The reduction of α-tocopherolquinone by human NAD(P)H: Quinone oxidoreductase: The role of α-tocopherolhydroquinone as a cellular antioxidant

Article abstract of DOI:10.1124/mol.52.2.300

α-Tocopherolquinone (TQ), a product of α-tocopherol oxidation, can function as an antioxidant after reduction to α-tocopherolhydroquinone (TQH₂). We examined the ability of human NAD(P)H:quinone oxidoreductase (NQO1) to catalyze the reduction o

Full text of DOI:10.1124/mol.52.2.300

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MOLECULAR PHARMACOLOGY, 52:300–305 (1997).
The Reduction of ␣-Tocopherolquinone by Human NAD(P)H:
Quinone Oxidoreductase: The Role of
␣
-Tocopherolhydroquinone as a Cellular Antioxidant
DAVID SIEGEL, EMIKO M. BOLTON, JEANNE A. BURR, DANIEL C. LIEBLER, and DAVID ROSS
Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver,
Colorado 80262 (D.S., E.M.B., D.R.), and Department of Pharmacology and Toxicology, College of Pharmacy and Cancer Center, University of
Arizona, Tucson, Arizona 85721 (J.A.B., D.C.L.)
Received March 18, 1997; Accepted April 30, 1997
SUMMARY
␣
-Tocopherolquinone (TQ), a product of ␣-tocopherol oxida- demonstrated a correlation between NQO1 activity and TQ
tion, can function as an antioxidant after reduction to ␣-toco- reduction to TQH . CHO cells with elevated NQO1 generated
2
pherolhydroquinone (TQH ). We examined the ability of human and maintained higher levels of TQH after treatment with TQ
2
2
NAD(P)H:quinone oxidoreductase (NQO1) to catalyze the re- relative to NQO1-deficient CHO cells. TQH2 generated from
duction of TQ to TQH2 in cell-free and cellular systems. In NQO1-mediated reduction of TQ prevented cumene hydroper-
reactions with purified human NQO1, TQ was reduced to TQH2. oxide-induced lipid peroxidation in rat liver microsomes. In
Kinetic parameters for the reduction of TQ by NQO1 (K ϭ 370 addition, cumene hydroperoxide-induced lipid peroxidation
m
3
minϪ1
Ϫ1
Ϫ1
␮M; kcat ϭ 5.6 ϫ 10
; kcat/Km ϭ 15 min ⅐ ␮M ) indicate was inhibited more efficiently by TQ in CHO cell lines with
that NQO1 can efficiently reduce TQ to TQH . A comparison of elevated NQO1 activity. These data demonstrate that NQO1
2
the rate of reduction of TQ and coenzyme Q10 by NQO1 can reduce TQ to TQH2 and that TQH2 can function as an
showed that TQ is reduced more efficiently than coenzyme Q10
.
efficient antioxidant. This work suggests that one of the phys-
Experiments with either Chinese hamster ovary (CHO) cells iological functions of NQO1 may be to regenerate antioxidant
stably transfected with human NQO1 or CHO cell sonicates forms of ␣-tocopherol.
NQO1 (EC 1.6.99.2), or DT-diaphorase, is an obligate two- preventive enzyme (6–9), which has been recognized for Ͼ30
electron reductase that catalyzes reduction of a broad range years (11).
of substrates (1, 2). It is a flavoprotein that exists as a
Whether NQO1 catalyzes the reduction of endogenous sub-
homodimer and is biochemically characterized by its unique strates remains unclear. A potential role for NQO1 in vita-
ability to utilize either NADH or NADPH as reducing cofac- min K metabolism has been suggested (12, 13), but studies
tors and by its inhibition by the anticoagulant dicumarol (1, with purified NQO1 do not support such a role (14). An
3
). The enzyme is generally considered as a detoxification important observation is that x-radiation and UV radiation,
enzyme because of its ability to detoxify reactive quinones which are known to generate oxidative stress, induced NQO1
and quinone-imines to less reactive and less toxic hydroqui- expression Ͼ30-fold in human cells (15). Our data show that
nones (1, 3). Such a two-electron reduction also bypasses hepatic loading of iron in rats generates oxidative stress and
1
semiquinone production and thus prevents the generation of a concomitant induction of NQO1. This suggests that in
reactive oxygen species derived from interaction of the addition to limiting oxygen radical formation from exogenous
semiquinone with molecular oxygen (4, 5). The ability of quinones, NQO1 may play an endogenous antioxidant role.
