From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084
Received for publication, July 26, 2000, and in revised form, October 27, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human aldo-keto reductase
AKR1C1 (20 Oxidative stress plays a detrimental role in a number of
pathological conditions, including cancer, atherosclerosis, and
several neurodegenerative disorders (1-5). One of the main classes of cytotoxic and genotoxic substances produced during oxidative stress is
Mammalian cells have developed multiple enzymatic pathways for the
detoxification of these lipid peroxidation by-products. The best
characterized of these enzymes include the GSTs, aldehyde dehydrogenase, and alcohol dehydrogenase (14, 15). GSTs catalyze conjugation of GSH to HNE via Michael addition at the C-3
carbon, thereby preventing further nucleophilic addition to this toxic compound. Aldehyde dehydrogenase catalyzes the oxidation of HNE to the
innocuous 4-hydroxy-2-nonenoic acid, while alcohol dehydrogenase catalyzes reduction of the terminal aldehyde to its alcohol, yielding the unreactive metabolite DHN. Recently two members of the
AKR2 superfamily, aldose
reductase and FR-1, were also shown to catalyze HNE reduction (16-19),
thereby expanding the repertoire of enzyme families that contribute to
detoxification of HNE in hepatic and extrahepatic tissues.
In previous studies, a 20 Several HSDs in the AKR1C subfamily also play roles in xenobiotic
metabolism by oxidizing PAH trans-dihydrodiol proximate carcinogens to reactive and redox-active o-quinones
(26-29). However, the roles of AKR1C1 in steroid and PAH metabolism
fail to provide a rationale for its isoform-specific induction by
electrophiles and ROS.
In the present studies, we sought to identify an activity that might
explain the robust induction of AKR1C1 during oxidative insult. We
found that, like its closely related rat homolog AKR1C9 (30), AKR1C1
weakly catalyzed reduction of DHA to regenerate ascorbic acid. However,
the low specific activity observed suggested that this was not the ROS
counter-response catalyzed by AKR1C1. Subsequent studies revealed that
HNE and other Chemicals and Reagents--
Cell culture media and reagents were
obtained from Life Technologies, Inc. Homogeneous recombinant AKR
proteins, including aldehyde reductase (AKR1A1), were overexpressed in
Escherichia coli and purified as described previously (28,
31, 32). Chemical Synthesis of 1,4-Dihydroxynonene from
4-Hydroxy-2-nonenal--
DHN was synthesized by NaBH4
reduction of HNE. Briefly, a 4-fold molar excess of NaBH4
(25 µmol) was added dropwise to a solution of 1 mg HNE (6.4 µmol)
in 4 ml of anhydrous methanol with continuous stirring over 10 min.
Aliquots were removed over time and analyzed by TLC to monitor the
progress of the reaction. Reactions were terminated by the addition of
1.0 ml of 0.1 M HCl to decompose unreacted
NaBH4. The solution was extracted with 3 × 20 ml of dichloromethane, and the extract was dried over anhydrous sodium sulfate and reduced under vacuum at room temperature. The chemically synthesized DHN was separated from the starting material by TLC, extracted from silica, and resuspended in CDCl3 for
subsequent NMR analysis.
Isolation and Characterization of the Product of HNE
Reduction--
Large scale enzymatic incubations were conducted in 20 ml of 100 mM potassium phosphate, pH 6.0, containing 3.2 mM NADPH and 320 µM HNE solubilized in 2%
methanol. Following the addition of the purified enzyme (100 µg) the
reactions were incubated at 37 °C for 4 h and then terminated
by extraction of the products with dichloromethane (three 20-ml
aliquots). The organic solvent was dried with anhydrous sodium sulfate
and then removed under reduced pressure. Following resuspension in
dichloromethane, the resulting extract was subjected to TLC separation.
DHN was separated from HNE by TLC in acetone-hexane (80:20, v/v).
Detection was performed with methanol/sulfuric acid (1:1) reagent
followed by heating. TLC plates were analyzed by densitometry on a
Uniscan densitometer (Analtech, Newark, DE) to determine the
chromatographic profile of HNE standard (Rf = 0.49), chemically synthesized DHN (Rf = 0.20),
and the enzymatic product. The enzymatic product was eluted from the
silica with methanol, evaporated to dryness, and resuspended in
CDCl3 for NMR analysis.
NMR Spectroscopy--
1H NMR spectra were obtained
on a Bruker AM-250 spectrometer operating at 250 MHz, using
CDCl3 as the solvent. All chemical shifts ( Spectrophotometric Assays and Kinetic
Characterization--
Km and kcat
values were obtained by varying the substrate concentration at constant
saturating cofactor concentration (180 µM NADPH or 2.3 mM NADP+) in 1.0-ml systems containing 100 mM potassium phosphate buffer, pH 7.0, at 37 °C. Initial
velocities at each substrate concentration were determined on a Beckman
DU640 spectrophotometer by measuring the change in absorbance of
pyridine nucleotide at 340 nm ( Cell Culture--
HepG2 hepatoma cells (passages 10-30) were
maintained in Eagle's minimal essential medium supplemented with 10%
heat-inactivated fetal bovine serum and antibiotics. Cells were
incubated at 37 °C in a humidified atmosphere containing 5%
CO2 and were passaged every 4 days at 1:10 dilution. For
induction studies, 48 h prior to treatment, 3 × 106 cells were seeded into 100-mm dishes containing fresh
medium. Two days later (~50-60% confluency), cells were
exposed to HNE. Aliquots (10 µl) of 1000× stock solutions in
acetonitrile were added to 10 ml of fresh culture medium, and cells
were incubated for the indicated times before harvesting.
RNA Isolation and Northern Analysis of AKR1C
mRNA--
Cellular RNA was isolated using the Trizol® reagent.
