Oxidation of proximal protein sulfhydryls by phenanthraquinone, a component of diesel exhaust particles

Article abstract of DOI:10.1021/tx0100993

Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and 2,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups, whereas minimal reactivities of this quinone with monothiol compounds such as GSH, 2-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary microsomes from rats and total membrane preparation isolated from bovine aortic endothelial cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies revealed that thiyl radical intermediates and reactive oxygen species were generated during interaction of phenanthraquinone with DTT. From these findings, it is suggested that phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation of presumably proximal thiols and the reduction of molecular oxygen.

Full text of DOI:10.1021/tx0100993

Chem. Res. Toxicol. 2002, 15, 483-489
483
Oxid a tion of P r oxim a l P r otein Su lfh yd r yls by
P h en a n th r a qu in on e, a Com p on en t of Diesel Exh a u st
P a r ticles
,
†
‡
‡
‡
§
Yoshito Kumagai,* Sachie Koide, Keiko Taguchi, Akiko Endo, Yumi Nakai,
|
†
Toshikazu Yoshikawa, and Nobuhiro Shimojo
Department of Environmental Medicine, Institute of Community Medicine, and Master’s
Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8575, J apan, Analytical Instruments Division, J EOL Ltd., 3-1-1 Musashino,
Akishima, Tokyo 196, J apan, and First Department of Internal Medicine, Kyoto Prefectural
University of Medicine, Kamigyo-ku, Kyoto 602, J apan
Received May 31, 2001
Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation
of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In
the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of
DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily
with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and
2
,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups,
whereas minimal reactivities of this quinone with monothiol compounds such as GSH,
-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused
2
by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular
oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary
microsomes from rats and total membrane preparation isolated from bovine aortic endothelial
cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol
group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of
phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies
revealed that thiyl radical intermediates and reactive oxygen species were generated during
interaction of phenanthraquinone with DTT. From these findings, it is suggested that
phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation
of presumably proximal thiols and the reduction of molecular oxygen.
In tr od u ction
and hydroxyl radical (5). Recent studies have indicated
that exposure of macrophages to organic chemicals
extracted from DEP resulted in induction of apoptosis
Exposure of experimental animals and humans to
1
diesel exhaust particles (DEP) is associated with lung
(6) and an increase in the gene expression of the oxidative
cancer, allergic inflammation, asthma, and cardiopulmo-
nary dieseases (1-3). It was reported previously that
intratracheal administration of DEP into mice caused a
marked mortality due to lung edema formation. This
condition was attenuated by pretreatment with super-
oxide dismutase (SOD) conjugated with poly(ethylene
glycol) (4). We found that quinones contained in DEP
were reduced by one electron by NADPH-cytochrome
P450 reductase, leading to overproduction of superoxide
stress-inducible protein heme oxygenase-1 (7). Taken
together, it is likely that quinones in DEP can play a
critical role in catalyzing the generation of reactive
oxygen species, resulting in cellular oxidative stress.
Although Schuetzle and co-workers (8, 9) reported previ-
ously that a variety of quinones have been identified as
DEP components, few studies on the involvement of the
quinones in oxidative stress-dependent DEP toxicity have
been reported.
Quinones are found in the diet and in contaminants
in urban air and are utilized as dye, antibiotics, and
anticancer drugs (8, 10). Due to their ubiquitous pres-
ence, the toxicology of quinones is an area of extreme
interest. The toxicity of quinones has been proposed to
result from their potential to (1) serve as alkylating
agents and (2) interact with, for example, flavoproteins
to generate reactive oxygen species (10-12). Thus, the
toxicity of many quinoid compounds is due to either or
both oxidative stress (i.e., oxidation of sulfhydryls) as well
as covalent modification of biological macromolecules.
Oxidation of cysteine residues in proteins is associated
with not only decreased sulfhydryl status but also
*
Address correspondence to this author at the Department of
Environmental Medicine, Institute of Community Medicine, University
of Tsukuba, Tsukuba, Ibaraki 305-8575, J apan. Tel: 81-298-53-3297,
Fax: 81-298-53-3039, E-mail: yk-em-tu@md.tsukuba.ac.jp.
†
Department of Environmental Medicine, University of Tsukuba.
Master’s Program in Environmental Sciences, University of Tsuku-
‡
ba.§
Analytical Instruments Division, J EOL Ltd.
First Department of Internal Medicine, Kyoto Prefectural Univer-
|
sity of Medicine.
1
Abbreviations: DEP, diesel exhaust particles; BAEC, bovine aorta
endothelial cells; DTT, dithiothreitol; NEM, N-ethylmaleimide; DTNB,
,5′-dithiobis(2-nitrobenzoic acid); BAL, 2,3-dimercapto-1-propanol;
DMPS, 2,3-dimercapto-1-propanesulfonic acid; BSA, bovine serum
albumin; SOD, superoxide dismutase; DMPO, 5,5′-dimethyl-1-pyrroline
N-oxide; Hepes, 4-(2-hydroxyethyl)-1-piperazineesulfonic acid.
5
1
0.1021/tx0100993 CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/28/2002

4
84 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Kumagai et al.
(pH 7.0)/0.1 mM EDTA to make a final concentration of 5.4-
6
.5 mg/mL. The microsomal preparation was stored at -70 °C
before use; under these conditions, a decline of protein sulfhydryl
-
1
content with a rate of 0.3 nmol of SH group (mg of protein)
-1
day was observed. Protein concentration was measured by the
method of Bradford (16) with bovine serum albumin as the
standard.
F igu r e 1. Structures of phenanthraquinone and NEM.