NQO1 to deactivate many reactive species, including quino- Recent work has suggested that NQO1 maintains ubiquinone
nes, quinone-imines, and azo compounds, demonstrates its (CoQ ) in its quinol form, which can act as an antioxidant to
10
importance as a chemoprotective enzyme (6–10). The other protect membranes from oxidative stress (16).
major protective effect of NQO1 is to function as a cancer
We examined the role of NQO1 in the metabolism of ␣-to-
This work was supported by United States Public Health Service Grants
CA51210 and CA59585.
1
D. Siegel and L. Valerio, unpublished observations.
ABBREVIATIONS: TQ, ␣-tocopherolquinone; TQH , ␣-tocopherolhydroquinone; NQO1, NAD(P)H:quinone oxidoreductase; CoQ10, coenzyme
2
Q10; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; GC-MS, gas chromatog-
raphy-mass spectrometry, DCPIP, 2,6-dichlorophenol-indophenol; TBARS, thiobarbituric acid reactive substances; HEPES, 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid.
300

Reduction of ␣-Tocopherolquinone by NQO1
301
copherol derivatives. In this report, we demonstrate that TQ equipped with a Carlo Erba 8000 series gas chromatograph and a
is an excellent substrate for human NQO1. TQ is produced Fisons on-column injector and operated in the electron ionization
mode at 70 eV. The HPLC peak corresponding to TQH was collected,
via free radical attack of ␣-tocopherol (vitamin E) and has no
intrinsic antioxidant activity (17–19). The product of NQO1-
2
TQH2 was extracted from the mobile phase with hexane, and the
hexane was evaporated in vacuo. TQH was converted to the tris-(O-
trimethylsilyl) derivative as described (25). The derivative was ana-
lyzed by GC-MS on a 30 m ϫ 0.25 mm DB-5ms column (J & W
Scientific, Folsom, CA) with cold on-column injection (25). The tris-
2
mediated reduction of TQ is TQH which, unlike TQ, is a
2
potent antioxidant (18, 19). Reduction of TQ to its TQH2
derivative has been demonstrated in cellular systems, but
the enzymes responsible have not been characterized (18–
(
O-trimethylsilyl) derivative of TQH2 coeluted with an authentic
standard and yielded a mass spectrum identical to that of the stan-
2
0). This work demonstrates a role for NQO1 in ␣-tocopherol
metabolism and suggests that one of the physiological func- dard: m/z 665 [Mϩ, 16%] 575 [21], 341 [8], 309 [100], 294 [20], 280
tions of NQO1 may be to regenerate antioxidant forms of [10], 236 [9], 220 [6]. This spectrum is essentially identical to that we
␣
-tocopherol.
previously reported for TQH (25). The molecular ion for the tris-(O-
trimethylsilyl) derivative appeared at m/z 665 due to mass defect;
the monoisotopic mass is 664.5 amu.
2
Materials and Methods
Kinetic analysis of TQ and CoQ10 reduction by NQO1. Ki-
Chemicals. NADH, NADPH, cholic acid, dicumarol, DCPIP, ara- netic analysis of TQ reduction by NQO1 was determined by spectro-
chidonic acid, butylated hydroxytoluene, ␣-tocopherol acetate, photometrically monitoring NADH oxidation. Reactions (1 ml) were
cumene hydroperoxide, 2-thiobarbituric acid, HEPES, and CoQ10 performed in 50 mM potassium phosphate buffer, pH 7.4, containing
were purchased from Sigma Chemical (St. Louis, MO). TQ was 5% (w/v) cholic acid, 0.4% (v/v) Tween-80, 200 ␮M NADH, 0.288 ␮g of
obtained from United States Biochemical (Arlington Heights, IL). NQO1, and 0.01–1.28 mM TQ. Reactions were monitored for 0–2 min
Stock solutions of TQ were prepared in 95% (v/v) ethanol unless at 27° at 340 nm. Kinetic parameters were determined using Enzfit-
otherwise stated. All other reagents were of analytical grade.
