 48105
Received September 3, 1999
Peroxynitrite (PN), a very reactive oxidant formed by the combination of superoxide and
nitric oxide, appears to play a role in producing tissue damage in a number of inflammatory
conditions. Pharmacological scavenging and decomposition of PN within these areas has
therapeutic value in several tissue injury models. Recently, we have been interested in nitroxide
free radical-containing compounds as possible scavengers of PN decomposition products.
Nitroxides can undergo redox reactions to the corresponding hydroxylamine anion or oxo-
ammonium cation in biological systems as shown by its ability to react with superoxide, leading
to the formation of hydrogen peroxide and molecular oxygen. We found that 4-hydroxy-2,2,6,6-
tetramethylpiperidine-1-oxyl (Tempol) inhibits PN-mediated nitration of phenolic compounds
in the presence of a large molar excess of PN, suggesting a catalytic-like mechanism. In these
experiments, Tempol inhibited PN-mediated nitration over the pH range of 6.5-8.5. This
inhibition was specific for nitration and had no effect on hydroxylation. After the inhibition of
PN-mediated nitration, Tempol was recovered from the reaction mixtures unmodified. In
addition, Tempol was effective in protecting PC-12 cells from death induced by SIN-1, a PN-
generating compound. The exact mechanism of Tempol’s interaction with PN is not clear;
however, we propose that an intermediate in this reaction may be a nitrogen dioxide radical-
Tempol complex. This complex could react with water to form either nitrite or nitrate, or with
a phenol radical to produce nitrophenol or nitrosophenol products and regenerate the nitroxide.
markers of oxidative damage are associated with areas
of neuronal degeneration (11-16). Nitrotyrosine ac-
cumulation is also evident in brains obtained from
patients suffering from multiple sclerosis (MS) (17, 18).
It is thought that PN production from infiltrating acti-
vated macrophages may play a part in the formation of
brain lesions in MS patients. In fact, a scavenger of PN,
uric acid, prevents the development of experimental
allergic encephalomyelitis in mice, suggesting possible
therapeutic applications in human MS (19, 20). PN is also
strongly implicated in head trauma (21), brain ischemia
(22-24), and amyotrophic lateral sclerosis (25). In addi-
tion, PN may amplify the immune response in these
diseases through the activation of prostaglandin biosyn-
thesis (26), causing increased collateral damage to sur-
rounding tissues.
In tr od u ction
Peroxynitrite (PN) is a very reactive oxidant formed
by the combination of superoxide with nitric oxide (1).
The reaction between superoxide and nitric oxide occurs
at the limit of diffusion control with a second-order rate
constant of 1.9 × 1010 M-1 s-1 (2), which is faster than
the enzymatic reaction of superoxide dismutase with
superoxide. Compared to other biological reactive oxygen
species, PN is relatively long-lived with a t1/2 of about 1
s at physiological pH and is consequently capable of
crossing cellular membranes and attacking extracellular
as well as intracellular targets (3, 4). PN can lead to
tissue damage via the nitration of tyrosine residues (5),
oxidation of methionine (6) and cysteine residues (7), and
DNA strand breakage (8-10). The consequence of these
reactions can compromise cellular homeostasis and lead
to cell death.
Under physiological conditions, PN can react with
carbon dioxide to form nitrosoperoxocarbonate anion
(ONOOCO2-) at a rate constant of 5.8 × 104 M-1 s-1 (27).
Considering the high concentration of carbon dioxide
(1-2 mM) in the extracellular and intracellular spaces
(28), it is likely that the majority of PN formed in vivo
reacts with CO2 to form ONOOCO2- (29). The CO2 adduct
of PN has altered reactivity, giving rise to an increased
level of nitration of aromatic residues and slower reactiv-
ity with antioxidants such as glutathione (27, 30, 31). In
the case of phenol nitration by PN, a 2-fold increase in
the level of nitrated products was obtained when bicar-
The production of PN has been shown to be associated
with multiple neurological diseases and may play a role
in the pathology of disease progression. In brains from
Alzheimer’s disease patients, nitrotyrosine and other
* To whom correspondence should be addressed: Neuroscience
Therapeutics, Parke-Davis Pharmaceutical Research, 2800 Plymouth
Rd., Ann Arbor, MI 48105. Phone: (734) 622-5216. Fax: (734) 622-
7178. E-mail: richard.carroll@wl.com.
† Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research,
Division of Warner-Lambert Co.