Mea su r em en t of Th iol Con ten t. Phenanthraquinone and
changes in oxidative stress-mediated signal transduction
other quinones used were dissolved in Me SO. All spectropho-
2
(
13, 14). If the sulfhydryls are modified, physiological
tometric measurements were performed using a Shimadzu UV-
1600 double-beam spectrophotometer (Kyoto, J apan). Thiol
contents of DTT preparations and in BAEC were determined
as described previously (17). The incubation mixture (1 mL)
containing different concentrations of phenanthraquinone (final
functions would be altered, thereby inducing oxidative
stress.
In this study, we chose phenanthraquinone as a model
compound because this chemical is known as a relatively
abundant quinone in DEP (8). Several previous reports
indicate that electrophiles such as N-ethylmaleimide
2
concentration of Me SO, 2%) was incubated with a variety of
sulfhydryls at 37 °C for 10-60 min. For anaerobic conditions,
argon gas was bubbled through the solution during each
incubation. In the case of nonprotein sulfhydryl compounds (i.e.,
DTT), an aliquot (0.5 mL) of the incubation mixture was mixed
with 10% trichloroacetic acid (0.5 mL), and then a portion (0.5
mL) of the mixture was mixed with 1 mL of 0.4 M Tris-HCl
(NEM) (Figure 1) can promote alkylation of protein thiols.
Unlike NEM, phenanthraquinone will not alkylate thiols
but has the potential to act as a redox cycling quinone,
generating thiol oxidants such as hydrogen peroxide. In
the present study, using NEM and phenanthraquinone
(pH 8.9)/20 mM EDTA and 25 µL of 10 mM DTNB. For the
protein sulfhydryl compound (with the BAEC preparation), an
aliquot (0.1 mL) of the incubation mixture was mixed with 0.3
mL of 0.2 M Tris-HCl (pH 8.2)/20 mM EDTA, followed by 20 µL
of 10 mM DTNB, 0.15 mL of 5% SDS. Each resulting mixture
was measured at 412 nm against a blank to determine the
(Figure 1), we (1) investigate the possibility of oxidation
of protein sulfhydryls by phenanthraquinone and (2) look
for the generation of reactive oxygen species and oxidized
thiol intermediates formed during interaction of these
compounds with sulfhydryls.
2
content of the thiol groups (with Me SO). For nonprotein
sulfhydryls, each thiol compound was used as the standard, and
quantitation of protein sulfhydryl content was determined using
Ma ter ia ls a n d Meth od s
-
1
-1
an extinction coefficient of 13.6 mM cm
.
Ma ter ia ls. The chemicals and proteins were obtained as
follows: Phenanthraquinone, DTT, NEM, and bovine serum
albumin (BSA) from Nacalai Tesque, Inc. (Kyoto, J apan); 5,5′-
dimethyl-1-pyrroline N-oxide (DMPO) from Labotech Co. (Tokyo,
J apan); catalase from Sigma Chemical Co. (St. Louis, MO); 2,3-
dimercapto-1-propanol (BAL) and 2,3-dimercapto-1-propane-
sulfonic acid (DMPS) from Aldrich Chemical. Co. Inc. (Milwau-
kee, WI); Econo-Pac 10DG column from Bio-Rad Laboratories
Detection of Th iyl Ra d ica l. Electron spin resonance (ESR)
studies were performed at 25 °C by using an J ES-FA200
spectrometer (J EOL Co. Ltd., Tokyo, J apan) as described
previously (5). Thiyl radicals generated during the chemical
reaction of phenanthraquinone with DTT were identified as its
DMPO adduct by the method of Yim et al. (18). The reaction
mixture (0.2 mL) containing 100 µM phenanthraquinone (in
2
Me SO, final concentration of 2%), 10 mM DTT, 100 mM DMPO,
(
Richmond, CA); cytochrome c from Wako Pure Chemical
Industries, Ltd. (Osaka, J apan). Cu,Zn-superoxide dismutase
Cu,Zn-SOD) was purified from human liver by the method of
and 100 mM potassium phosphate buffer (pH 7.4) was incubated
at 25 °C for 30 s. The spectrometer settings are indicated in
the figure legends.
(
Kumagai et al. (15). All other chemicals used were obtained from
commercial sources and were of the highest grade available.
Gen er a tion of Rea ctive Oxygen Sp ecies. Superoxide
production was determined by Cu,Zn-SOD-inhibitable reduction
of cytochrome c as described previously (5) except that cyto-
chrome c was used instead of its acetylated form and reduction
of cytochrome c was measured at 550 nm using an extinction
P r ep a r a tion of En zym e. Bovine aortic endothelial cells
(BAEC) were obtained from Dainippon Pharmaceutical Indus-
trial Co. (Tokyo, J apan). BAEC were maintained in Dulbecco’s
modified Eagle’s medium: Nutrient mixture F-12 supplemented
with 15% heat-inactivated fetal bovine serum, penicillin (100
units/mL), streptomycin (100 µg/mL), fibroblast growth factor-
acidic (5 ng/mL), and heparin (10 units/mL). Cells were incu-
-1
-1
coefficient of 21.1 mM cm . The incubation mixture (1.5 mL)
consisted of 0.1 µM phenanthraquinone, 0.1 mM DTT, 0.05 mM
cytochrome c, and 50 mM Tris-HCl (pH 7.5) in the absence and
presence of Cu,Zn-SOD (2400 units). The reaction was initiated
by addition of the quinone to a sample cuvette. Hydrogen
peroxide was determined according to the method of Hilde-
brandt and Roots (19). The incubation mixture (1.5 mL)
contained 0.1 µM phenanthraquinone, 100 µM DTT, and 0.1 M
potassium phosphate buffer (pH 7.5). Reaction was performed
at 37 °C for different time periods and terminated by addition
of 1 mL of 2.5% perchloric acid. After the reaction mixture was
centrifuged at 14000g for 5 min, the supernatant (0.2 mL) was
mixed with 1 mL of water, 0.24 mL of 10 mM ferroammonium
sulfate, and 0.12 mL of 2.5 M potassium thiocyanate. Then, each
resulting mixture was measured at 480 nm. For the calibration
curve, hydrogen peroxide was used as the standard.