Total RNA (10 µg) was separated by electrophoresis on 1.0%
agarose/formaldehyde gels and transferred overnight to Duralon-UV
membranes (Stratagene). Membranes were prehybridized in hybridization
buffer (50% formamide, 10% dextran sulfate, 1 M NaCl, and
1% SDS) with 100 µg/ml sheared salmon sperm DNA at 42 °C for
2 h. After prehybridization, membranes were hybridized to
107 dpm of an [ Quantitative Isoform-specific Reverse Transcription PCR of AKR1C1
mRNA--
Total RNA (1 µg) from untreated and HNE-treated HepG2
cells was reverse transcribed into cDNA and subjected to 18 cycles
of reverse transcription PCR amplification (94 °C for 45 s
(denaturation), 60 °C for 45 s (annealing); 72 °C for 2 min
(extension)) using a 1 µM concentration of the following
isoform-specific primers: AKR1C1 (forward, 5'-dGTAAAGCTTTAGAGGCCAC-3';
reverse, 5'-dCACCCATGGTTCTTCTCGG-3') and AKR1C1 and AKR1C3 Possess Low but Detectable Dehydroascorbate
Reductase Activities--
ROS can lead to uncontrolled GSH depletion,
ascorbic acid oxidation, lipid peroxidation, and DNA damage unless
cells mount an effective counter-response. A previous study reported
that rat liver 3 Reduction of 4-Hydroxy-2-nonenal by Multiple AKR Family
Members--
One of the most common cytotoxic products of lipid
peroxidation is HNE. Like prostaglandins, HNE is derived from
arachidonic acid and possesses several structural characteristics in
common with prostaglandins (Fig. 1). HNE
(200 µM) was thus screened as a substrate for several
human AKR1C subfamily members that share high sequence identity with
bovine prostaglandin F synthase, which is involved in
prostaglandin metabolism (Fig. 2).
The ROS- and Michael acceptor-inducible AKR1C1 (20 Catalytic Efficiency of AKR1C1 for HNE--
Determination of
kinetic constants for AKR1C1 at pH 7.0 and 37 °C revealed a
Km of 34 µM and a
kcat of 8.8 min Substrate Specificity of AKR1C1 for Km of Recombinant Human AKR1C1 for NADP+ and
NADPH Cofactors--
To determine the nucleotide cofactor specificity
of AKR1C1-catalyzed reduction of HNE, the ability of the enzyme to
catalyze NADH- versus NADPH-dependent reduction
of HNE was assessed (Fig. 3). AKR1C1
catalyzed HNE reduction with a 100-fold increase in activity using
NADPH as cofactor compared with NADH. Kinetic constants of AKR1C1 for
NAD(P)(H) were generated using 20 Identification of 1,4-Dihydroxy-2-nonene as the Product of the
AKR1C1-dependent Reduction of
4-Hydroxy-2-nonenal--
Since members of the AKR1 family can possess
either double bond reductase or aldehyde reductase activities (37), it
was unclear whether AKR1C1 was catalyzing the reduction of the
To establish the structure of the AKR1C1-derived metabolite, the
enzymatic product was extracted, isolated by TLC, and subjected to NMR
spectroscopy (Fig. 5). The product of the
enzymatic reaction gave identical chemical shifts to those assigned to
the synthetic standard (Fig. 5, compare B and C).
Importantly, there was loss of the aldehydic proton ( Induction of Human AKR1C1 by HNE--
Since human AKR1C1 is
inducible by extracellular ROS (H2O2),
intracellular ROS (2,3-dimethoxynaphthalene-1,4-dione) and exogenous Michael acceptors (ethacrynic acid), we also sought to determine whether the ROS-derived, endogenous Michael acceptor HNE could also
stimulate the expression of AKR1C1. Exposure of HepG2 cells to HNE for
24 h caused a concentration-dependent induction of AKR1C mRNA expression as measured by Northern blot analysis (Fig. 6A). Equal loading of total
RNA was verified by reprobing the blot for the expression of GAPDH. A
concurrent time-dependent induction of AKR1C enzyme
activity was observed in HepG2 cell lysates following HNE treatment as
measured by 1-acenapthenol oxidation (Fig. 6B).
To verify that the ROS- and Michael acceptor-inducible AKR1C1 was
responsible for the global increases in AKR1C mRNA caused by HNE,
first strand cDNA libraries from untreated and HNE-treated HepG2
mRNA were PCR-amplified with isoform-specific primers for AKR1C1
and Oxidative stress induces the expression of a battery of human
enzymes by signaling to antioxidant response elements/electrophilic response elements present in the 5'-flanking region of their respective genes. Regulated genes include GSTs (38, 39), Several antioxidant vitamins are protective during oxidative stress,
including By contrast, HNE appears to be a physiological substrate of AKR1C1 with
a catalytic efficiency similar to that of the 20 AKR1C1 also demonstrated specificity for other The Km value of AKR1C1 for HNE (34 µM)
is substantially lower than those of isozymes I and II of alcohol
dehydrogenase, 250-1430 and 100 µM, respectively (54),
which have long been considered to be the sole source of reductive HNE
metabolism in many tissues. These Km values are
important when it is considered that under normal conditions the HNE
concentration is <1.0 µM but may rise significantly and
approach concentrations of 100 µM under conditions of
oxidative stress. These arguments would suggest that the
Km constant for AKR1C1 is more favorable to handle
physiological concentrations of HNE than that observed with isozymes I
and II of alcohol dehydrogenase. The Km of AKR1C1
for HNE is, however, similar to that reported for the HNE-specific GST
isoform GSTA4-4 (15) and the NADH-specific human class IV alcohol
dehydrogenase (55). This warrants a comparison of the catalytic
efficiencies of these enzymes to determine which is most important in
HNE elimination.
Catalytic efficiencies of GSTA4-4 are 500-fold greater than that
observed with AKR1C1. Although GSTA4-4 has been characterized in human
fetal brain, it has been argued that this isozyme is ubiquitously
expressed in a manner similar to its rodent counterparts (15). In
addition, GST-catalyzed conjugation of HNE has been described in human
liver, heart, cornea, and retina (56). Thus, GST-catalyzed conjugation
of GSH to HNE is undoubtedly an important elimination pathway. However,
under conditions of oxidative stress, there is a concomitant decrease
in GSH leading to a change in redox state (increased GSSG:GSH) making
non-GSH-dependent pathways an important second barrier to
the toxic effects of HNE. Importantly, under conditions of oxidative
stress (H2O2) and drugs that deplete GSH
(e.g. ethacrynic acid), AKR1C1 can be induced up to 10-fold, and this induction may be a component of an effective counter-response to this stress.
The recently described human class IV alcohol dehydrogenase possesses a
kcat which is 2 orders of magnitude higher than
that of the AKR1C1 isoform, suggesting that this enzyme is the better catalyst (57). However, the cytosolic concentrations of nicotinamide cofactors favor NAD+ over NADH and NADPH over
NADP+, suggesting that NADPH-dependent
reductases (i.e. AKRs) may be "primed" with the
appropriate cofactor for HNE reduction. Calculation of the
Km constant of AKR1C1 for NADPH
(Km NADPH) demonstrates that AKR1C1 will be
saturated with NADPH under physiological conditions and will probably
possess sufficient affinity for HNE to contribute to its overall
reductive elimination. Together, these observations suggest an
important role of AKR1C1 in the elimination of the toxic HNE.