ter kinetic software (Biosoft, Cambridge, UK) from the mean of
CHO cell lines and NQO1 purification. CHO cell lines stably triplicate determinations. A comparison of the rate of reduction of
transfected with human NQO1 cDNA were developed as previously TQ and CoQ10 (5, 50 ␮M) by NQO1 was determined by monitoring
described (21). CHO cell lines were grown in Ham’s F-12 (GIBCO NADH oxidation as described above. Reactions (1 ml) were per-
BRL, Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine formed in 50 mM potassium phosphate buffer, pH 7.4, containing 5%
serum and 2 mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml cholic acid and 0.4% Tween-80, 200 ␮M NADH, and 7.2 ␮g of NQO1.
streptomycin (GIBCO BRL). Recombinant human NQO1 protein was For these experiments, initial stock solutions of TQ and CoQ10 (10
purified from Escherichia coli using Cibacron Blue affinity chroma- mM) were dissolved in N,N-dimethylformamide before dilution in
tography as previously described (22). The specific activity of the buffer.
purified NQO1 protein was 625 ␮mol of DCPIP/min/mg. NQO1 spe-
Lipid peroxidation in rat liver microsomes and CHO cells.
cific activity in the CHO cell lines and purified NQO1 protein was Lipid peroxidation was measured by analysis of TBARS as previ-
determined using DCPIP reduction as previously described (23). ously described (26). Reactions with rat liver microsomes were
Protein concentrations were determined according to the method of performed as follows: 200 ␮M NADH, 3.6 ␮g of NQO1, 50 ␮M TQ,
Lowry (24).
Formation of TQH2 in CHO sonicates and cultured cells. phate buffer, pH 7.4, containing 1% cholic acid (final volume, 0.5
The reduction of TQ by CHO sonicates was performed by the addition
ml) at 27°. After 30 min, cumene hydroperoxide (1 mM) was added
of 650 ␮g of CHO sonicate to 50 mM potassium phosphate buffer, pH to initiate lipid peroxidation. After 1 hr, the reaction was stopped
.4, containing 1% (w/v) cholic acid, 200 ␮M NADH, and 50 ␮M TQ
by the addition of 100 ␮M butylated hydroxytoluene and 2 ml of
and 750 ␮g of microsomes were added to 50 mM potassium phos-
7
(
final volume, 250 ␮l) at 27°. After 30 min, the reactions were 0.25 N hydrochloric acid containing 15% (w/v) trichloroacetic acid
terminated, and TQ and TQH analyses were performed as described and 0.37% (w/v) 2-thiobarbituric acid. The sample was heated to
2
below. The conversion of TQ to TQH in the cultured CHO cell lines 80° for 20 min and then centrifuged at 2500 rpm for 10 min, and
2
was determined using the following methods: CHO cell lines were the absorbance at 535 nm was determined. Results were ex-
grown to Ͼ90% confluency, the medium was removed, and fresh pressed as TBARS equivalents using a molar extinction coefficient
5
medium containing 50 ␮M TQ was added. After 6, 12, or 24 hr, the of 1.56 ϫ 10 (26). Lipid peroxidation studies with CHO cell lines
reaction was terminated by removal of the TQ-containing medium, were performed as follows: CHO cell lines were grown to Ͼ90%
washing of the cells with 10 ml of PBS twice, and scraping of the cells confluency on 100-mm tissue culture plates. Before the initiation
into 1 ml of PBS. The cells were pelleted by centrifugation, the of lipid peroxidation, the medium was exchanged with fresh me-
supernatant was removed, and the cell pellet was snap-frozen in dium containing 250 ␮M arachidonic acid and 0, 1, or 5 ␮M TQ.
liquid nitrogen and stored at Ϫ70° until analysis by HPLC.
