Synthesis and cellular effects of an intracellularly activated analogue of 4-hydroxynonenal

Article abstract of DOI:10.1021/tx010115w

4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation and has been suggested to play a role in the pathogenesis in several common diseases as well as injuries caused by environmental toxicants. Although formed intracellul

Full text of DOI:10.1021/tx010115w

40
Chem. Res. Toxicol. 2002, 15, 40-47
Syn th esis a n d Cellu la r Effects of a n In tr a cellu la r ly
Activa ted An a logu e of 4-Hyd r oxyn on en a l
M. Diana Neely,* Venkataraman Amarnath, Carl Weitlauf, and
Thomas J . Montine
Department of Pathology and the Centers for Molecular Neuroscience and Molecular Toxicology,
Vanderbilt University Medical School, MCN U4216, 21st Avenue South,
Nashville, Tennessee 37232-2561
Received J uly 16, 2001
4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation
and has been suggested to play a role in the pathogenesis in several common diseases as well
as injuries caused by environmental toxicants. Although formed intracellularly in vivo, for
practical reasons this molecule is applied extracellularly in order to analyze its biological effects.
The focus of this study was to develop an approach that would enable intracellular HNE
production in the absence of the many other products and processes that occur in cells
experiencing generalized oxidative stress. To this end, we synthesized 1,1,4-tris(acetyloxy)-
2(E)-nonene (HNE[Ac]3), a triester analogue of HNE that is itself unreactive but could be
hydrolyzed intracellularly presumably by lipases and/or esterases into the highly reactive HNE.
In vitro lipase rapidly converted HNE(Ac)3 initially to 4-acetyloxy-2-nonenal (HNE[Ac]1) and
then to HNE. Neuro 2A cell lysate also caused a rapid hydrolysis of HNE(Ac)3 into HNE(Ac)1
and HNE. Incubation of BSA with HNE(Ac)3 resulted in protein-adduct formation only in
the presence of lipase. We demonstrated adduction of HNE to proteins in Neuro 2A cells exposed
to HNE(Ac)3 by immunoblotting and immunocytochemistry using antibodies specific for HNE-
Michael adducts on proteins. We have previously shown that microtubule organization is very
sensitive to HNE. Analysis of Neuro 2A cell microtubules showed that this cytoplasmic organelle
is similarly sensitive to HNE and HNE(Ac)3.
impairment of mitochondrial respiration, inhibition of
DNA-, RNA-, and protein synthesis, stimulation of neu-
trophil chemotaxis, modulation of platelet aggregation
and changes in responses of second messenger pathways
(17, 19, 20).
In tr od u ction
There is increasing evidence that oxidative stress, and
lipid peroxidation in particular, contributes to disease
states as diverse as atherosclerosis, neurodegenerative
diseases, and to injuries resulting from environmental
toxicants (1-6). Reactive aldehydes generated during the
process of lipid peroxidation are believed to play impor-
tant roles in mediating the pathophysiological effects of
oxidative stress (7-16). 4-Hydroxy-2-nonenal (HNE)1 is
one of the main aldehydes formed during lipid peroxi-
dation and has received considerable attention due to its
bioactivity. This R,â-unsaturated aldehyde is a strong
electrophile which readily forms adducts with cellular
nucleophiles such as glutathione, cysteine, lysine, and
histidine of proteins, and also with nucleic acids (17, 18).
Analysis of HNE modified proteins in cells exposed to
HNE or experiencing oxidative stress has not revealed a
consistent major cellular target, but rather suggests an
abundance of targets the identities of which likely vary
with cell type and type of oxidative insult. Microsomal,
mitochondrial, plasma membrane, and cytoplasmic tar-
gets have been identified (17). High concentrations of
HNE are cytotoxic, whereas at lower concentrations, this
aldehyde can cause disruption of cytoskeletal integrity,
To study the effects of HNE, cells or organelles can be
subjected to oxidative insults and the formation of HNE
correlated with change in a particular activity; alterna-
tively, exogenous HNE can be applied to cells or or-
ganelles and changes in cellular functions or enzyme
activities measured. Esterbauer and colleagues have
compared the inhibition of glucose-6-phosphatase by
endogenous HNE formed in microsomes subjected to
oxidative insults and by HNE externally applied to
microsomes. They measured an ID50 of 1.5 µM HNE in
oxidatively stressed microsomes; however, in microsomes
exposed to exogenous HNE, the ID50 for glucose-6-
phosphatase inhibition was 100-200-fold higher (150-
300 µM) (21). One interpretation of these experiments is
that endogenously generated HNE and exogenously
applied HNE may have access to different targets.