‡ Neuroscience Chemistry, Parke-Davis Pharmaceutical Research,
Division of Warner-Lambert Co.
10.1021/tx990159t CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

Tempol Inhibits Phenol Nitration
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 295
Tem p ol Recover y a fter Its In h ibition of P h en ol Nitr a -
tion . Solutions of phenol (1 mM, Aldrich) were made in 100 mM
sodium phosphate (pH 7.5) containing 50 mM sodium bicarbon-
ate and either 0, 6.25, or 12.5 µM Tempol. Phenol solutions (100
µL) were mixed with an equal volume of PN solution (1 mM in
H2O) to give a final PN concentration of 0.5 mM. Controls were
run by mixing phenol solutions with an equal volume of H2O.
Reactions were performed in a 96-well plate, and nitration was
followed spectrophotometrically at 405 nm using a microplate
reader (n ) 6, Molecular Devices, Thermomax).
bonate was added to the reaction mixture (29). These
observations suggest that scavenging the CO2 adduct of
PN would be useful in reducing tissue damage in areas
of high nitric oxide production.
The focus of this work was to characterize a new type
of PN scavenger. The nitroxide, 4-hydroxy-2,2,6,6-tetra-
methylpiperidine-1-oxyl (Tempol), was found to act as an
inhibitor PN-mediated nitration and an inhibitor of cell
death induced by the PN generator SIN-1.
The mixtures described above were examined for Tempol
content using LC/MS/MS. The system consisted of a Waters
2790 Separations Module for compound separation prior to
analysis by a Quattro Ultima tandem mass spectrometer
(Micromass). Separation was carried out using a TSK-GEL
column (TosoHaas, ODS-80TS, 2 mm × 15 cm, 5 µm particle
size). Samples were maintained at 4 °C prior to injection.
Chromatography was carried out isocratically at 30 °C using a
mobile phase consisting of 15% acetonitrile in 0.1% acetic acid
and a flow rate of 0.270 mL/min.
Ma ter ia ls a n d Meth od s
Syn th esis of P er oxyn itr ite. The synthesis of PN was
performed as previously described with slight modifications (32).
Briefly, a pH 12 solution of 0.2 M NaN3 (100 mL) in a 100 mL
glass bubbler was ozonized at 0 °C using a Welsbach ozonator
(model T-23) operating with an oxygen pressure of 8 psi. A flow
rate of 0.05 SCFM and an electric discharge of 90 V were
employed. This typically gave 80 mM PN solutions in about 25-
30 min. The concentration of PN was determined spectrophoto-
metrically by diluting the sample 100-fold in water (pH 12) and
measuring its absorbance at 302 nm (ꢀ302 ) 1670 M-1 cm-1).
Nitr a tion of 4-Hyd r oxyp h en yla ceta te by P er oxyn itr ite.
Solutions of 4-hydroxyphenylacetate (HPA, 1 mM, Aldrich) were
made in 100 mM sodium phosphate at the indicated pH and
sodium bicarbonate concentrations. HPA solutions (100 µL) were
mixed with an equal volume of PN (2.5 mM in H2O) to give a
final PN concentration of 1.25 mM. Reactions were performed
in a 96-well plate, and nitration was followed spectrophoto-
metrically at 405 nm using a microplate reader (Molecular
Devices, Thermomax). HPA nitration inhibition experiments
were performed by mixing Tempol, at the indicated concentra-
tions, in the HPA solutions prior to PN addition. The concentra-
tion of 4-hydroxy-3-nitrophenylacetate was determined spec-
trophotometrically (ꢀ430 ) 4400 M-1 cm-1) after the pH of the
reaction mixtures had been increased to 10-11 with 1 N NaOH
(33).