After 14 hr, the TQ-containing medium was removed, the cells
Analysis of TQ and TQH2. HPLC was used to separate TQ and were washed with 10 ml of PBS, and 10 ml of Krebs-HEPES
TQH2 in reactions with either purified NQO1, CHO sonicates or buffer, pH 7.2, was added. Lipid peroxidation was initiated by the
CHO cell lines. Reactions (purified NQO1, CHO sonicates) were addition of cumene hydroperoxide (1 mM), and after 2.5 hr, the
stopped with an equal volume of cold acetonitrile containing 0.01% cells were scraped into the medium and collected by centrifuga-
(
w/v) butylated hydroxytoluene and 200 ␮M ␣-tocopherol acetate tion. The supernatant was removed, and the cell pellet was resus-
(
internal standard) and then centrifuged at 10,000 rpm for 2 min. pended in 1 ml of PBS. An aliquot (10 ␮l) of cell suspension was
The cell pellet (see above) was resuspended in 150 ␮l of acetonitrile removed for protein determination according to the method of
containing 0.003% (w/v) butylated hydroxytoluene and 67 ␮M ␣-to- Lowry (24), and lipid peroxidation was measured using the
copherol acetate and then centrifuged at 10,000 rpm for 2 min. The TBARS assay in the remaining cell suspension as described above.
supernatant was analyzed on a LiChrosorb RP-18 column (5 ␮m, 25
Statistical analysis. Results are expressed as mean Ϯ standard
cm, Merck, Darmstadt, Germany) with the following gradient: buffer deviation. Statistical significance between sets of data was deter-
A, 1% (v/v) acetic acid; buffer B, 100% acetonitrile; gradient, 70–95% mined using the Student’s t test.
buffer B over 10 min and then hold at 95% buffer B for 25 min. Flow
rate was maintained at 1.5 ml/min with UV detection at 280 nm.
Results
HPLC retention times were TQH , 14 min; TQ, 22.1 min; and inter-
2
nal standard, 33.3 min. TQH was analyzed by capillary GC-MS on
TQ as a substrate for purified NQO1. Initial experi-
2
a Fisons MD800 instrument (Fisons Instruments, Beverly, MA) ments were performed to determine whether TQ could serve

3
02
Siegel
as a substrate for purified NQO1. HPLC analysis of a reac- TABLE 1
tion containing TQ, NADH, and NQO1 showed loss of the TQ Kinetic parameters for the reduction of TQ by NQO1
peak (tR ϭ 22.1 min) and formation of a more polar product Reactions conditions are described in the text.
(
tR ϭ 14 min; Fig. 1A). The product from the reduction of TQ
K
m
k
cat
kcat/K
m
by NQO1 was confirmed by GC-MS as TQH (for details, see
2
minϪ1
minϪ1 ⅐ ␮MϪ1
␮
M
Materials and Methods). The reaction was NADH or NADPH
dependent and dicumarol inhibitable (Fig. 1B). Kinetic val-
ues for the reduction of TQ by NQO1 were determined spec-
trophotometrically by monitoring the oxidation of NADH.
3
3
70
5.6 ϫ 10
15
TABLE 2
Comparative rate of reduction of CoQ10 and TQ by NQO1
Reaction conditions are described in the text.
Values for K , kcat, and kcat/Km are presented in Table 1 and
m
suggest that NQO1 efficiently reduces TQ to TQH . Kinetic
2
Substrate con-
centration
parameters for the reduction of CoQ10 by NQO1 could not be
determined due to the poor solubility of CoQ . Comparative
CoQ10
TQ
10
␮
M
nmol of NADH/min/mg of protein
studies on the reduction of TQ and CoQ10 by NQO1 were
performed at substrate concentrations of 5 and 50 ␮M (Table
5
0
7.9 Ϯ 0.1
52.0 Ϯ 1.3
290 Ϯ 7.4
3000 Ϯ 60
5
2
). The rate of TQ reduction was ϳ36-fold greater at 5 ␮M and
Values are mean Ϯ standard deviation from three separate determinations.