Several studies have reported that HNE is unequally
distributed between the lipid and aqueous phase (22, 23)
and HNE concentrations in the millimolar range have
been measured in biological membranes exposed to
oxidative stress (21, 24). This suggests that the majority
of endogenously formed HNE remains with intracellular
membranes and encounters intracellular targets before
it diffuses out of the cell and has access to targets on the
cell surface. Targets of exogenously applied HNE are
* To whom correspondence should be addressed. Phone: (615) 322-
3699. Fax: (615) 343-9825. E-mail: diana.neely@mcmail.vanderbilt.edu.
1
Abbreviations: BSA, bovine serum albumin; FBS, fetal bovine
serum; HNE, 4-hydroxy-2-nonenal; HNE(Ac)1, 4-acetyloxy-2-nonenal;
HNE(Ac)3, 1,1,4-tris(acetyloxy)-2(E)-nonene; PBS, phosphate buffered
saline.
10.1021/tx010115w CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/15/2001

An Intracellularly Activated HNE Analogue
Chem. Res. Toxicol., Vol. 15, No. 1, 2002 41
identified the fractions containing the pure triester by gas
chromatography, then combined and evaporated them to obtain
4-[3H]HNE(Ac)3. The specific activity of 4-[3H]HNE(Ac)3 was
12.3 µCi/µM.
HNE(Ac)3 Hyd r olysis. We analyzed spontaneous and lipase-
induced hydrolysis of HNE(Ac)3. HNE(Ac)3 (28.6 mM) in phos-
phate buffer (20 mM) was incubated in the absence or presence
of 2.9 mg/mL lipase (isolated from Pseudomonas fluorescens,
3414 units/mg, Fluka, NY, no. 62321) at 37 °C and 1 µL aliquots
were removed from the reaction mixture every 15 min for
likely different and probably involve extracellular do-
mains of proteins and lipids in the plasma membrane to
a greater extent. This suggests that the effects of endo-
genously formed HNE may not always be accurately
mimicked in experiments with externally added HNE.
To develop a tool that would allow us to compare
cellular effects of exogenously applied and endogenously
formed HNE, we synthesized 1,1,4-tris(acetyloxy)-2(E)-
nonene (HNE[Ac]3). This triester analogue of HNE is
itself not reactive, but once it diffuses into cells it is
hydrolyzed to the reactive HNE and its analogue, 4-acetyl-
oxy-2-nonenal (HNE[Ac]1). This molecule therefore,
allowed us to compare the effects of intracellularly formed
and exogenously applied HNE on a cytoplasmic organelle,
the microtubule network.
analysis by gas-liquid chromatography performed with
a
Hewlett-Packard 5890 Series II GC-FID equipped with a HP-
INNOWax capillary column (15 m × 0.53 mm) (Hewlett-
Packard, Wilmington, DE). The carrier gas flow (helium) was 8
mL/min and the temperature was programmed to hold at 80
°C for 2 min and then increase to 200 °C at 20 °C/min and then
hold at 200 °C. Acetic acid (CH3COOH), 4-acetyloxy-2-nonenal
(HNE[Ac]1), HNE, and HNE(Ac)3 eluted after 3.4, 6.7, 7.9, and
9.1 min, respectively (see Figures 1 and 2). To analyze HNE-
(Ac)3 hydrolysis by cell lysate, we cultured Neuro 2A cells exactly
as described below, then washed them twice with PBS, and
harvested and sonicated (2 times for 20 s at 20 W) the cells of
two T-25 flasks in 1.2 mL of PBS. Five microliters of a 100 mM
HNE(Ac)3 stock solution were added to 400 µL of this cell lysate
(1.4-2.0 mg/mL protein) and incubated at 37 °C. We removed
70 µL samples at different times and extracted them with 100
µL of ethyl acetate. The extracts were analyzed by gas-liquid
chromatography as described above.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. Chemicals required for the synthesis of 4-hydroxy-
2(E)-nonenal (HNE) and 1,1,4-tris(acetyloxy)-2(E)-nonene
(HNE[Ac]3) were from Aldrich (Milwaukee, WI). Materials used
for cell culture were from Life Technologies (Grand Island, NY).