The eluent from the LC system was then analyzed using an
electrospray ionization (ESI) technique. The mode examined was
positive ionization (ESP+) ion selection and multiple reaction
monitoring (MRM). The parent ion (M + 1) had a mass of m/z
173.10; the daughter ion that was selected had a mass of m/z
158.04. The dwell time for the scan was 0.25 s. The cone voltage
was 28 V, and the collision energy was 14 eV. The source block
temperature was 150 °C, and the desolvation temperature was
270 °C. The cone gas rate was set at 204 L/h, while the de-
solvation gas rate was set at 588 L/h. The LC/MS/MS run time
was 10 min. The retention time of Tempol was 6.95 ( 0.5 min.
Raw data were processed using the MassLynx software
package. A calibration curve was created by running Tempol
standards at 1, 0.1, 0.01, and 0 µg/mL. The areas of the analyte
peaks were then measured, and their concentrations in micro-
grams per milliliter were found using the calibration curve as
a reference.
Tem p ol-Med ia ted Nitr ite F or m a tion fr om P er oxyn i-
tr ite. Solutions of 2 mM HPA were made in 100 mM sodium
phosphate (pH 7.5) containing 50 mM sodium bicarbonate
containing the indicated amount of Tempol. HPA solutions (100
µL) were mixed with an equal volume of 1.0 mM PN to give a
final PN concentration of 0.5 mM. Control experiments were
performed using the buffer described above with the indicated
Tempol concentration and no HPA. The control samples were
also mixed with an equal volume of 1 mM PN. Reactions were
performed in a 96-well plate and the mixtures allowed to set
for 5 min at room temperature. The samples were then diluted
1:10 in H2O. The diluted samples were then assayed for nitrite
concentration using a modified method of the Griess assay (35).
Briefly, 100 µL of diluted sample was mixed with 50 µL of 1%
sulfanilamide (Sigma) in 0.1 M HCl and 50 µL of 0.1% N-(1-
naphthyl)ethylenediamine (Sigma) in H2O. Samples were al-
lowed to set at room temperature for 5 min, and the absorbances
were read at 550 nm. Sample concentrations were extrapolated
from a standard curve made from nitrite standards ranging from
0 to 250 µM. Contaminating nitrite in the PN solutions was
evaluated by mixing 1 mM PN with an equal volume of 5%
phosphoric acid prior to nitrite analysis. The background level
of contaminating nitrite was then subtracted from all experi-
ments.
HP LC An a lysis of P h en ol a n d Its P N-Med ia ted Nitr a -
tion P r od u cts. Solutions of phenol (1 mM, Aldrich) were made
in 100 mM sodium phosphate at the indicated pH containing
50 mM sodium bicarbonate with and without 25 µM Tempol.
Phenol solutions (100 µL) were mixed with an equal volume of
PN (2.5 mM in H2O) to give a final PN concentration of 1.25
mM. Reactions were performed in a 96-well plate, and nitration
was followed spectrophotometrically at 405 nm using a micro-
plate reader (n ) 6, Molecular Devices, Thermomax). Product
analysis was performed using a modified procedure described
by Lemercier et al. (34). Briefly, reaction products were analyzed
using a Rainin HPXL gradient system (Rainin Instrument Co.,
Inc.) with a dual-wavelength SpectroMonitor 4100 detector
(Thermoseparations Products). Separations were carried out on
a Nucleosil 5 C18 100A column (150 mm × 4.6 mm, Phenom-
enex) using mobile phases A [27 mM acetate/30 mM citrate
buffer (pH 3.2)] and B (acetonitrile). A gradient of 15 to 52% B
was employed over the course of 18 min at a flow rate of 1 mL/
min. The column was then washed for 2 min with 100%
acetonitrile and equilibrated at 15% B for 15 min. The peak area
(milliabsorbance units per second) was measured from chro-
matographs obtained from absorbance measurements at 280
nm, and retention times were compared to standards for peak
identification.