ϳ57-fold greater at 50 ␮M compared with the rate of CoQ10
reduction. These data demonstrate that NQO1 can reduce
TABLE 3
TQ considerably more efficiently than CoQ .
10
Reduction in TQ by NQO1-transfected CHO sonicates
Reduction of TQ by NQO1-transfected CHO cell soni-
cates. The reduction of TQ by sonicates prepared from CHO
cell lines stably transfected with NQO1 and expressing dif-
ferent amounts of NQO1 activity was examined by HPLC
Reaction conditions are described in the text.
Cell line
CHO-glyA
CHO-815
CHO-812
nmol of DCPIP/min/mg of protein
a
NQO1 activity
TQ reduced
N.D.
98
1300
11.0 Ϯ 0.03
(
Table 3). A relationship between TQ reduction and NQO1
b
0.3 Ϯ 0.27
5.0 Ϯ 0.54
activity was apparent. Sonicates prepared from the CHO cell
line with the highest NQO1 activity (CHO812) catalyzed the
greatest amount of TQ reduction. Less TQ reduction was
observed in sonicates from the CHO cell line CHO815, which
contains intermediate levels of NQO1, whereas sonicates
a
Not detectable above control (minus sonicate) levels. Limit of detection Ͻ5
nmol of DCPIP/min/mg of protein.
b
nmol of TQ reduced in 30 min.
Values are mean Ϯ standard deviations from three separate determinations.
from the NQO1-deficient CHO parent cell line (CHOglyA
)
catalyzed only minimal TQ reduction. The reduction of TQ by
the CHO812 and CHO815 sonicates was NADH or NADPH
dependent and dicumarol inhibitable. The product of the
reduction of TQ by CHO sonicates cochromatographed with
TQH prepared from the reduction of TQ by purified NQO1
2
(
data not shown).
Reduction of TQ by NQO1-transfected CHO cells in
culture. Experiments were performed to examine TQ reduc-
tion and TQH formation in NQO1-transfected CHO cells in
2
culture (Fig. 2). Previous studies have shown that there were
no significant differences between the CHO812 and CHOglyA
cell lines in the levels of one-electron reductases; NADPH:
cytochrome P450 reductase, and NADH:cytochrome b reduc-
5
tase (21). The NQO1-rich CHO812 and NQO1-deficient
CHOglyA cell lines were exposed to 50 ␮M TQ in culture, and
the amounts of TQ and TQH2 were determined by HPLC
after 6 and 12 hr. The results are expressed as the ratio of
TQH to TQ and show that the high-NQO1 cell line, CHO812,
2
had significantly higher TQH -to-TQ ratios compared with
2
the NQO1-deficient cell line, CHOglyA. These data indicate
that cells with high NQO1 can generate and maintain higher
levels of TQH than NQO1-deficient cells.
2
Antioxidant actions of TQH . Experiments were per-
2
formed to determine the stability of TQH . HPLC analysis
2
confirmed that TQH formed in the reaction with NQO1 and
2
stoichiometric amounts (50 ␮M) of TQ and NADH was stable
Fig. 1. HPLC analysis of the reduction of TQ by purified NQO1. Reac- under aerobic conditions. No autoxidation of TQH back to
2
tion conditions were 50 ␮M TQ, 200 ␮M NADH, and 7.2 ␮g of NQO1 in
TQ could be detected after 3 hr in 50 mM potassium phos-
phate buffer, pH 7.4, containing 1% cholic acid. The oxidation
of TQH to TQ, however, could readily be induced by unsat-
urated fatty acid peroxidation (Fig. 3A). In reactions in which
50 mM potassium phosphate buffer, pH 7.4, containing 1% cholic acid.
Reactions were performed in a total volume of 1 ml at 27° for 30 min in
the absence (A) and presence (B) of 20 ␮M dicumarol. HPLC conditions
are described in Materials and Methods.