2
bated in a humidified atmosphere of 95% air/5% CO . The
medium was changed every 2-3 days, and cells were routinely
passaged by trypsin/EDTA with a split ratio of 1:4. BAEC
between passages 3 and 6 were scraped from the culture plate
and homogenized in 50 mM Tris-HCl (pH 7.4)/0.1 mM EDTA/
0
.1 mM EGTA/1 mM phenylmethylsulfonyl fluoride/leupeptin
(1 µg/mL). The homogenate was centrifuged at 100000g for 60
min to isolate the total membrane fraction. The resulting pellets
were suspended in the homogenate buffer containing 2.5 mM
CaCl . Suspensions obtained were frozen under liquid nitrogen
2
and kept at -70 °C before use. For pulmonary preparations,
lungs of Wistar rats (4 weeks) were homogenized in 3 volumes
of 10 mM Tris-HCl (pH 7.0)/0.1 mM EDTA. The homogenates
were centrifuged at 9000g for 20 min. The supernatants were
recentrifuged at 105000g for 60 min. Cytosol fraction (3 mL)
was applied to a Econo-Pac 10DG column to remove low
molecular weight thiol compound such as GSH. Pellets obtained
were washed with 100 mM potassium pyrophosphate buffer (pH
HP LC. Separation of phenanthraquinone and NEM was
accomplished using a Shimadzu HPLC system (Kyoto, J apan).
After reaction, an aliquot (20-100 µL) was applied to a YMC
packed column AM-type (250 × 4.6 mm i.d., 5 µm particle size,
Yamamura Labs, Kyoto, J apan) at a flow rate of 1 mL/min.
Water/acetonitrile (1:1, v/v) was used as the mobile phase, and
detection was performed at 255 nm. Under these conditions, the
7
.4) to remove hemoglobin and recentrifuged at 105000g for 60
min. The pellets were suspended in 10 mM Tris-HCl

Oxidation of Sulfhydryls by Phenanthraquinone
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 485
Ta ble 1. Con su m p tion of SH Gr ou p s in Mon oth iol a n d
a
Dith iol Com p ou n d s by P h en a n th r a qu in on e
consumption of SH group (nmol)
compound
monothiol
GSH
1 nmol
10 nmol
2.7 ( 0.5
0
0
1.8 ( 2.3
0.3 ( 1.0
0.3 ( 2.0
2
-mercaptoethanol
N-acetyl-L-cysteine
dithiol
F igu r e 2. Consumption of thiol content in DTT during incuba-
tion with phenanthraquinone under aerobic and anaerobic
conditions. Incubation mixture (1 mL) consisted of phenan-
thraquinone (0.1, 1, and 10 nmol), DTT (100 nmol) in 0.1 M
potassium phosphate buffer (pH 7.5). Reactions were carried
out at 37 °C for different time periods. For the anaerobic
condition, argon gas was bubbled through the solution during
each incubation. Then the consumption of the thiol group was
determined under conditions described under Materials and
Methods. Each value is the mean of three determinations.
Note: the SD value of the mean for each data point was less
than 5%.
DTT
BAL
DMPS
103.0 ( 0.3
43.9 ( 1.0
17.1 ( 2.1
103.0 ( 0.0
98.0 ( 0.6
89.1 ( 1.0
a
The incubation mixture (1 mL) consisted of phenanthraquino-
ne (1 and 10 nmol) and various thiol compounds (100 nmol) in
00 mM potassium phosphate buffer (pH 7.5). Reactions were
carried out at 37 °C for 10 min under aerobic conditions. Each
value is the mean ( SD of three determinations.
1
Ta ble 2. Con su m p tion of Th iol Con ten ts in BAEC d u r in g
a
In cu ba tion w ith P h en a n th r a qu in on e a n d NEM
SH groups consumed (nmol)
retention times of phenanthraquinone and NEM were 9.7 and
phenanthraquinone
added (nmol)
NEM added
(nmol)
4
.6 min, respectively. For the reaction of phenanthraquinone
with the BAEC preparation, the reaction mixture was filtered
with a membrane filter (4 mm i.d., 0.5 µm pore size) and the
filtrate immediately analyzed by the HPLC system described
above.
time (min)
1
10
25
50
10
30
3.7 ( 4.4 12.9 ( 8.1 15.4 ( 6.1 25.7 ( 3.9
19.8 ( 4.8 23.9 ( 4.2 22.4 ( 3.6 28.6 ( 1.3
26.8 ( 5.1 34.2 ( 5.0 19.8 ( 1.3 32.0 ( 5.2
6
0
a
The incubation mixture (1 mL) consisted of BAEC preparation
with phenanthraquinone, 67.28 nmol of SH; with NEM, 48.16
Resu lts
(
nmol of SH) and different amounts of phenanthraquinone or NEM
in 0.1 M potassium phosphate buffer (pH 7.5). Reactions were
carried out at 37 °C for different time periods under aerobic
conditions. Then the consumption of the thiol group was deter-
mined under conditions described under Materials and Methods.
Each data is the mean ( SD of three determinations.