Reagents for sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and immunoblotting were obtained
from Bio-Rad (Hercules, CA). All other chemicals were from
Sigma (St. Louis, MO), unless otherwise indicated.
Syn th esis of 4-Hyd r oxy-2(E)-n on en a l (HNE). Unlabeled
HNE was synthesized using Gardner’s procedure (25). To
synthesize 4-[3H]HNE, we prepared 1,1-dimethoxynon2-en-4-
one as described (26) and purified it by column chromatography
(4:1 hexanes-ethyl acetate). The above ketone (100 mg, 0.5
mmol) was reduced with NaB3H4 (26) (2 mCi/mg, American
Radiolabeled Chemicals, Inc., St. Louis, MO), hydrolyzed with
2.5% H2SO4 in tetrahydrofuran, and purified by column chro-
matography (silica, 2:1 hexanes-ethyl acetate). The specific
activity was 15.5 µCi/µM.
In Vitr o Rea ctivity of HNE(Ac)3. We examined the in vitro
reactivity of HNE(Ac)3 by analyzing the modification of bovine
serum albumin (BSA) (fraction V, Sigma, St. Louis, MO) after
incubation with HNE(Ac)3 or HNE in the presence and absence
of lipase by immunoblotting and autoradiography. For analysis
by immunoblotting BSA (1.6 mg/mL) in 20 mM phosphate buffer
was incubated with HNE(Ac)3 or HNE both at 4.5 mM for 2 h
at 37 °C in the presence or absence of lipase (3 mg/mL). For
analysis by autoradiography BSA was incubated with [3H]HNE-
(Ac)3 at 4.5 mM or [3H]HNE at 3.6 mM. We stopped the
reactions by adding Laemmli sample buffer and subjected the
samples to SDS-PAGE (31). Immunoblotting was performed
as described below using an antibody specific for HNE-Michael
protein adducts (Calbiochem, CA, no. 393205). For autoradiog-
raphy, we treated the SDS-PAGE gels with an autoradiography
enhancer (New England Nuclear, Massachusetts, no. NEF 992)
according to the manufacturer’s instructions, dried them and
exposed them to film (Kodak X-OMAT AR, Sigma, Missouri) for
3 days at - 80 °C.
Cell Cu ltu r e. Neuro 2A neuroblastoma cells (American Type
Culture Collection, Rockville, MD) were grown in growth
medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture
F-12 (1:1) (DMEM/F12) with 10% (v/v) FBS and penicillin-
streptomycin at 100 units/mL and 100 µg/mL respectively. Two
days before the experiment, we subcultured the cells in growth
medium and left them to adhere overnight. For immunocy-
tochemical analysis Neuro 2A cells were subcultured at 5 × 104
cells/mL in the wells of 35 mm glass bottom microwell dishes
(MatTek Corp., MA) and for analysis of HNE(Ac)3 hydrolysis
or protein modification in T-25 culture flasks at 20 × 104 cells/
mL. The next morning, we removed the serum containing
growth medium and replaced it with N2-medium (DMEM/F12
medium containing penicillin-streptomycin and N-2 supplement
[Life Technologies, Grand Island, NY]). After 24 h culturing in
N2-medium, we exposed the cells to HNE or HNE(Ac)3 for 1 h
at 37 °C and 5% CO2.
Syn t h esis
of
1,1,4-Tr is(a cet yloxy)-2(E )-n on en e
(HNE[A]3). Although extensive work has been done on protect-
ing aldehydes as acetals, only a handful of reports are available
for converting an aldehyde group to 1,1-diacetate (27-30), and
they all involve treatment with acetic anhydride in the presence
of Lewis acids. When we added HNE to a solution of acetic
anhydride and BF3-O(C2H5)2 at room temperature (27), we were
unable to isolate a distinct product from the dark mixture. The
hydroxyl group at the 4-position likely was oxidized, and the
resulting 4-oxononenal underwent further reactions. This was
avoided when the initial mixing was done in ether at low
temperature to form the 4-acetyloxy-2-nonenal that was con-
verted to 1,1-diacetate on further reaction.