To date, the only other enzymes with a similar low
Km constant for HNE are other AKR1 family members,
aldose reductase and FR-1, which gave Km values of
22 and 9 µM, respectively (17-19). By contrast, AKR1A1
is not saturatable with these high concentrations of HNE (4 mM) and is unlikely to play a significant role in the
reductive elimination of this metabolite. AKR1C1 is expressed at
relatively high levels in multiple tissues associated with xenobiotic
metabolism/oxidative stress, including lung, liver, and mammary gland
(25). Importantly, AKR1C1 is the only known AKR with a low
Km for HNE that is robustly induced by ROS and by
agents that deplete GSH.
Given the multiplicity of potential HNE-derived metabolites, it was
important to determine the identity of the product of the
AKR1C1-dependent reduction of HNE. Certain AKR1 family
members function as double bond reductases, and a single point mutation (H117E) is sufficient to introduce a double bond reductase activity into AKR1C9 (37). Whether AKR1C1 can function as a double bond reductase is unknown. Chromatographic separation and subsequent NMR
spectroscopy confirmed the structure of the enzymatic product to be
that of DHN. These findings verified that human AKR1C1 catalyzes reduction of the terminal aldehyde in HNE. Other experiments
demonstrated that AKR1C1 does not function as an oxidoreductase with
respect to HNE; it fails to oxidize DHN to HNE (Fig.
8). Thus, AKR1C1 is expected to yield a
net accumulation of the innocuous metabolite DHN within the cell.
(3
)-hydroxysteroid dehydrogenase) is induced by
electrophilic Michael acceptors and reactive oxygen species (ROS) via a
presumptive antioxidant response element (Burczynski, M. E., Lin,
H. K., and Penning, T. M. (1999) Cancer Res. 59, 607-614). Physiologically, AKR1C1 regulates progesterone action by
converting the hormone into its inactive metabolite
20
-hydroxyprogesterone, and toxicologically this enzyme activates
polycyclic aromatic hydrocarbon trans-dihydrodiols to
redox-cycling o-quinones. However, the significance of its potent induction by Michael acceptors and oxidative stress is unknown.
4-Hydroxy-2-nonenal (HNE) and other
,
-unsaturated aldehydes produced during lipid peroxidation were reduced by AKR1C1 with high
catalytic efficiency. Kinetic studies revealed that AKR1C1 reduced HNE
(Km = 34 µM,
kcat = 8.8 min
1) with
a kcat/Km similar to that
for 20
-hydroxysteroids. Six other homogeneous recombinant AKRs were
examined for their ability to reduce HNE. Of these, AKR1C1 possessed
one of the highest specific activities and was the only isoform induced
by oxidative stress and by agents that deplete glutathione (ethacrynic
acid). Several hydroxysteroid dehydrogenases of the AKR1C subfamily
catalyzed the reduction of HNE with higher activity than aldehyde
reductase (AKR1A1). NMR spectroscopy identified the product of the
NADPH-dependent reduction of HNE as 1,4-dihydroxy-2-nonene.
The Km of recombinant AKR1C1 for nicotinamide
cofactors (Km NADPH ~6 µM,
Km(app) NADH >6 mM) suggested that
it is primed for reductive metabolism of HNE. Isoform-specific
reverse transcription-polymerase chain reaction showed that exposure of
HepG2 cells to HNE resulted in elevated levels of AKR1C1 mRNA.
Thus, HNE induces its own metabolism via AKR1C1, and this enzyme may
play a hitherto unrecognized role in a response mounted to counter
oxidative stress. AKRs represent alternative
GSH-independent/NADPH-dependent routes for the reductive elimination of HNE. Of these, AKR1C1 provides an inducible cytosolic barrier to HNE following ROS exposure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated aldehydes generated endogenously as a break-down product of lipid peroxidation of
-6 polyunsaturated fatty acids (6).
Of these, HNE1 is one of the
major unsaturated aldehydes produced and may be more deleterious than
the initial production of ROS. This metastable reactive metabolite
possesses a longer half-life than ROS and can diffuse from its sites of
origin in the cellular membranes. Since HNE is a Michael acceptor, it
attacks nucleophilic targets intracellularly and can act as a potent
cytotoxin (for a review, see Ref. 6). In vivo, HNE
immunoreactive adducts have been detected in atherosclerotic
plaques (7), in the degenerating neurons of the substantia nigra
affected by Parkinson's disease (8), and in the neurofibrillary
tangles associated with Alzheimer's disease (9). HNE is also
genotoxic, since it will readily form propano-dGuo adducts, which can
lead to promutagenic lesions (10, 11). Related etheno adducts may be
derived from the more reactive bifunctional electrophile
4-oxo-2-nonenal (12, 13), and whether this can be metabolically derived
from HNE is unknown.
(3
)-hydroxysteroid dehydrogenase
(AKR1C1), a member of the AKR1C subfamily, was found to be potently induced by Michael acceptors and ROS in human colon and hepatoma cell
lines through an apparent antioxidant response element-type mechanism
(20-22). However, the physiologic reason for the robust induction of
this enzyme by electrophiles and ROS has remained unclear. The HSDs of
this subfamily play important roles in steroid target tissues (for a
review, see Ref. 23). Human AKR1C1-AKR1C4 are plastic enzymes and
display 3-, 17-, and 20-ketosteroid reductase activity and
3
-, 17
-, and 20
-hydroxysteroid oxidase activity (24, 25). This
plasticity indicates that these enzymes can interconvert potent
androgens, estrogens, and progestins with their cognate inactive
metabolites and have the potential to regulate occupancy of steroid
hormone receptors based on their tissue distribution. In the case of
AKR1C1, all of these reactions occurred, but the most catalytically
efficient is the interconversion of progesterone to its inactive
metabolite, 20
-hydroxyprogesterone, suggesting that this is its
physiological role (25).
,
-unsaturated aldehydes produced during lipid
peroxidation were substrates of the inducible AKR1C1 isoform and
identified DHN as the product of the AKR1C1-catalyzed HNE reduction.
HNE was also found to directly induce AKR1C1 expression. The induction
of AKR1C1 by HNE and ROS suggests that it may belong to a battery of
genes (GST,
-glutamylcysteine synthetase, etc.) that mount a
counter-response to ROS during periods of electrophilic and oxidative stress.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NADPH nucleotide cofactor was obtained from Roche
Molecular Biochemicals. DHA, alkenals, and
2,3-dimethoxynaphthalene-1,4-dione were purchased from Sigma, and HNE
was obtained from Calbiochem. Because HNE will self-polymerize on
storage, only new lots of HNE were used in these experiments. All other
chemicals used were of the highest grade available.