2

Reduction of ␣-Tocopherolquinone by NQO1
303
TQH was formed after the reduction of TQ by NQO1, the TABLE 4
2
2ϩ
addition of arachidonic acid, Fe , and hydrogen peroxide Inhibition of cumene hydroperoxide-induced microsomal lipid
catalyzed the oxidation of TQH back to TQ. However, when
arachidonic acid was absent or replaced by stearic acid, a
peroxidation by TQH2
2
Reaction conditions are described in the text.
saturated fatty acid, minimal oxidation of TQH to TQ could
Treatment
TBARS
nmol
2
be observed (Fig. 3, B and C). These data suggest that TQH2
can be oxidized back to TQ via products of unsaturated fatty
acid peroxidation.
Studies were performed to examine the antioxidant effects
of TQH2 on microsomal and cellular lipid peroxidation.
Cumene hydroperoxide-induced lipid peroxidation was mea-
sured under aerobic conditions in microsomes supplemented
with TQ, NADH, and NQO1 (Table 4). Minimal amounts of
cumene hydroperoxide-induced lipid peroxidation were de- 0.05.
tected in reactions pretreated with NADH, NQO1, and TQ. In
control reactions, however (minus NADH, NQO1, or TQ),
substantial amounts of lipid peroxidation were detected.
NADH alone did exhibit some antioxidant activity, which
could be expected based on its known ability to scavenge
organic radicals (27). These results indicate that TQH2
formed from the reduction of TQ by NQO1 can function as an
antioxidant and inhibit lipid peroxidation in microsomal in-
cubations.
Control reactions
ϪNADH
ϪTQ
ϪNQO1
Complete reaction
NADH, TQ,
NQO1)
4.2 Ϯ 0.7
2.2 Ϯ 0.2
2.1 Ϯ 0.4
0.4 Ϯ 0.1
a
(
Complete system was significantly different from control (Ϫ NQO1) at p Ͻ
Values are mean Ϯ standard deviation from three separate determinations.
a
2
Complete reaction to generate TQH .
reduce TQ to TQH and that TQH can then function as an
2
2
antioxidant and inhibit cumene hydroperoxide-induced lipid
peroxidation.
Discussion
The data presented in this report indicate that TQ can be
Experiments were then performed to examine the antiox- reduced to TQH in cell-free and cellular systems by NQO1
2
idant properties of TQH in a cultured cell system (Fig. 4). and that TQH2 can function as an effective antioxidant.
2
Because of the low unsaturated fatty acid content in cultured Previous work has shown that TQ can be metabolized to
cells (28), the CHO cell lines were supplemented with ara- TQH by rat liver microsomal, mitochondrial, and cytosolic
2
chidonic acid. The high-NQO1-expressing cell line, CHO812
,
preparations (18–20). The reduction of TQ to TQH in these
2
and the NQO1-deficient cell line, CHOglyA, were loaded with preparations was either NADPH or NADH dependent, which
arachidonic acid (250 ␮M) and 0, 1, or 5 ␮M TQ for 14 hr, after suggests that many cellular reductases are capable of cata-
which the cells were exposed to cumene hydroperoxide (1 mM) lyzing this reaction. There are limited data available that
in culture. The cells were harvested, and lipid peroxidation
was measured. Essentially identical levels of lipid peroxida-
tion were detected in the absence of TQ in the two cell lines
(
Fig. 4). Loading with TQ, however, inhibited lipid peroxida-
tion 3-fold at 1 ␮M and 5-fold at 5 ␮M in the CHO812 cell line
compared with the CHOglyA cell line (Fig. 4). These studies
indicate that cells with high NQO1 levels can efficiently
Fig. 3. HPLC analysis of the oxidation of TQH2 by arachidonic acid
peroxidation. Reaction conditions were 50 ␮M TQ, 50 ␮M NADH, and
Fig. 2. The formation of TQH2 in NQO1-transfected CHO cell lines. 7.2 ␮g of NQO1 in 50 mM potassium phosphate buffer, pH 7.4, con-
CHOGlyA and CHO812 cell lines were treated with 50 ␮M TQ in culture; taining 1% cholic acid. After 30 min, we added (A) 5 mM arachidonic
at 6 and 12 hr, the cells were harvested, and the amounts of TQH and
acid, 50 ␮M ferrous sulfate, and 500 ␮M hydrogen peroxide; (B) 50 ␮M
2
TQ were determined by HPLC. Data represent mean Ϯ standard devi- ferrous sulfate and 500 ␮M hydrogen peroxide; and (C) 5 mM stearic
ation values from three separate determinations (o, CHOGlyA; f, acid, 50 ␮M ferrous sulfate, and 500 ␮M hydrogen peroxide, and the
CHO812). ء, CHO812 cells were significantly different from CHOGlyA cells reactions were allowed to proceed for 90 min. HPLC conditions are
(
p Ͻ 0.05).