In ter a ction of P h en a n th a qu in on e a n d NEM w ith
Su lfh yd r yls. The interaction of both phenanthraquinone
and NEM with the thiol groups of DTT and the thiol
groups contained in the BAEC preparation was exam-
ined. When NEM (25 and 50 µM) was incubated with
DTT (100 µM) in 0.1 M potassium phosphate buffer (pH
Ta ble 3. Con su m p tion of SH Gr ou p s in Lu n g Micr osom es
a
7
.5) at 37 °C for 10 min under anaerobic conditions, the
SH groups of DTT consumed were 7.2 ( 2.0 and 20.5 (
.3 µM, respectively (n ) 3). The level of thiol modifica-
of Ra ts d u r in g In ter a ction w ith P h en a n th r a qu in on e
phenanthraquninone
added (nmol)
SH groups
consumed (nmol)
2
tion by NEM reached a plateau after 10 min. The rate of
thiol consumption by phenanthraquinone was also pH-
dependent. Not unexpectedly, the rate of DTT oxidation
increased with increasing pH [i.e., 6.9 ( 3.4, 48.3 ( 1.0,
and 103.8 ( 0.3 µM modified thiol was formed after 10
min in the presence of phenanthraquinone (1 µM) at pH
0
0
2
0
.02
.2
0 ( 0.1
2.4 ( 0.4
2.7 ( 0.5
5.3 ( 1.1
2
a The incubation mixture (1.5 mL) consisted of lung microsomes
from rats (27.75 nmol of SH group) and different amounts of
phenanthraquinone in 100 mM potassium phosphate buffer (pH
.5). Reactions were carried out at 37 °C for 60 min under aerobic
conditions. Each value is the mean ( SD of three determinations.
5
.5, 6.5, and 7.5, respectively, n ) 3]. As shown in Figure
7
2
, the SH group of DTT was sensitive to dissolved oxygen
under the conditions examined. However, the destruction
of DTT was found to be catalytic with respect to phenan-
thraquinone since complete thiol loss in a 100 µM DTT
solution was seen with the addition of <100 µM phenan-
thraquinone under anaerobic conditions (Figure 2). For
example, phenanthraquinone at only 10 µM resulted in
the complete loss of free thiol groups in a 100 µM DTT
solution at pH 7.5 after 30 min, whereas molecular
oxygen stimulated phenanthraquinone-mediated con-
sumption of thiols in DTT markedly. The ability of
phenanthraquinone to oxidize thiols was not mediated
by a metal-dependent process since buffer treated with
the metal chelator dithizone did not affect the reaction
products. Interestingly, SH groups of dithiol compounds
such as DTT, BAL, and DMPS were selectively oxidized
by phenanthraquinone, whereas those of monothiol com-
pounds such as GSH, 2-mercaptoethanol, and N-acetyl-
L-cysteine were not (Table 1).
Compared to DTT oxidation, the thiols contained in the
membrane fraction of BAEC were less susceptible to
phenanthraquinone-mediated modification. However, an
excess amount of the protein sulfhydryls in BAEC
preparation was consumed after incubation with phenan-
thraquinone, but not NEM, suggesting a redox cycling
of phenanthraquinone in the presence of protein thiol
(Table 2). As shown in Table 3, the unusual stoichiomet-
ric relationship between protein sulfhydryls consumed
and phenanthraquinone added (0.2 nmol) was also seen
with rat lung microsomes. In contrast, phenanthraquino-
ne (1 and 10 nmol) was unaffected by a reactive mono-
thiol group of BSA (100 nmol) although the thiol content
(data not shown). However, treatment of thiols with 1,4-
benzoquinone, 2-methyl-1,4-benzoquinone, 2-chloroben-
zoquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone, pyr-
roloquinoline quinone, 2-anilino-1,4-naphthoquinone,
lapachol, 2-chloroanthraquinone, 5,12-naphthacenequino-
ne, 9,10-anthraquinone, or mytomycin c at a concentra-
tion of 1 µM gave negligible amounts of thiol oxidation

4
86 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Kumagai et al.
F igu r e 3. Measurement of phenanthraquinone or NEM re-
maining during incubation with DTT: (A) phenanthraquinone;
(
B) NEM. Open and closed bars indicate amounts of phenan-
thraquinone and NEM in the absence and presence of DTT
BAEC), respectively. The incubation mixture (1 mL) consisted
F igu r e 5. Superoxide generation during reaction of phenan-
thraquinone with DTT. The incubation mixure (1.5 mL) consist-
ing of phenanthraquinone (0.15 nmol) was incubated with DTT
(
of phenanthraquinone (10 nmol) or NEM (50 nmol), DTT (100
nmol), or BAEC preparation (40.37 nmol of SH) in 0.1 M
potassium phosphate buffer (pH 7.5). Reactions were carried
out at 37 °C for 30 min under aerobic conditions. Then, the
remaining phenanthraquinone or NEM was determined under
conditions described under Materials and Methods. The amount
of each compound remaining is expressed as its peak height.
Each value is the mean ( SD of three determinations.
(150 nmol), cytochrome c (50 nmol) in 50 mM Tris-HCl (pH 7.5)
in the presence and absence of Cu,Zn-SOD (2400 units) under
aerobic conditions. Reactions were performed at 25 °C under
aerobic conditions. Each value is the average of duplicate
determinations.
F igu r e 6. Effects of reactive oxygen scavengers on phenan-
thraquinone-mediated consumption of protein sulfhydryls. PQ,
phenanthraquinone; SOD, Cu,Zn-SOD; CAT, catalase. The
incubation mixture (1.5 mL) consisted of phenanthraquinone
(
150 nmol) and lung microsomes from rats (104 nmol of SH
group) in 0.1 M potassium phosphate buffer (pH 7.5) in the
absence and presence of Cu,Zn-SOD or catalase (1000 units).