E ffect s of Tem p ol on P N-In d u ced P h en ol H yd r oxyl-
a tion . Solutions of phenol (10 mM, Aldrich) were made in 100
mM sodium phosphate (pH 6.5) with and without 25 µM Tempol.
Phenol solutions (100 µL) were mixed with an equal volume of
PN (1.0 mM in H2O) to give a final PN and phenol concentra-
tions of 500 µM and 5 mM, respectively. Reactions were
Cell Cu ltu r e a n d P er oxyn itr ite-In d u ced Cytotoxicity.
Cytotoxicity was induced in PC12 cells following the procedures
of Estevez et al. (36) with minor modifications. In collagen-
coated 96-well plates, PC12 cells were plated at a density of 1.5
× 105 cells/cm2 in OptiMem (Gibco BRL) containing 4% fetal
bovine serum and 5 µg/mL gentamicin (Gibco BRL) and allowed
to adhere overnight. Cultures were then washed once with
phosphate-buffered saline containing 5 mM glucose (pH 7.4) and
then placed in 200 µL of OptiMem containing 5 µg/mL gentami-
cin. 3-Morpholinosydnonimine (SIN-1, Cayman Chemicals), a
PN generator, was dissolved in 50 mM phosphate (pH 5.0) just
performed in
a 96-well plate, and nitration was followed
spectrophotometrically at 405 nm using a microplate reader (n
) 6, Molecular Devices, Thermomax). Reaction products were
analyzed by HPLC as described above using the modified
procedure described by Lemercier et al. (34).

296 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Carroll et al.
Ta ble 1. HP LC P ea k Ar ea s of P r od u cts Obta in ed fr om
P N Nitr a tion of P h en ola
pH 4-nitrosophenol phenol 4-NP 2-NP [Tempol] (µM)
8.5
8.0
7.5
7.0
6.5
8.5
8.0
7.5
7.0
6.5
215
46
13
861
826
729
609
580
955
902
942
975
968
58
121
526
765
846
N/D
N/D
N/D
N/D
41
128
265
1010
1130
1148
43
N/D
N/D
N/D
36
0
0
0
0
N/Db
N/D
396
284
171
139
133
0
12.5
12.5
12.5
12.5
12.5
a
Reaction conditions are identical to those reported in the
legend of Figure 2. Peak areas (millivolts per second) were
obtained from HPLC chromatograms while monitoring absorbance
b
at 280 nm. N/D, not determined. Peaks fell below our ability of
detection for accurate quantitation.
F igu r e 1. Inhibition of PN-mediated nitration of 4-hydroxy-
phenylacetate by Tempol. The reactions were carried out using
0.5 mM HPA, 1.25 mM PN, and 25 mM bicarbonate in 50 mM
sodium phosphate. Each point represents six iterations plotted
with the standard deviation. Symbols represent the pH at which
the reactions were carried out: (O) pH 6.5, (0) 7.0, (]) 7.5, (9)
8.0, and ([) 8.5.
prior to use, and 5 µL was added to each well to give the
indicated concentration. The cells were allowed to incubate for
24 h, and cytotoxicity was determined using a detection kit
(Boehringer Mannheim) that measured lactate dehydrogenase
(LDH) activity released from damaged cells. In experiments with
the PN scavenger, Tempol was added at the indicated concen-
trations to each culture 15 min prior to SIN-1 (1 mM final
concentration) addition. SIN-1 at a concentration of 1 mM has
been reported to generate nitric oxide and superoxide at rates
of 3.68 and 7.02 µmol/min, respectively (37). The percent
inhibition of PN-mediated cytotoxicity was determined by the
percent reduction in LDH activity release as compared to that
of SIN-1-treated cultures. As a control, 50 units/mL SOD
(Sigma, S-2515) was added to SIN-1-treated cultures to prevent
the formation of PN.