described in Materials and Methods.

3
04
Siegel
tion. TQ pretreatment has been shown to protect cultured
cells from lipid peroxidation and cytotoxicity, presumably
due to its reduction to TQH (30–33). Our data demonstrate
2
that cells with high NQO1 expression had higher levels of
TQH2 and were more resistant to lipid peroxidation than
were cells lacking NQO1. The ability of cells to reduce TQ to
TQH via NQO1 therefore represents an effective protective
2
mechanism against lipid peroxidative injury.
Beyer et al. (16) have shown recently that purified rat
NQO1 is capable of reducing coenzyme Q derivatives to their
quinol forms and that these reduced compounds have sub-
stantial antioxidant properties. In their study, the quinol
forms of coenzyme Q (CoQ , CoQ ) were shown to protect
9
10
multilamellar vesicles against lipid peroxidation and isolated
hepatocytes against Adriamycin-induced oxidative stress.
We extended this work from rat to human NQO1 and com-
pared the rate of reduction of CoQ10 and TQ. Under the
Fig. 4. Inhibition of cumene hydroperoxide-induced lipid peroxidation conditions used in our study, the rate of TQ reduction by
in NQO1-transfected CHO cell lines by pretreatment with TQ. CHOGlyA
and CHO812 cell lines were pretreated with 250 ␮M arachidonic acid
and 0, 1, or 5 ␮M TQ and then exposed to 1 mM cumene hydroperoxide
in culture. Reaction conditions are described in Materials and Methods.
NQO1 was markedly greater (36–57-fold) than that observed
with CoQ . The results obtained in both our work and that
10
of Beyer et al. (16) clearly demonstrate that NQO1 can main-
Data represents three separate determinations (Ϯ standard deviation: tain endogenous quinones in a reduced antioxidative state.
o, CHOGlyA; f, CHO812). ء, CHO812 cells were significantly different
from CHOGlyA cells (p Ͻ 0.05).
It is conceivable that one of the functions of NQO1 may be
to regenerate antioxidant forms of ␣-tocopherol (Fig. 5). A
role for NQO1 in maintaining physiological levels of ␣-to-
characterize the enzymes responsible for the reduction of TQ copherol from the reduction of ␣-tocopherones by NQO1 has
to TQH . One study has shown that purified NADPH-cyto- previously been postulated (34). Oxidation of ␣-tocopherol by
2
chrome P450 reductase can reduce TQ to TQH (20), and a peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which
2
second study has suggested that TQ is a substrate for puri- may either hydrolyze to TQ or be reduced to regenerate
fied human brain carbonyl reductase (29).