Reactions were carried out at 37 °C for 60 min under aerobic
conditions. Each value is the mean ( SD of three determina-
tions.
F igu r e 4. ESR spectra of radical species generated. Closed
circles, thiyl-DMPO adduct; open circles, methyl-DMPO adduct.
tained in the BAEC preparation, it was consumed
(Figure 3B).
(
A) Horseradish peroxidase (10 µg) was incubated with 10 mM
DTT, 0.05 mM hydrogen peroxide, and 20 mM Hepes buffer (pH
.2). (B) 0.1 mM phenanthraquinone was incubated with 10 mM
DTT, 100 mM DMPO, and 0.1 M potassium phosphate buffer
pH 7.5). Phenanthraquinone was dissolved in Me SO (final
concentration of 2%). Each reaction was performed at 25 °C for
.5 min under aerobic conditions. The instrument settings were
7
Th iyl Ra d ica l Gen er a tion . Thiyl radicals can be
trapped with a radical trapping agent such as DMPO (24,
(
2
2
5). Figure 4A shows a typical ESR spectrum of the thiyl
0
radical formed in a system of DTT, horseradish peroxi-
dase, hydrogen peroxide, and DMPO. This DMPO adduct
has a distinctive ESR spectrum consisting of four rela-
as follows: modulation width, 100 kHz-0.2 mT; sweep time, 1
min; time constant, 0.03 s; microwave power, 7.99 mW; micro-
wave frequency, 9.4124 GHz; magnetic field, 335.3 ( 5 mT.
N
tively broad lines with coupling constants of R ) 1.51
of commercial BSA used in the study was 0.52 mol of SH
group/mol.
mT and RH
(
) 1.60 mT, as reported by Mason and Rao
20). When phenanthraquinone (100 µM) was mixed with
â
The fact that phenanthraquinone can destroy thiols
in a catalytic fashion could indicate that there is no direct
chemical reaction between phenanthraquinone and
thiols. That is, phenanthraquinone serves as a catalyst
for the generation of the ultimate thiol-degrading
species. Consistent with this idea, it was found that
phenanthraquinone was not consumed during the thiol-
destroying reaction (Figure 3A). On the other hand,
NEM is known to react directly with thiol functions,
leading to covalent and irreversible modification. Indeed,
when NEM was reacted with DTT or the thiols con-
DTT (10 mM) at pH 7.4, signals which were identical to
those generated by the thiyl radical generating system
were observed (Figure 4B). Moreover, a radical species
corresponding to a methyl-DMPO adduct was also de-
tected (RN ) 1.63 mT and RH ) 2.32 mT) (21). The
detection of the methyl-DMPO adduct is indicative of
hydroxyl radical production since it has been demon-
strated that the reaction of hydroxyl radical with
Me
2
SO (used to solubilize phenanthraquinone) in the
presence of DMPO results in the formation of this adduct

Oxidation of Sulfhydryls by Phenanthraquinone
22) (Figure 4B). These signals were not detected when
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 487
resulting in the overall oxidation of thiols by O
(
2
:
phenanthraquinone or DTT alone was incubated (data
not shown).
•
-
+
Q (phenanthraquinone) + R-SH f Q + H +
•
P r od u ction of Rea ctive Oxygen Sp ecies. The de-
struction of thiols in the presence of catalytic amounts
of phenanthraquinone and the generation of thiyl and
reduced oxygen intermediates (as evidenced by the
generation of the DMPO adducts) are consistent with a
process whereby phenanthraquinone serves as a catalyst
for the oxidation of thiols by the electron acceptor,
molecular oxygen. Such a process would generate, as an
intermediate, the reduced oxygen species superoxide. As
expected, a reaction of phenanthraquinone (0.1 nmol)
with DTT (100 nmol) caused a time-dependent generation
of superoxide as determined by Cu,Zn-SOD-inhibitable
reduction of cytochrome c (Figure 5). Under the condi-
tions, production of hydrogen peroxide with a rate of 44
nmol per 5 min was also observed. To confirm whether
these reactive oxygen species could contribute to phenan-
thraquinone-mediated oxidation of protein sulfhydryls,
we examined effects of scavenging agents for superoxide
and hydrogen peroxide on consumption of microsomal
protein sulfhydryls from rat lung caused by phenan-
thraquinone (Figure 6). Addition of an excess amount of
catalase (1000 units) suppressed the phenanthraquin-
one-mediated consumption of microsomal protein sulf-
hydryls. However, Cu,Zn-SOD was without effect on the
oxidation.
R-S (1)
•
• -
R-S + R-SH f R-S-S-R
(2)
•
-
• -
R-S-S-R + Q f Q + R-S-S-R
(3)
(4)
(5)
(6)
•
•
R-S + Q f R-SQ
•
• -
R-SQ + R-SH f Q + R-S-S-R
Q• - + O f Q + O
• -
2
2
O2• - + O2• - + 2H f H O + O
+
(7)
(8)
2
2
2
Fe + O2 f Fe2+ + O2
3
+
• -
Fe2+ + H O f Fe + ‚OH + OH
3+
-
(9)
2
2
Me SO + ‚OH f ‚CH + MeSO(OH)
(10)
2
3
There is ample precendence for all of the above reactions.