H) are in ppm
downfield from tetramethylsilane. The spectra were referenced to
residual protonated solvent residues as internal standards; for
CDCl3,
H = 7.3 ppm. Analysis of HNE gave the
following spectral assignments:
0.89 (t, 3H, 9-CH3);
1.25-1.67 (m, 4H and m, 4H, 5-CH2, 6-CH2,
7-CH2, 8-CH2);
3.65-3.76 (m, 1H, 4-CH);
5.83-6.05 (d, 1H, 2-CH JAB 3.0 Hz);
6.13-6.38 (t, 1H, 3-CH, JBA 1-2 Hz and
JBX 1-2 Hz);
9.55-9.61 (d, 1H, 1-CHO).
Analysis of chemically synthesized DHN and the AKR1C1 product gave the following spectral assignments, which were identical with the previously characterized spectral shifts of DHN (33):
0.89 (t, 3H,
9-CH3);
1.25-1.69 (m, 10H, 5-CH2,
6-CH2, 7-CH2, 8-CH2, and 2-OH);
4.10-4.21 (m, 3H, 1-CH2, 4-CH);
5.69-5.90 (m, 2H, 2-CH, 3-CH; gave the expected 10-line hyperfine splitting pattern based
on a JAB, JABY,
JABZ and JBA,
JBX, coupling system). A residual CH2Cl2 shift (
= 5.29) was
present in the chemically synthesized DHN, and a residual
CH3OH shift (
= 3.44) was detected in
the enzymatic product.
= 6220 M
1
cm
1). Actual values of the kinetic constants
were determined using ENZFITTER to fit untransformed data (substrate
concentration versus initial rate) to a hyperbolic equation
to provide estimates of Km and
Vmax and their associated S.E. values (34). Km(app) values for nicotinamide cofactors were
obtained by varying NADH/NADPH concentration at a constant progesterone concentration (50 µM, limit of solubility) or by varying
NAD+/NADP+ concentration at a constant
20
-hydroxyprogesterone concentration (50 µM, limit of solubility).
-32P]dATP probe
corresponding to an 855-base pair EcoRI fragment of the
human colon DD1 cDNA (pBluescript-hcDD kindly provided by Dr. Paul
Ciaccio and Dr. Ken Tew). Signal intensities were measured using the
PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA), and
blots were exposed to x-ray film at
80 °C overnight. For purposes
of normalization, blots were stripped and reprobed with a 780-base pair
PstI/XbaI fragment of human GAPDH labeled by
random priming as above.
-actin amplimers
CLONTECH (25). Amplified products from various
cycle numbers were fractionated on ethidium bromide-agarose gels and
photographed under UV for comparison.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD (AKR1C9) functions as a dehydroascorbate
reductase (30). Thus, human HSDs of the AKR1C subfamily were initially screened for their ability to catalyze the reduction of 1.5 mM DHA as a potential antioxidant activity. Interestingly,
only two human isoforms that are induced by ROS and Michael acceptors
(AKR1C1 and, to a lesser extent, AKR1C3) catalyzed the reduction of DHA to ascorbic acid. The low specific activities observed for the reduction of DHA (less than 8 nmol/min/mg) at millimolar substrate concentrations suggested that DHA was not the physiological substrate for the inducible AKR1C isoforms during periods of oxidative stress.
(3
)-HSD) and
its closely related (>98% amino acid identity) AKR1C2 (type 3 3
-HSD, bile acid-binding protein) homolog possessed the highest
specific activities for HNE reduction of the six homogeneous
recombinant AKRs studied. AKR1C3 (type 2 3
-HSD) and AKR1A1 (aldehyde
reductase) possessed intermediate specific activities for HNE
reduction, while the major 3
-HSD activities in human and rat liver,
AKR1C4 (type 1 3
-HSD) and AKR1C9 (rat 3
-HSD), respectively,
possessed low activity toward HNE. Of these enzymes, AKR1C3 has
recently been shown to be identical to human prostaglandin F synthase
(35). In this comparison, AKR1C1 had a specific activity that was 3 times greater than that seen with AKR1A1. In previous studies, AKR1C1
was shown to have a specific activity 300 times less than AKR1A1 (36), raising the issue as to why this discrepancy exists. In the earlier studies, assays were conducted with nonphysiologic amounts of HNE (4 mM). This led to a comparison of the catalytic efficiencies of AKR1C1 and AKR1A1 for HNE and an examination of whether HNE could
inactivate either enzyme by Michael addition.
View larger version (10K):
[in a new window]
Fig. 1.
Prostaglandins and 4-hydroxy-2-nonenal arise
from arachidonic acid. Enzymatic and nonenzymatic processes
convert arachidonic acid to prostaglandins and HNE, respectively.
View larger version (14K):
[in a new window]
Fig. 2.
Reduction of 4-hydroxy-2-nonenal by
AKRs. Homogeneous recombinant AKR1 enzymes (2 µg) were incubated
in cuvettes containing 200 µM HNE as described under
"Experimental Procedures." Reactions were initiated by the addition
of enzyme. No detectable rate was observed in the minus cofactor and
minus enzyme controls. Specific activities are expressed in
nmol/min/mg.
1 for
HNE. The catalytic efficiency for HNE
(kcat/Km = 0.27 × 106 M
1
min
1) observed with AKR1C1 was considerably
higher than that anticipated with AKR1A1. AKR1A1 was not saturable at 1 mM HNE, and in fact v versus [S]
plots were entirely linear, indicating that under the conditions of the
highest substrate concentrations employed the velocity was still
pseudo-first order. Thus, the catalytic efficiency is not measurable
and must be considerably lower than that observed with AKR1C1.
Preincubation of AKR1C1 and AKR1A1 with 4 mM HNE (under
assay conditions reported in Ref. 36) led to a
time-dependent inactivation of AKR1C1 only (data not
shown). Thus, an explanation for the inadvertently high specific
activity reported for AKR1A1 can be explained by its lack of saturation and lack of time-dependent inactivation, both of which are
observed with AKR1C1 at 4 mM HNE. Importantly, we find that
AKR1C1 is not inactivated in the presence of NADP+. Since
cofactor protects AKR1C1 against this event, no inactivation occurs
over the time course of the enzyme assay used to generate the
steady-state kinetic parameters. When the catalytic efficiency of
AKR1C1 for HNE is compared with its values for the
NADP(H)-dependent oxidation/reduction of various
physiological (20
-hydroxysteroids, prostaglandins) and xenobiotic
(PAH trans-dihydrodiols) substrates (Table
I), HNE is reduced with a catalytic
efficiency similar to that of 20
-hydroxysteroids, which are the
presumed substrates of AKR1C1 in vivo.