␣-tocopherol (35). The regeneration of ␣-tocopherol from ␣-to-
Previous experiments using rat liver microsomes and pu- copherones occurs via a two-electron reduction, which could
rified NADPH cytochrome P450 reductase have suggested conceivably be catalyzed by a reductase such as NQO1 be-
that TQH2 is unstable under aerobic conditions, although cause this enzyme can catalyze the direct two-electron reduc-
paradoxically the authors also demonstrated that TQH2 tion of a broad range of substrates. Experiments are under
could be detected after aerobic incubations of isolated hepa- way to examine the potential role of NQO1 in the reduction of
tocytes with TQ (20). In our experiments, reduction of TQ to ␣-tocopherones. In addition, the conversion of TQ into ␣-to-
TQH by NQO1 proceeded readily under aerobic conditions, copherol in humans has recently been demonstrated (36); the
2
and the TQH that was formed was stable with no significant authors suggest that ␣-tocopherol is regenerated from TQ
2
autoxidation detected after 3 hr of incubation. The require- after enzymatic reduction to TQH , although the enzyme(s)
2
ment for anaerobic conditions to detect significant quantities responsible have not yet been identified.
of TQH after metabolism of TQ in previous studies using rat
Recent work in our laboratory has shown that NQO1 ex-
2
liver microsomes and purified cytochrome P450 reductase pression is polymorphic due to an A-T substitution in exon 6
probably reflects the instability of the ␣-tocopherol semiqui- of the NQO1 gene (37, 38). This base pair substitution leads
none radical to oxygen. Indeed, we have found that incuba- to a proline-to-serine change in the amino acid structure,
tion of rat liver microsomes with NADPH and TQ to generate which results in a total lack of NQO1 protein expression in
the ␣-tocopherol semiquinone radical results in extensive individuals homozygous for this mutation (37, 38). The poly-
oxygen uptake under aerobic conditions (data not shown). morphism demonstrates mendelian transmission (39) and
Because NQO1 is a two-electron reductase, it can directly
form TQH2 without the intermediacy of the ␣-tocopherol
semiquinone radical.
The stability of TQH is important because the hydroqui-
2
none form of TQ is responsible for the antioxidant function
(
18, 19). The current data show that TQH is stable and does
2
not undergo substantial autoxidation. However, the addition
2ϩ
of an unsaturated fatty acid (arachidonic acid), Fe , and
hydrogen peroxide did catalyze oxidation of TQH back to
2
TQ. These data indicate that TQH , like ␣-tocopherol, is
2
reactive primarily toward lipid-derived radicals. In a study
2ϩ
comparing the inhibition of ascorbate/Fe -induced lipid per-
oxidation in liposomes, TQH was 5-fold more effective than
2
Fig. 5. Role of NQO1 in the regeneration of antioxidant forms of ␣-to-
␣
-tocopherol (18). The ability of a cell to maintain TQ in its
⅐
copherol (␣-T). ␣-T , ␣-tocopherol radical; ␣-TQ, ␣-tocopherolquinone;
reduced form will result in substantial antioxidant protec- ␣-TQH , ␣-tocopherolhydroquinone.
2

Reduction of ␣-Tocopherolquinone by NQO1
305
occurs with a prevalence of ϳ6% in whites and ϳ18% in the
Chinese (40). Our data suggest that individuals lacking ex-
pression of NQO1 may have a decreased capacity to protect
against cellular oxidative damage, and this may have impli-
cations for chemoprotection and chemoprevention.
In summary, NQO1 has classically been considered a de-
toxification enzyme because of its role in the reduction of
exogenous quinone substrates to their hydroquinone forms,
thus bypassing reactive semiquinone radical formation. The
␣-tocopherol a reservoir for ␣-tocopheryl hydroquinone? Free Rad. Biol.
Med. 19:197–207 (1995).
0. Hayashi, T., A. Kanetoshi, M. Nakamura, M. Tamura, and H. Shirahama.
Reduction of ␣-tocopherolquinone to ␣-tocopherolhydroquinone in rat
hepatocytes. Biochem Pharmacol. 44:489–493 (1992).
1. Gustafson, D. L., H. D. Beall, E. M. Bolton, D. Ross, and C. A. Waldren.
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2. Beall, H. D., R. T. Mulcahy, D. Siegel, R. D. Traver, N. W. Gibson, and D.
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2
2
2
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2
mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage
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2
4. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein
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Send reprint requests to: Dr. David Siegel, School of Pharmacy C238,
UCHSC, 4200 E. 9th Avenue, Denver, CO 80206. E-mail: david.siegel@
uchsc.edu
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