Resu lts
In h ibition of 4-Hyd r oxyp h en yla ceta te Nitr a tion
by Tem p ol. The ability of Tempol to act as a scavenger
for PN-mediated nitration was examined using the HPA
nitration assay. Tempol protected 500 µM HPA from
nitration by 1.25 mM PN in a concentration-dependent
manner (Figure 1). Under these conditions, Tempol was
able to prevent PN-mediated nitration with IC50 values
of 2.5, 3.2, 3.7, 5.5, and 8.4 µM at pH 6.5, 7.0, 7.5, 8.0,
and 8.5, respectively. Maximal nitration occurred at pH
6.5 and resulted in the nitration of approximately 105
µM HPA. The 20-fold turnover of Tempol under these
conditions suggests that it is acting in a catalytic-like
fashion.
Effect of Alk a lin e p H on Tem p ol’s In ter a ction s
w ith P h en ol a n d P er oxyn itr ite. Phenol nitration and
nitrosation by PN were evaluated by HPLC at pH 6.5,
7.0, 7.5, 8.0, and 8.5 (Table 1). The maximum amount of
phenol nitration in this experiment occurred at pH 6.5
and decreased with increasing pH. Conversely, increased
nitrosation of phenol occurred as the pH increased. In
our experiments, 4-nitrosophenol represented approxi-
mately 17, 4, and 1% of the total peak areas at pH 8.5,
8.0, and 7.5, respectively (Figure 2A). When Tempol was
added to the reaction mixtures, the nitration of phenol
was almost completely abolished under all conditions
(Figure 2B). However, Tempol caused increases in the
extent of formation of nitrosophenol of 28, 24, 15, 13, and
11% at pH 8.5, 8.0, 7.5, 7.0, and 6.5, respectively (Table
F igu r e 2. HPLC profiles of products obtained from PN
reactions with phenol. Reactions were carried out using equal
volumes of 1.0 mM phenol in 50 mM sodium phosphate and 50
mM sodium bicarbonate at the indicated pH with 2.5 mM PN.
The reaction mixtures contained no Tempol (A) or 12.5 µM
Tempol (B). Peak identities were as follows: peak 1, 4-nitroso-
phenol; peak 2, phenol; peak 3, 4-nitrophenol; and peak 4,
2-nitrophenol. Product identification was made by comparison
of HPLC standards separated under identical conditions.
1). An increase in the amount of nitrosated product
implies the formation of a complex between Tempol and
one or more of the decomposition products of PN.
Effect of Tem p ol on P N-Med ia ted Hyd r oxyla tion
of P h en ol. When using a 10-fold molar excess of phenol
over PN at pH 6.5, hydroxylated products of phenol, as
well as some minor nitration products, were identified
by HPLC (Figure 3). However, these hydroxylated prod-
ucts were not formed in the presence of added bicarbon-
ate (Table 2). When 12.5 µM Tempol was added to the
reaction mixture, the formation of nitrated products (2-
and 4-nitrophenol) was inhibited and was independent
of whether bicarbonate was added to the reaction mix-
ture. Conversely, the extent of formation of hydroxy-
phenol products slightly increased in the presence of
Tempol (Figure 3 and Table 2). Tempol also increased
the extent of formation of nitrosophenol with or without
the addition of bicarbonate to the reaction mixture (Table
2).

Tempol Inhibits Phenol Nitration
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 297
F igu r e 4. Tempol recovery after its inhibition of phenol
nitration. Reactions were carried out using equal volumes of
1.0 mM phenol in 100 mM sodium phosphate and 50 mM sodium
bicarbonate (pH 7.5) containing either 6.25 or 12.5 µM Tempol
with 1 mM PN. The percent recovery of Tempol, as compared
to those of the non-peroxynitrite-treated samples, was deter-
mined by HPLC/MS/MS (solid bars). The extent of inhibition of
phenol nitration in these reactions was calculated by the
decrease in absorbance at 405 nm as compared to those of non-
Tempol-treated reactions (hatched bars). Each sample repre-
sents an average of six iterations with the standard deviation.