Reduction of quinones by thiolate (eq 1), reaction of thiyl
radical with thiolate (23) (or quinones) to give the
corresponding disulfide radical anion (18) (eq 2) and
quinone-thiol radical, which can react with thiolate to
form semiquinone radical anion and disulfides (eqs 4 and
5
), reduction of quinones to these semiquinone radical
Discu ssion
anions by disulfide radical anion (eq 3), reduction of O
2
The present results indicated that protein sulfhydryls
undergo facile modification by phenanthraquinone, a
component of DEP. We find that like NEM, phenan-
thraquinone is capable of destroying the thiols of both
DTT and those contained in a BAEC preparation (Figure
by a semiquinone radical anion (eq 6), and chemical
disproportion of superoxide to hydrogen peroxide and
molecular oxygen (eq 7) are all established processes.
Hydrogen peroxide can undergo a one-electron reduction
to hydroxyl radical in the presence of trace metals (e.g.,
iron contaminated in phosphate buffer) via a metal-
catalyzed Haber-Weiss reaction (24) as shown in eqs 8
and 9. However, there were signals of methyl-DMPO
instead of OH-DMPO (Figure 4). Because the concentra-
2
and Table 2). However, the mechanisms of thiol
destruction appear to be different for the two chemical
agents. Phenanthraquinone-mediated modification of thi-
ols is catalytic since thiol destruction occurs without loss
of phenanthraquinone whereas thiol loss via the NEM-
mediated process results in simultaneous loss of NEM
tion of Me
radical (25), in the reaction mixture was approximately
60 mM, a reasonable explanation is that the hydroxyl
radical formed reacts better with Me SO than with
DMPO, resulting in trapping the methyl radical (18, 21,
6). In ESR experiments, no signals of the semiquinone
2
SO, a potent scavenging agent for hydroxyl
2
(Figure 3). It is well recognized that quinones have two
2
chemical properties consisting of (1) electrophilic attack
to nucleophiles, resulting in thiol adduct formation, and/
or (2) redox cycling, wherein there is rapid and sequential
reduction to the quinone, leading to production of reactive
oxygen species (11). Therefore, we conclude that the
reactivity of phenanthraquinone toward DTT is classified
into the latter case.
2
radical of phenanthraquinone (eqs 3 and 5) were detected.
This discrepancy may result from instability of o-semi-
quinones (27) and/or rapid oxidation by molecular oxygen
(eq 6).
Using synthetic thiol species, it was found that phenan-
The catalytic destruction of thiols by phenanthraquino-
ne occurred even in anaerobic media; however, such a
phenomenon was further accelerated in the presence of
molecular oxygen. That is, phenanthraquinone acts as a
thraquinone-mediated oxidation of sulfhydryls occurs
specifically with proximal thiol systems (Table 1). Con-
sistent with this idea, the reactive monothiol group
(Cys34) of BSA (28) was not modified by reactions with
2
catalyst for the thiol-mediated reduction of O , which
phenanthraquinone. From these findings, we postulate
that only proximal sulfhydryls in proteins will be oxidized
by interaction with phenanthraquinone. However, it was
found that phenanthraquinone-mediated oxidation of the
protein sulfhydryls of rat lung microsomes was dimin-
ished by addition of catalase, but not Cu,Zn-SOD (Figure
6). Although hydrogen peroxide formed during reaction
of phenanthraquinone with DTT (see Results) is shown
to oxidize thiol oxidation of BSA (29), this quinone could
not modify the monothiol group of BSA in the present
leads to the generation of reactive oxygen species and
thiol oxidation. This idea is supported by our observation
that both reduced oxygen species and the oxidized thiol
species, thiyl radical, are observed by ESR (Figure 4).
Furthermore, the reaction of phenanthraquinone with
thiols results in the generation of a species, such as
superoxide, which can reduce cytochrome c in an SOD-
inhibitable fashion (Figure 5). Taken together, these data
are consistent with the following chemical reactions

4
88 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Kumagai et al.
study. Indeed, it is indicated that interaction of phenan-
thraquinone with proximal thiols, but not monothiol, is
essential to initiate sulfhydryl oxidation. Nevertheless,
it should be noted that although reactions (eqs 1-5)
caused by interaction of thiols with phenanthraquinone
play a main role in the oxidation of protein sulfhydryls,
hydrogen peroxide produced as a byproduct of the reac-
tion with sulfhydryls may partially contribute to the
sulfhydryl oxidation as reported by others (29). The
reactivity of phenanthraquinone with the dithiol group
in preference to the monothiol group may reflect the
lower redox potential of the dithiol as compared to
monothiols (30).
Intracellular thiol oxidation (i.e., conversion of thiols
to disulfides) can be reversed by the thioredoxin/thiore-
doxin reductase system in the presence of NADPH (31).
These proteins have vicinal dithiol functions at the active
center (31). Interestingly, using a partially purified
protein preparation, we have found that thioredoxin
activity was completely inhibited by 10 µM phenan-
thraquinone (Taguchi, K., et al., unpublished observa-
tions), suggesting that oxidation of the active dithiol
group is caused by this quinone. This implies that
dysfunction of thioredoxin by phenanthraquinone may
cause not only reduction of antioxidant status but also
alteration in the redox-dependent signal transduction
Yuji Ishii, Institute of Community Medicine, University
of Tsukuba, for preparation of the manuscript. This
research was supported in part by grants-in-aid (11877398
and 13672340, Y.K.) for scientific research from the
Ministry of Education, Science and Culture of J apan, by
the Naito Foundation (Y.K.), and by a fund (University
Research Project, Y.K.) from the University of Tsukuba.
Refer en ces
(1) McClellan, R. O. (1987) Health effects of exposure to diesel
exhaust particles. Annu. Rev. Pharmacol. Toxicol. 27, 279-300.
(2) Dockery, D. W., Pope, A. C., III, Xu, X., Spengler, J . D., Ware, J .