Comparison of catalytic efficiencies of AKR1C1 for various PAH
trans-dihydrodiols, steroids, and 4-hydroxy-2-nonenal
,
-Unsaturated Aldehydes
of Increasing Chain Length--
To investigate the specificity of the
AKR1C1-catalyzed reduction of HNE, a series of unsaturated aldehydes
(alkenals) of increasing chain length were also screened as substrates
of AKR1C1. Calculation of catalytic efficiencies revealed that
kcat/Km increased over
20-fold as the length of the carbon chain increased from 5 to 9 carbons
(Table II). The predominant effects were
on Km and were reflected in
kcat/Km. Interestingly, the
presence of the 4' hydroxyl group on 2-nonenal raises the
Km ~5-fold, while kcat
remained unchanged.
Substrate specificity of AKR1C1 for unsaturated aldehydes of increasing
chain length
-hydroxyprogesterone and
progesterone as a reversible oxidation/reduction substrate pair (Table
III). AKR1C1 appears to be specific for
the triphosphate-containing nicotinamide cofactors, with
Km constants for NADP+ and NADPH in the
low micromolar range. Since AKR1C1 fails to oxidize DHN using
NADP+ as cofactor (data not shown), AKR1C1 appears to be a
unidirectional NADPH-specific
,
-unsaturated aldehyde reductase.
This suggests that AKR1C1 is primed to catalyze the
NADPH-dependent reduction of
,
-unsaturated
aldehydes.
View larger version (12K):
[in a new window]
Fig. 3.
Specificity of AKR1C1 for NADPH. Using a
standard spectrophotometric assay, increasing concentrations of
homogeneous recombinant AKR1C1 (0.01-0.05 mg) were incubated with 200 µM HNE in the presence of 180 µM NAD(P)H.
Initial rates for each enzyme concentration with each cofactor were
calculated and converted to total activity in nmol/min.
Km constants of homogeneous recombinant AKR1C1 for
nicotinamide cofactors
2-double bond or the terminal aldehyde in HNE or both.
To distinguish between these possibilities, DHN was chemically
synthesized, and its chromatographic profile was compared with that of
the enzymatic product of the AKR1C1-catalyzed reaction. Thin layer
chromatographic separation revealed that the AKR1C1-derived product
(Fig. 4C) possessed an
Rf value (0.20) identical to that of chemically
synthesized DHN (Fig. 4B), and the products of both
reactions coeluted when the products were mixed (Fig.
4D).
View larger version (22K):
[in a new window]
Fig. 4.
Chromatographic identity of the product
formed by the reduction of HNE catalyzed by AKR1C1 with the chemically
synthesized DHN. HNE (320 µM) was incubated with
NADPH (3.2 mM) in the presence of AKR1C1 (0.5 mg) until the
reaction was 50% complete. The products of the reaction were then
extracted and separated by TLC, and Rf values
were compared with those obtained for the chemically synthesized DHN.
A, HNE starting material. B, HNE and chemically
synthesized DHN standards. C, AKR1C1-dependent
reaction at 50% completion. D, co-chromatography of the
AKR1C1 product and DHN.
~9.5 ppm)
and retention of the vinylic protons of the
2-double
bond (
~5.8 ppm), yielding a 10-line hyperfine splitting pattern
characteristic of a JAB,
JABY, JABZ and
JBA, JBX, coupling system (Fig. 5, B and C), establishing the
identity of the enzymatic product as DHN.
View larger version (26K):
[in a new window]
Fig. 5.
Confirmation that DHN is the enzymatic
product of AKR1C1 reduction of HNE by NMR spectroscopy. The
product of the AKR1C1-dependent reaction was extracted,
subjected to TLC purification, and then analyzed by high field proton
NMR. In the NMR spectrum of both chemically and enzymatically
synthesized DHN, note especially the loss of the aldehydic proton at
~10 ppm and the retention of the vinylic protons at ~5.8-6.2 ppm
with new resonances, which gave the predicted 10-line splitting pattern
for a JAB, JABY,
JABZ and JBA,
JBX, coupling system. A, HNE
starting material. B, chemically synthesized DHN standard.
C, enzymatically synthesized product. (The inset
in A shows the structure of HNE and the splitting patterns
for its methine protons; the inset in B
shows the structure of DHN and the splitting patterns for its vinylic
protons.)
View larger version (30K):
[in a new window]
Fig. 6.
Induction of AKR1C mRNA and enzymatic
activity by HNE. A, Northern blot analysis of AKR1C
mRNA. HepG2 cells (3 × 106) were seeded into
10-cm dishes, and 1 day later they were replaced with serum-free
medium. Following serum withdrawal for 24 h, HNE (0-100
µM) was added in a final concentration of 0.1% ethanol.
At various time points, total RNA was harvested. RNA (5 µg) from the
various treatments was electrophoresed, transferred to membranes, and
sequentially analyzed for AKR1C and GAPDH expression as described under
"Experimental Procedures." Only the levels of the AKR1C transcript
are shown. B, induction of AKR1C1 enzymatic activity by HNE.
HepG2 cells were exposed to 10 µM HNE, and lysates were
prepared over time and assayed for 1-acenapthenol oxidation (22).
Protein content for purposes of normalization was determined by the
Bradford method. Data are expressed as -fold induction of
AKR1C1-specific activity relative to the time 0 control.
-actin (Fig. 7). Isoform-specific
semiquantitative reverse transcription PCR demonstrated that AKR1C1 was
the AKR isoform induced following a 6-h exposure of HepG2 cells to 10 µM HNE. By contrast, primer pairs for AKR1C3, an isoform
that shows modest induction by ROS, failed to detect induction of this isoform by HNE (data not shown). In previous experiments, we have shown
that AKR1A1 is not induced by ROS or Michael acceptors (22).
View larger version (13K):
[in a new window]
Fig. 7.