F igu r e 3. HPLC profiles of PN-mediated hydroxylation of
phenol. Solutions of 100 mM sodium phosphate (pH 6.5)
containing 10 mM phenol were made without (A) and with (B)
25 µM Tempol. Phenol solutions were mixed 1:1 with 1.0 mM
PN 10 min prior to HPLC analysis. Product identification was
made by comparison of HPLC standards separated under
identical conditions (C). Peak identities were as follows: peak
1, hydroquinone; peak 2, catechol; peak 3, 4-nitrosophenol; peak
4, phenol; peak 5, 4-nitrophenol; and peak 6, 2-nitrophenol.
Ta ble 2. HP LC P ea k Ar ea s of P r od u cts Obta in ed fr om
P N Hyd r oxyla tion of P h en ola
hydroxy-
HCO3 quinone catechol
4-nitroso-
phenol
[Tempol]
(µM)
4-NP 2-NP
0
0
25
25
40
46
62
89
N/D
N/D
N/D
22
N/D
55
24
N/D
153
34
44
N/D
292
65
0
12.5
0
N/Db
N/D
12.5
a
Reaction conditions are identical to those reported in the
legend of Figure 3. Peak areas (millivolts per second) were
obtained from HPLC chromatograms while monitoring absorbance
b
at 280 nm. N/D, not determined. Peaks fell below our ability of
detection for accurate quantitation.
Tem p ol Recover y a fter In h ibition of P N-Med i-
a ted P h en ol Nitr a tion . To help determine the mech-
anism by which Tempol prevents phenol nitration, Tem-
pol was analyzed for potential modification after inhibiting
PN-mediated nitration of phenol. Tempol, at 6.25 and
12.5 µM, inhibited nitration by 67 and 72%, respectively
(Figure 4). In this reaction, PN was in a 40- and 80-fold
molar excess over Tempol. After inhibition, Tempol was
recovered by LC/MS/MS and compared to controls that
had no PN added (Figure 4). Peroxynitrite treatment had
no significant effect on the recovery of Tempol, demon-
strating that it is not being consumed during its inhibi-
tion of PN-mediated nitration.
Tem pol-Mediated Nitr ite For m ation fr om P er oxy-
n itr ite. Upon the decomposition of PN, nitrate and
nitrite are formed under physiological conditions. The
influence of Tempol on this decomposition was evaluated
by measuring the extent of nitrite formation in the
presence and absence of a HPA. Under these reaction
conditions, a solution containing 1 mM HPA and 500 µM
PN resulted in the formation of 186 µM nitrite. When
12.5 µM Tempol was added prior to PN addition, the
concentration of nitrite that was detected increased to
279 µM. This increase of approximately 93 µM nitrite
primarily occurred at Tempol concentrations of <2 µM
(Figure 5, b). In the absence of added HPA, an even more
dramatic increase was observed. In experiments where
F igu r e 5. Tempol-mediated nitrite formation from peroxy-
nitrite. Peroxynitrite (500 µM) was allowed to decompose in 100
mM sodium phosphate and 50 mM sodium bicarbonate (pH 7.5)
with 500 µM HPA (b) and without HPA ([). The formation of
nitrite was assessed in experiments using the indicated con-
centrations of Tempol. Each sample represents an average of
four iterations; error bars representing standard deviations are
smaller than the symbols.
no Tempol was added, 154 µM nitrite was detected after
PN addition. When 12.5 µM Tempol was included in the
experiment, the concentration of nitrite increased by 176
µM to a final concentration of 330 µM (Figure 5, [).
In h ibition of SIN-1-In d u ced Cytotoxicity by Tem -
p ol. The ability of Tempol to protect cells from PN-
induced cell death was assessed in culture. Cytotoxicity
of PC-12 cells by PN was initiated by the addition of SIN-
1, a compound that decomposes, at neutral pH, into
superoxide and nitric oxide that combine to form PN.
Cytotoxicity was determined by measuring the amount
of LDH released from cells 24 h after the addition of SIN-
1. Cell damage was induced with as little as 0.25 mM
SIN-1 and reached its maximum at 2.0 mM (Figure 6).