H., Fay, M. E., Ferris, B. G., J r., and Speizer, F. E. (1993) An
association between air pollution and mortality in six U.S. cities.
N. Engl. J . Med. 329, 1753-1759.
(
3) Nel, A. E., Diaz-Sanchez, D., and Li, N. (2001) The role of
particulate pollutants in pulmonary inflammation and asthma:
evidence for the involvement of organic chemicals and oxidative
stress. Curr. Opin. Pulm. Med. 7, 20-26.
(
4) Sagai, M., Saito, H., Ichinose, T., Kodama, M., and Mori, Y. (1993)
Biological effects of diesel exhaust particles. I. In vitro production
of superoxide and in vivo toxicity in mouse. Free Radical Biol.
Med. 14, 37-47.
(5) Kumagai, Y., Arimoto, T., Shinyashiki, M., Shimojo, N., Nakai,
Y., Yoshikawa, T., and Sagai, M. (1997) Generation of reactive
oxygen species during interaction of diesel exhaust particle
components with NADPH-cytochrome P450 reductase and in-
volvement of the bioactivation in the DNA damage. Free Radical
Biol. Med. 22, 479-487.
(
6) Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999)
Chemicals in diesel exhaust particles generate reactive oxygen
radicals and induce apoptosis in macrophages. J . Immunol. 163,
5582-5591.
(14, 32).
We have recently shown that phenanthraquinone is a
potent inhibitor of endothelial nitric oxide synthase, and
thus suppresses endothelium-dependent relaxation of rat
aortas by acetylcholine and increases blood pressure in
rats (33). It was also found that the inhibitory action of
phenanthraquinone on endothelial nitric oxide synthase
activity was, in part, suppressed by addition of DTT (34).
It was reported that sulfhydryl groups in endothelial cells
play a critical role in nitric oxide formation (35-38). Patel
et al. (39) reported that modification of the thiol groups
in the transporter for L-arginine was associated with a
down-regulation of overall L-arginine transport into
endothelial cells. Taken together, our data suggest that
phenanthraquinone may alter the sulfhydryl status for
both the L-arginine transporter and endothelial nitric
oxide synthase, both of which will result in a loss of nitric
oxide biosynthesis.
(7) Li, N., Venkatesan, M. I., Miguel, A., Kaplan, R., Gujuluva, C.,
Alam, J ., and Nel, A. (2000) Induction of heme oxygenase-1
expression in macrophages by diesel exhaust particle chemicals
and quinones via the antioxidant-responsive element. J . Immunol.
165, 3393-3401.
(8) Schuetzle, D. (1983) Sampling of vehicle emissions for chemical
analysis and biological testing. Environ. Health Perspect. 47, 65-
8
0.
(
9) Schuetzle, D., Lee, F. S. C., Prater, T. J ., and Tejada, S. B. (1981)
The identification of polynuclear aromatic hydrocarbon (PAH)
derivatives in mutagenic fractions of diesel particulate extracts.
Int. J . Environ. Anal. Chem. 9, 93-144.
(
10) Monks, T. J ., Hanzlik, R. P., Cohen, G. M., Ross, D., and Graham,
D. G. (1992) Quinone chemistry and toxicology. Toxicol. Appl.
Pharmacol. 112, 2-16.
(11) O’Brien, P. J . (1991) Molecular mechanisms of quinone cytotox-
icity. Chem.-Biol. Interact. 80, 1-41.
(12) Bolton, J . L., Trush, M. A., Penning, T. M., Dryhurst, G., and
Monks, T. J . (2000) Role of quinones in toxicology. Chem. Res.
Toxicol. 13, 135-160.
Our previous results indicate that quinones in DEP are
good substrates for NADPH-cytochrome P450 reductase
and that superoxide and hydroxyl radical generated
during redox cycling of the quinones by this flavin
enzyme participate in the DEP-promoted oxidative stress
(
13) Stamler, J . S., and Slivka, A. (1996) Biological chemistry of thiols
in the vasculature and in vascular-related disease. Nutr. Rev. 54,
1-30.
(
14) Finkel, T. (2000) Redox-dependent signal transduction. FEBS
Lett. 476, 52-54.
(5). Nel and co-workers (3, 6, 7) have proposed that such
(
15) Kumagai, Y., Shinyashiki, M., Sun, G. F., Shimojo, N., and Sagai,
M. (1994) An efficient method for purification of cuprozinc
superoxide dismutase from bovine erythrocytes. Experimentia 50,
an overproduction of reactive oxygen species is associated
with allergic inflammation and induction of apoptosis by
DEP exposure. However, the chemicals associated with
this toxicity remain uncharacterized. The study indicated
that phenanthraquinone (a relatively abundant quinone
in DEP) can act as a catalytic oxidizing agent for
proximal protein sulfhydryls in addition to its ability to
interact with NADPH-cytochrome P450 reductase, lead-
ing to the overproduction of reactive oxygen species (5,
3
73-376.
(
16) Bradford, M. M. (1976) A rapid and sensitive method for the
quantitation of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72, 248-254.
(
17) Kumagai, Y., Todaka, T., and Toki, S. (1990) A new metabolic
pathway of morphine: In vivo and in vitro formation of morphi-
none and morphinone-glutathione adduct in guinea pig. J . Phar-
macol. Exp. Ther. 255, 504-510.
(
18) Yim, M. B., Chae, H. Z., Rhee, S. G., Chock, P. B., and Stadtman,
E. R. (1994) On the protective mechanism of the thiol-specific
antioxidant enzyme against the oxidative damage of biomacro-
molecules. J . Biol. Chem. 269, 1621-1626.