Isoform-specific induction of the ROS- and
Michael acceptor-inducible AKR1C1 isoform by HNE. HepG2 cells were
treated with Me2SO ( ) or 10 µM HNE (+), and
total RNA was harvested after 6 h. RNA (1 µg) from each sample
was reverse-transcribed into cDNA and then subjected to linear PCR
amplification (12-24 cycles) using specific primer pairs for AKR1C1
and
-actin. PCRs were electrophoresed and visualized with ethidium
bromide under UV light.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamylcysteine synthetase (40, 41), metallothionein (42, 43), and NQO1 (44, 45). The
proteins encoded by these inducible genes often represent either phase
II (de)toxification enzymes (NQO1, GST) or enzymes involved in
antioxidant defense (
-glutamylcysteine synthetase, metallothionein).
The pattern of induction of AKR1C1 by monofunctional and bifunctional
inducers and oxidative insult strongly implies that AKR1C1 is another
member of the antioxidant response element/electrophilic response
element-inducible battery of genes (22). However, the reason for the
robust induction of AKR1C1 by GSH-depleting electrophiles
(e.g. ethacrynic acid) and/or ROS was unclear. Based on
these observations, we determined whether AKR1C1 and/or other closely
related human AKRs might either 1) regenerate endogenous antioxidants
in the cell or 2) detoxify endogenous toxic metabolites generated
during oxidative stress.
-tocopherol (46) and ascorbic acid (47, 48). The closely
related 3
-HSD from rat liver (AKR1C9) was previously shown to
catalyze the reduction of DHA to ascorbic acid (30). Since such an
activity could contribute to the regeneration of an endogenous
antioxidant in cells, we determined whether any human AKRs might also
catalyze the reduction of DHA to ascorbic acid. Interestingly, DHA
reduction was identified as an enzymatic activity of the inducible
AKR1C1 and AKR1C3 isoforms. However, the low specific activity (~8
nmol/min/mg) observed at high (1.5 mM) concentrations of
DHA made it unlikely that DHA represents a physiologically relevant
substrate of AKR1C1 in vivo.
-hydroxysteroids. The various AKR1C isoforms displayed significant variability in their
ability to catalyze HNE reduction, illustrating a notable dissociation
of this function within the AKR1C subfamily. It is not surprising that
HNE is also a substrate of AKR1C2, since AKR1C1 and AKR1C2 are 98%
identical, with only a single conservative amino acid substitution
within the active site. AKR1C1 may be preferentially induced by ROS and
Michael acceptors over AKR1C2, which has a slightly higher specific
activity for two reasons. First, in liver, AKR1C2 (bile acid-binding
protein) will be saturated with bile acids, which act as potent
inhibitors of AKR1C2 but are far weaker inhibitors of AKR1C1 (49, 50).
Thus, AKR1C1 can continue to eliminate HNE in the presence of normal
bile acid concentrations. As an aside, under conditions of cholestatic
liver damage and/or bile acid duct ligation (51, 52), hepatic HNE metabolism is impaired, and this may result from inhibition of AKR1C1
by elevated bile acids. Second, AKR1C1 undergoes
time-dependent inactivation by HNE and may serve a
sacrificial role to eliminate HNE at high concentrations. Induction of
AKR1C1 may be necessary to replenish AKR1C1 under these conditions.
,
-unsaturated
aldehydes. The catalytic efficiency of AKR1C1 increased over 20-fold as
the length of the hydrocarbon backbone increased from five to nine
carbons. This is almost entirely due to a decrease in
Km and possibly reflects increased interaction of the hydrophobic substrate with hydrophobic residues predicted to line
the substrate binding site (53).
View larger version (16K):
[in a new window]
Fig. 8.
AKR1C1 catalyzed reduction of HNE to
DHN. The role of AKR1C1 in the reductive elimination of HNE is
shown.
DHN is a well characterized metabolite of HNE in multiple tissues in vivo. DHN appears to represent between 10 and 20% of the total metabolic fate of HNE in hepatocytes, enterocytes, and in perfused kidney (58-60). It is likely that while alcohol dehydrogenases catalyze NADH-dependent reduction of HNE, various members of the AKR superfamily are responsible for the fraction of NADPH-dependent reduction of HNE in these and other tissues. Indeed, recent reports have identified aldose-reductase and/or closely related oxidoreductases as major activities involved in the detoxification of HNE in cardiac tissue homogenates (61).
HNE was also shown to be an inducer of AKR1C1. HNE has also been reported to weakly induce both aldose reductase and GST-P (3- and 1.7-fold, respectively), two other enzymes that are capable of catalyzing its metabolic elimination (62, 63). Thus, in addition to its cytotoxic and genotoxic properties, HNE appears to act as a toxic "second messenger," which induces the expression of enzymes involved in its own metabolism. Recently, HNE present in oxidized LDL was shown to be sufficient to stimulate/activate the EGF receptor present in endothelial cells (64). It is unknown whether this is the mechanism whereby HNE exerts its induction of AKR1C1 expression. Genestein, but not equimolar concentrations of the inactive analog dadzien, inhibits the ability of the redox-cycling quinone 2,3-dimethoxynaphthalene-1,4-dione to induce AKR1C1 mRNA levels in HepG2 cells.3 However, it is unclear whether this inhibition is mediated by the nonspecific antioxidant properties of genestein (65) or by a specific inhibitory effect on the EGFR.
Finally, the identification of HNE as a substrate of the ROS-inducible
AKR1C1 isoform may explain, at least in part, its closely associated
pattern of regulation with certain GST isoforms by GSH-depleting agents
and GST inhibitors (20-22). While GST undoubtedly represents an
important pathway in the detoxification of HNE, studies in
H2O2-resistant hamster fibroblasts imply that
GSH-dependent processes are not the sole determinants of
H2O2 resistance (66). The studies presented
here as well as by others (17-19) imply that members of the AKR
superfamily act as a cytosolic barrier to the products of lipid
peroxidation in the absence of GSH. Of these, AKR1C1 is the isoform
that is induced by ROS and HNE as the counter-response to oxidative
stress is mounted.
![]() |
FOOTNOTES |
---|
* This research was supported by National Institutes of Health Grants CA55711 and CA39504 (to T. M. P.) and a Pharmaceutical Research and Manufacturers of America Foundation Advanced Predoctoral Fellowship (to M. E. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Bristol Myers Squibb Young Investigator Award for a
preliminary account of this work at the 90th Annual Meeting of the
American Association for Cancer Research, Philadelphia, PA, April
10-14, 1999. Present address: R. W. Johnson Pharmaceutical Research Inst., Route 202, P.O. Box 300, Raritan, NJ 08869.
§ To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1186; Fax: 215-573-2236; E-mail: penning@pharm.med.upenn.edu.