When cells were treated with 1 mM SIN-1, cytotoxicity
could be blocked by Tempol (Figure 7). The IC50 of
Tempol, determined by the percent reduction in LDH
activity, was 8.1 µM, and complete protection was
observed at 100 µM. The addition of 50 units of SOD/mL

298 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Carroll et al.
(eq 1). This compound then decomposes into a carbonate
radical anion and a nitrogen dioxide radical (eq 2).
-
ONOO- + CO2 f ONOOCO2
(1)
(2)
ONOOCO2- f ONO• + CO3
•
Due to resonance, the upaired electron in nitrogen dioxide
can exist on the oxygen or the nitrogen atom. Thus,
Tempol can react with either the oxygen atom, forming
a Tempol-ONO ester, or at the nitrogen atom, forming
a Tempol-nitrate ester (eqs 3 and 4). In addition, this
reaction must occur at a faster rate than that between
the nitrogen dioxide radical and phenol for Tempol to be
an effective inhibitor.
F igu r e 6. SIN-1-induced cytotoxicity in PC-12 cells. Cytotox-
icity was measured using the LDH release assay, which gives
rise to an increase in absorbance at 490 nm as LDH levels
increase. Each point represents an average of six iterations
plotted with the standard deviation.
Tempol• + ONO• f Tempol-ONO
Tempol• + O2N• f Tempol-NO2
(3)
(4)
These esters could then react with water to generate
hydroperoxy-Tempol (eq 5) and hydroxylamine-Tempol
(eq 6) as well as a molecule of nitrite or nitrate (eqs 5
and 6).
Tempol-ONO + H2O f HNO2 + Tempol-OH (5)
Tempol-NO2 + H2O f HNO3 + Tempol-H (6)
The large increase in nitrite production observed experi-
mentally suggests that the reaction shown in eq 5 is
predominant. Since products derived from Tempol-
nitrate esters were not observed, further chemistry of this
intermediate will not be discussed.
The carbonate radical anion could then abstract a
hydrogen atom from the peroxy-Tempol derivative to
generate the corresponding radical (eq 7).
F igu r e 7. Inhibition of SIN-1-induced cytotoxicity by Tempol.
Cytotoxicity was measured using the LDH release detection
assay. Inhibition of cytotoxicity by Tempol was assessed using
the percent inhibition of LDH activity. Each point represents
six iterations plotted with the standard deviation.
Tempol-OH + CO3• f Tempol-O• + HCO3 (7)
-
completely inhibited SIN-1-induced toxicity. These data
suggest that the formation of PN is needed for cellular
toxicity. In addition, Tempol was not toxic in our assays
at any of the concentrations that were studied (data not
shown).
The combination of two molecules of Tempol-O• would
produce an intermediate that could rapidly decompose
into two molecules of Tempol• and a molecule of oxygen
(eq 8).
Tempol-O• + Tempol-O• f
Discu ssion
Tempol-O-O-Tempol f 2Tempol• + O2 (8)
The nitroxide Tempol has the ability to inhibit PN-
mediated nitration of phenolic compounds in a catalytic-
like fashion. Considering that Tempol did not effect the
hydroxylation of phenol suggests that it does not react
directly with the parent molecule, but with one or more
of the free radical decomposition products of PN. A
probable candidate would be the nitrogen dioxide radical.
The trapping of this radical by Tempol would explain the
inhibition of nitration, increased production of nitrite, the
promotion of nitrosation, and the lack of an effect on
phenolic hydroxylation. The IC50 values of Tempol at pH
6.5, 7.0, and 7.5 suggest that it must react with at least
a 15-20-fold molar excess of nitrogen dioxide radical.
Considering Tempol was recovered unmodified from
these experiments, as determined by LC/MS/MS, it is
likely that the nitroxide is undergoing redox chemistry
with the generated free radicals.