4
0). This chemical reactivity indicates that phenan-
thraquinone may be, at least partially, responsible for
DEP-induced oxidative stress.
(
19) Hildebrandt, A. G., and Root, I. (1975) Reduced nicotinamide
adenine dinucleotide phosphate (NADPH)-dependent formation
and breakdown of hydrogen peroxide during mixed function
oxidation reactions in liver microsomes. Arch. Biochem. Biophys.
171, 385-397.
Ack n ow led gm en t. We thank Dr. J on M. Fukuto,
Department of Molecular and Medical Pharmacology,
UCLA School of Medicine, for helpful discussion and Dr.

Oxidation of Sulfhydryls by Phenanthraquinone
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 489
(
20) Mason, R. P., and Rao, D. N. R. (1990) Thiyl free radical
metabolites of thiol drugs, glutathione, and proteins. Methods
Enzymol. 186, 318-329.
(32) Morel, Y., and Barouki, R. (1999) Repression of gene expression
by oxidative stress. Biochem. J . 342, 481-496.
(33) Kumagai, Y., Hayashi, T., Miyauchi, T., Endo, A., Iguchi, A.,
Kiriya-Sakai, M., Sakai, S., Yuki, K., Kikushima, M., and Shimojo,
N. (2001) Phnanthraquinone inhibits eNOS activity and sup-
presses vasorelaxation. Am. J . Physiol. 281, R25-R30.
(34) Taguchi, K., Kumagai, Y., Endo, A., Kikushima, M., Ishii, Y., and
Shimojo, N. (2001) Phenanthraquinone affects endothelial nitric
oxide synthase activity through modification of the thiol groups:
an alternative inhibition mechanism. J . Health Sci. 47, 571-574.
(35) Murphy, M. E., Piper, H. M., Watanabe, H., and Sies, H. (1991)
Nitric oxide production by cultured aortic endothelial cells in
response to thiol depletion and replenishment. J . Biol. Chem. 266,
19378-19383.
(36) Zembowicz, A., Hatchett, R. J ., Radziszewsi, W., and Gryglewsi,
R. J . (1993) Inhibition of endothelial nitric oxide synthase by
ebselen. Prevention by thiol suggests the inactivation by ebselen
of a critical thiol essential for the catalytic activity of nitric oxide
synthase. J . Pharmacol. Exp. Ther. 267, 1112-1117.
(37) Marczin, N., Ryan, U. S., and Catravas, J . D. (1993) Sulfhydryl-
depleting agents, but not deferoxamine, modulated EDRF action
in cultured pulmonary arterial cells. Am. J . Physiol. 265, L220-
L227.
(38) Patel, J . M., and Block, E. R. (1995) Sulfhydryl-disulfide modifica-
tion and the role of disulfide oxidoreductases in regulation of the
catalytic activity of nitric oxide synthase in pulmonary artery
endothelial cells. Am. Respir. Cell Mol. Biol. 13, 352-359.
(
21) Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986)
Do human neutrophils make hydroxyl radical? Determination of
free radicals generated by human neutrophils activated with a
soluble or particulate stimulus using electron paramagnetic
resonance spectrometry. J . Biol. Chem. 261, 4426-4431.
22) Finkelstein, E., Rosen, G. M., and Rauckman, E. J . (1982)
Production of hydroxyl radical by decomposition of superoxide
spin-trapped adducts. Mol. Pharmacol. 21, 262-265.
23) Gutierrez, P. L., and Siva, S. (1995) Diaziquinone-glutathione
conjugates: Characterization and mechanisms of formation.
Chem. Res. Toxicol. 8, 455-464.
24) Cadenas, E. (1989) Biochemistry of oxygen toxicity. Annu. Rev.
Biochem. 58, 79-110.
(
(
(
(
25) Klein, S. M., Cohen, G., and Cederbaum, A. I. (1981) Production
of formaldehyde during metabolism of dimethyl sulfoxide by
hydroxyl radical generating system. Biochemistry 20, 6006-6012.
26) Ashwood-Smith, M. J . (1975) Current concepts concerning radio-
protective and cryoprotective properties of dimethyl sulfoxide in
cellular systems. Ann. N.Y. Acad. Sci. 43, 246-256.
(
(
27) Kalyanaraman, B., Sealy, R. C., and Sivarajah, K. (1984) An
electron spin resonance study of o-semiquinones formed during
the enzymatic and autoxidation of catechol estrogens. J . Biol.
Chem. 259, 14018-14022.
(
(
(
(
28) Sadler, P. J ., and Tucker, A. (1992) Proton NMR studies of bovine
serum albumin. Assignment of spin systems. Eur. J . Biochem.
(
39) Patel, J . M., Abeles, A. J ., and Block, E. R. (1996) Nitric oxide
exposure and sulfhydryl modulation alter L-arginine transport in
cultured pulmonary artery endothelial cells. Free Radical Biol.
Med. 20, 629-637.
2
05, 631-643.
29) Radi, R., Beckman, J . S., Bush, K. M., and Freeman, B. A. (1991)
Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of
superoxide and nitric oxide. J . Biol. Chem. 266, 4244-4250.
30) Wardman, P. (1989) Reduction potentials of one-electron couples
involving free radicals in aqueous solution. J . Phys. Chem. Ref.
Data 18, 1637-1755.
(
40) Kumagai, Y., and Shimojo, N. (2001) Induction of oxidatives stress
and dysfunction of nitric oxide-dependent vascular tone caused
by quinones contained in diesel exhaust particles. J . Health Sci.
4
7, 439-445.
31) Holmgren, A. (1985) Thioredoxin. Annu. Rev. Biochem. 54, 237-
2
71.
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