Published, JBC Papers in Press, November 11, 2000, DOI 10.1074/jbc.M006655200
2 The nomenclature for the aldo-keto reductase superfamily was proposed by Jez et al. and adopted at the 8th International Symposium on Enzymology and Molecular Biology of Carbonyl Metabolism in Deadwood, June 29 to July 3, 1996 (16). It is also described on the AKR superfamily home page on the World Wide Web.
3 M. E. Burczynski and T. M. Penning, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HNE, 4-hydroxy-2-nonenal;
AKR, aldo-keto reductase;
ADH, alcohol
dehydrogenase;
DHA, dehydroascorbate;
DHN, 1,4-dihydroxy-2-nonene;
GST, glutathione S-transferase;
HSD, hydroxysteroid
dehydrogenase;
3-HSD, 3
-hydroxysteroid dehydrogenase (EC
1.1.1.213: A-face specific);
20
-hydroxyprogesterone, 20
-hydroxypregn-4-en-3-one;
PAH, polycyclic aromatic hydrocarbon(s);
NQO1, NAD(P)H:quinone oxidoreductase 1;
ROS, reactive oxygen species;
PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Trush, M. A., and Kensler, T. W. (1991) Free Radic. Biol. Med. 10, 201-209[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ross, R. (1993) Nature 362, 801-809[CrossRef][Medline] [Order article via Infotrieve] |
3. | Poulsen, H. E., Prieme, H., and Loft, S. (1998) Eur. J. Cancer Prev. 7, 9-16[Medline] [Order article via Infotrieve] |
4. | Sun, A. Y., and Chen, Y. M. (1998) J. Biomed. Sci. 5, 401-414[CrossRef][Medline] [Order article via Infotrieve] |
5. | Facchinetti, F., Dawson, V. L., and Dawson, T. M. (1998) Neurobiology 18, 667-682 |
6. | Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Free Radic. Biol. Med. 11, 81-128[CrossRef][Medline] [Order article via Infotrieve] |
7. | Uchida, K., Toyokuni, S., Nishikawa, K., Kawakishi, S., Oda, H., Hiai, H., and Stadtman, E. R. (1994) Biochemistry 33, 12487-12494[Medline] [Order article via Infotrieve] |
8. |
Yoritaka, A.,
Hattori, N.,
Uchida, K.,
Tanaka, M.,
Stadtman, E. R.,
and Mizuno, Y.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2696-2701 |
9. | Markesbery, W. R., and Carney, J. M. (1999) Brain Pathol. 9, 133-146[Medline] [Order article via Infotrieve] |
10. | Yi, P., Zhan, D. J., Samokyszyn, V. M., Doerge, D. R., and Fu, P. P. (1997) Chem. Res. Toxicol. 10, 1259-1265[CrossRef][Medline] [Order article via Infotrieve] |
11. | Nair, J., Barbin, A., Velic, I., and Bartsch, H. (1999) Mutat. Res. 424, 59-69[CrossRef][Medline] [Order article via Infotrieve] |
12. | Rindgen, D., Nakajima, M., Wehrli, S., Xu, K., and Blair, I. A. (1999) Chem. Res. Toxicol. 12, 1195-1204[CrossRef][Medline] [Order article via Infotrieve] |
13. | Lee, S. H., Rindgen, D., Bible, R. H., Hajdu, E., and Blair, I. A. (2000) Chem. Res. Toxicol. 17, 565-574[CrossRef] |
14. | Hartley, D. P., Ruth, J. A., and Petersen, D. R. (1995) Arch. Biochem. Biophys. 316, 197-205[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hubatsch, J., Ridderstrom, M., and Mannervik, B. (1998) Biochem. J. 330, 175-179[Medline] [Order article via Infotrieve] |
16. | Jez, J. M., Flynn, T. G., and Penning, T. M. (1997) Biochem. Pharmacol. 54, 639-647[CrossRef][Medline] [Order article via Infotrieve] |
17. | Vander Jagt, D. L., Kolb, N. S., Vander Jagt, T. J., Chino, J., Martinez, F. J., Hunsaker, L. A., and Royer, R. E. (1995) Biochim. Biophys. Acta 1249, 117-126[Medline] [Order article via Infotrieve] |
18. | Srivastava, S., Chandra, A., Bhatnagar, A., Srivastava, S. K., and Ansari, N. H. (1995) Biochem. Biophys. Res. Commun. 217, 741-746[CrossRef][Medline] [Order article via Infotrieve] |
19. | Srivastava, S., Harter, T. M., Chandra, A., Bhatnagar, A., Srivastava, S. K., and Petrash, J. M. (1998) Biochemistry 37, 12909-12917[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Ciaccio, P. J.,
Jaiswal, A. K.,
and Tew, K. D.
(1994)
J. Biol. Chem.
269,
15558-15562 |
21. | Ciaccio, P. J., Shen, H., Jaiswal, A. K., Lyttle, M. H., and Tew, K. D. (1995) Mol. Pharmacol. 48, 639-647[Abstract] |
22. |
Burczynski, M. E.,
Lin, H. K.,
and Penning, T. M.
(1999)
Cancer Res.
59,
607-614 |
23. |
Penning, T. M.
(1997)
Endocrine Rev.
18,
281-305 |
24. |
Lin, H-K.,
Jez, J. M.,
Schlegel, B. P.,
Peehl, D. M.,
Pachter, J. A.,
and Penning, T. M.
(1997)
Mol. Endocrinol.
11,
1971-1984 |
25. | Penning, T. M., Burczynski, M. E., Jez, J. M., Hung, F-C., Lin, H-K., Ma, H., Moore, M., Palackal, N., and Ratnam, K. (2000) Biochem. J. 351, 67-77[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Smithgall, T. E.,
Harvey, R. G.,
and Penning, T. M.
(1986)
J. Biol. Chem.
261,
6184-6191 |
27. |
Smithgall, T. E.,
Harvey, R. G.,
and Penning, T. M.
(1988)
J. Biol. Chem.
263,
1814-1820 |
28. | Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1998) Biochemistry 37, 6781-6790[CrossRef][Medline] [Order article via Infotrieve] |
29. | Burczynski, M. E., Palackal, N. T., Harvey, R. G., and Penning, T. M. (1999) Polycyclic Arom. Cmpds. 16, 205-214 |
30. | Del Bello, B., Maellaro, E., Sugherini, L., Santucci, A., Comporti, M., and Casini, A. F. (1994) Biochem. J. 304, 385-390[Medline] [Order article via Infotrieve] |
31. |
Pawlowski, J. E.,
and Penning, T. M.