The formation of Tempol-ONO in eq 3 can also explain
the increase in the level of nitrosation that is observed
when phenol is treated with PN in the presence of
Tempol. Phenol could initially be oxidized by the carbon-
ate radical anion to a phenolic radical that subsequently
reacts with the Tempol-ONO ester (eq 9). The products
of this reaction would be nitrosophenol and a peroxy-
Tempol radical.
Ph-O• + Tempol-ONO f
phenol-NO + Tempol-O• (9)
The peroxy-Tempol radical could then react with a
second molecule, as in eq 8, which would lead to the
formation of two molecules of Tempol• and a molecule of
oxygen. It is unlikely that the phenolic radical reacts
directly with the Tempol-NO2 adduct from eq 4. The
product of such a reaction would be nitrated phenol.
Experimentally, we only see the inhibition of nitration
The following mechanism is proposed to explain the
inhibition of phenol nitration by Tempol and is presented
in Scheme 1. It is known that PN anion readily combines
with carbon dioxide to form nitrosoperoxocarbonate anion

Tempol Inhibits Phenol Nitration
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 299
Sch em e 1. P r op osed Mech a n ism for th e Sca ven gin g of P er oxyn itr ite Decom p osition P r od u cts by Tem p ol
and not the enhancement of it. This may also indicate
that the reaction between H2O and Tempol-NO2 to form
nitrate is much faster than the corresponding reaction
with the phenolic radical. A more likely explanation
would be that the formation of Tempol-ONO (eq 3)
predominates over the formation of Tempol-NO2 (eq 4),
as suggested by the increase in nitrite production. Also,
as the pH was increased, a greater proportion of the
phenoxide anion would exist in solution. One could then
expect chemistry related to the anion to be an alternative
source of 4-nitrosophenol (eq 10)
products produced from two molecules of superoxide
during this reaction are oxygen and hydrogen peroxide.
A similar one-electron redox reaction for peroxynitrite
decomposition products is proposed in Scheme 1.
The ability of Tempol to inhibit PN cellular toxicity was
evaluated in a cell-based model. SIN-1, a generator of
the PN precursors superoxide and nitric oxide, induced
PC-12 cell death in a dose-dependent manner between 0
and 1 mM (Figure 6). When Tempol was introduced in
this model, it inhibited cell death induced from 1 mM
SIN-1 with an IC50 of 8 µM (Figure 7). The mechanism
by which Tempol works in this model may be twofold. It
can react with the decomposition products of PN as
described above, or it may be reacting with the PN
precursor superoxide or nitric oxide. Tempol has been
shown to be a SOD mimic (38, 39) and may contribute to
the protection of PC-12 cells through this mechanism by
preventing PN formation. This conclusion was supported
experimentally when SOD was added to the SIN-1-
treated cells and complete protection was observed. These
data suggest multiple utilities of Tempol in preventing
oxidative damage due to superoxide and PN. However,
further evaluation of this compound will be needed to
determine its therapeutic relevance.
PhO- + Tempol-ONO f
ON-Ph-O- + Tempol-OH (10)
Nitroxides have been shown to perform similar redox
chemistry when reacting with superoxide as a superoxide
dismutase mimic, which is shown in the following reac-
tions (38, 39).
RR′NOH + •O2- + H+ f RR′NO• + H2O2 (11)
RR′NO• + •O2- f RR′NO- + O2
(12)
The reaction between Tempol and superoxide is catalytic
and cycles between the hydroxylamine and nitroxide. The
In conclusion, we have identified Tempol as an effective
inhibitor of PN-mediated nitration. This inhibition is

300 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Carroll et al.
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thought to involve the capture of the nitrogen dioxide
radical, leading to the formation of nitrite or nitrosated
phenol. The blockade of PN-mediated reactivity was also
extended to a cell-based model. Here, cell death induced
by SIN-1 was completely inhibited by Tempol. These
findings suggest that nitroxides may be a suitable
molecular backbone for potent antioxidants capable of
destroying PN reactivity while acting as a SOD mimetic.
Ack n ow led gm en t. We thank Kevin W. Anderson
and Kari R. Schmidt for their excellent technical as-
sistance.
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