(1994)
J. Biol. Chem.
269,
13502-13510 |
32. | Palackal, N. T., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1999) Proc. Am. Assoc. Cancer Res. 40, Abstr. 4883 |
33. | Bates, R. W., Dietz-Martin, D., Kerr, W. J., Knight, J. G., Ley, S. V., and Sakellaridis, A. (1990) Tetrahedron 46, 4063-4082[CrossRef] |
34. | Leatherbarrow, R. J. (1987) ENZFITTER: A Non-Linear Regression Data Analysis Program for the IBM PC (and True Compatibles) , Biosoft, Cambridge, UK |
35. | Suzuki-Yamamoto, T., Nishizawa, M., Fukui, M., Okuda-Ashitaka, E., and Nakajima, T. (1999) FEBS Lett. 462, 335-340[CrossRef][Medline] [Order article via Infotrieve] |
36. | O'Connor, C., Ireland, L. S., Harrison, D. J., and Hayes, J. D. (1999) Biochem. J. 343, 487-504[CrossRef][Medline] [Order article via Infotrieve] |
37. | Jez, J. M., and Penning, T. M. (1998) Biochemistry 37, 9695-9703[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Nguyen, T.,
and Pickett, C. B.
(1992)
J. Biol. Chem.
267,
13535-13539 |
39. | Pinkus, R., Weiner, L. M., and Daniel, V. (1995) Biochemistry 34, 81-88[Medline] [Order article via Infotrieve] |
40. |
Shi, M. M.,
Kugelman, A.,
Iwamoto, T.,
Tian, L.,
and Forman, H. J.
(1994)
J. Biol. Chem.
269,
26512-26517 |
41. |
Mulcahy, R. T.,
Wartman, M. A.,
Bailey, H. H.,
and Gipp, J. J.
(1997)
J. Biol. Chem.
272,
7445-7454 |
42. | Dalton, T., Palmiter, R. D., and Andrews, G. K. (1994) Nucleic Acids Res. 22, 5016-5023[Abstract] |
43. | Klaassen, C. D., and Liu, J. (1998) Environ. Health Perspect. 106, 297-300[Medline] [Order article via Infotrieve] |
44. |
Favreau, L. V.,
and Pickett, C. B.
(1991)
J. Biol. Chem.
266,
4556-4561 |
45. |
Li, Y.,
and Jaiswal, A. K.
(1993)
J. Biol. Chem.
268,
21454-21459 |
46. | Burczynski, J. M., Hayes, J. R., Longhurst, P. A., and Colby, H. D. (1999) Free Radic. Biol. Med. 26, 987-991[CrossRef][Medline] [Order article via Infotrieve] |
47. | Wells, W. W., and Xu, D. P (1994) J. Bioenerg. Biomembr. 26, 369-377[Medline] [Order article via Infotrieve] |
48. | Winkler, B. S., Orselli, S. M., and Rex, T. S. (1994) Free Radic. Biol. Med. 17, 333-349[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Stolz, A.,
Hammond, L.,
Lou, H.,
Takikawa, H.,
Ronk, M.,
and Shively, J. E.
(1993)
J. Biol. Chem.
268,
10448-10457 |
50. | Matsuura, K., Deyashiki, Y., Sato, K., Ishida, N., Miwa, G., and Hara, A. (1997) Biochem. J. 323, 61-64[Medline] [Order article via Infotrieve] |
51. | Leonarduzzi, G., Parola, M., Muzio, G., Garramone, A., Maggiora, M., Robino, G., Poli, G., Dianzani, M. U., and Canuto, R. A. (1995) Biochem. Biophys. Res. Commun. 214, 669-675[CrossRef][Medline] [Order article via Infotrieve] |
52. | Parola, M., Leonarduzzi, G., Robino, G., Albano, E., Poli, G., and Dianzani, M. U (1996) Free Radic. Biol. Med. 20, 351-359[CrossRef][Medline] [Order article via Infotrieve] |
53. | Jez, J. M., Bennett, M. J., Schlegel, B. P., Lewis, M., and Penning, T. M. (1997) Biochem. J. 326, 625-636[Medline] [Order article via Infotrieve] |
54. | Sellin, S., Holmquist, B., Mannervik, B., and Vallee, B. L. (1991) Biochemistry 30, 2514-2518[Medline] [Order article via Infotrieve] |
55. | Allali-Hassani, A., Peralba, J. M., Martras, S., Farres, J., and Pares, X. (1998) FEBS Lett. 426, 362-366[CrossRef][Medline] [Order article via Infotrieve] |
56. | Awasthi, Y. C., Zimniak, P., Singhal, S. S., and Awasthi, S. (1995) Biochem. Arch. 11, 47-54 |
57. | Esterbauer, H., Zollner, H., and Lang, J. (1985) Biochem. J. 228, 363-373[Medline] [Order article via Infotrieve] |
58. | Grune, T., Siems, W., Kowalewski, J., Zollner, H., and Esterbauer, H. (1991) Biochem. Int. 25, 963-971[Medline] [Order article via Infotrieve] |
59. | Grune, T., Siems, W. G., and Petras, T. (1997) J. Lipid Res. 38, 1660-1665[Abstract] |
60. | Siems, W. G., Zollner, H., Gruyne, T., and Esterbauer, H. (1997) J. Lipid Res. 38, 612-622[Abstract] |
61. |
Srivastava, S.,
Chandra, A.,
Wang, L.,
Seifert Jr, W. E.,
Dague, B. B.,
Ansari, N. H.,
Srivastava, S. K.,
and Bhatnagar, A.
(1998)
J. Biol. Chem.
273,
10893-10900 |
62. | Fukuda, A., Nakamura, Y., Ohigashi, H., Osawa, T., and Uchida, K. (1997) Biochem. Biophys. Res. Commun. 236, 505-509[CrossRef][Medline] [Order article via Infotrieve] |
63. |
Spycher, S. E.,
Tabataba-Vakili, S.,
O'Donnell, V. B.,
Palomba, L.,
and Azzi, A.
(1997)
FASEB J.
11,
181-188 |
64. |
Suc, I.,
Meilhac, O.,
Lajoie-Mazenc, I.,
Vandaele, J.,
Jurgens, G.,
Salvayre, R.,
and Negre-Salvayre, A.
(1998)
FASEB J.
12,
665-671 |
65. | Peterson, G. (1995) J. Nutr. 125 (suppl.), 784-789 |
66. | Spitz, D. R., Kinter, M. T., and Roberts, R. J. (1995) J. Cell. Physiol. 165, 600-609[Medline] [Order article via Infotrieve] |