Therefore, acetic anhydride (0.36 mL, 3.6 mmol) and BF3-
OEt2 (30 µL) were stirred in dry ether (5 mL) and cooled to -
40 °C in dry ice-2-propanol bath. HNE (160 µL, 1 mmol) was
added and the resulting solution was warmed to room temper-
ature. The reaction mixture was heated to reflux in an oil bath
kept at 50 °C. After 2 h, we mixed the reaction solution with 10
mL of 1 M phosphate buffer (pH 8.0), separated the layers, and
extracted the aqueous layer with ether (2 × 10 mL). The
combined ether layers were dried, concentrated, and purified
by column chromatography (silica: hexane, 3:1 hexanes-ethyl
1
acetate); yield 120 mg (40%); H NMR δ 7.10 (d, 1 H, J ) 6.0
Hz, C1-H), 5.92 (dd, 1 H, J ) 15. 7 and 6.0 Hz, C3-H), 5.68 (dd,
1 H, J ) 15.7 and 6.0 Hz, C2-H), 5.26 (dt, 1 H, J ) 6.2, C4-H),
2.05 (s, 9 H, CH3CdO), 1.6-0.8 (11 H, alkyl chain); 13C NMR δ
170.2 (C4OCdO), 168.6 (C1OC)O), 135.0 (C3), 124.6 (C2), 88.6
(C1), 72.6 (C4), 33.9 (C5), 31.4 (C7), 24.5 (C6), 22.4 (C8), 21.1 and
20.8 (CH3CdO), 13.9 (C9); GC-MS m/z 300 (1, M+), 241 (2, M -
CH3CO2), 198 (25, 241 - CH3CO), 169 (50, 198 - CHO), 156
and 155 (100, HNE). The conversion of 4-[3H]HNE to 4-[3H]-
HNE(Ac)3 paralleled the above preparation on a smaller scale.
The purification of the crude product was carried out on a small
(10 × 2 cm) column of silica (3:1 hexanes-ethyl acetate). We
Distr ibu tion of Ra d ioa ctivity in to P r otein a n d Lip id
F r a ct ion of Neu r o 2A Cells E xp osed t o [3H]HNE or
[3H]HNE(Ac)3. Cellular protein and lipid were extracted from
Neuro 2A cells exposed to 250 µM [3H]HNE or [3H]HNE(Ac)3
for 1 h. After exposure, the cells were pelleted and washed two
times with Hank’s balanced salt solution. We dissolved the final
cell pellet in 2 mL of chloroform:methanol (2:1, vol:vol) and
incubated it at 4 °C for 1 h with occasional vortexing. Then, we

42 Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Neely et al.
added 4 mL of ice-cold ether to the solution and, after rigorous
vortexing, removed the upper (ether) phase and stored it on ice.
The proteins in the chloroform/methanol phase were pelleted
and re-extracted with 2 mL of ice cold ether. We combined the
two ether fractions, evaporated the ether, and redissolved the
lipids in hexane. Protein pellets were dissolved in Laemmli
sample buffer, and radioactivity associated with lipids and
proteins was determined using a Packard 1900CA liquid scintil-
lation counter (Packard, Meriden, CA).
Im m u n oblot An a lysis a n d Au tor a d iogr a p h y. After ex-
posure to HNE or HNE(Ac)3 Neuro 2A cells were scraped from
the culture flask with a rubber policeman, pelleted, and solu-
bilized in Laemmli sample buffer. The proteins were then
separated by SDS-PAGE (31) and transferred to a PVDF
membrane (Immobilon-P, Millipore, Bedford, MA), and the
membrane was incubated with “blotto” (4% dry milk in tris-
buffered saline with 0.05% Tween 20) for at least 2 h at room
temperature to block unspecific antibody binding sites. We then
immunoreacted the proteins with anti-HNE Michael adducts
antibody (Calbiochem, San Diego, CA, no. 393205) diluted 4000-
fold in “blotto” overnight at 4 °C. After incubation with an HRP-
conjugated anti-rabbit IgG (Sigma, St. Louis, MO, no. A0545)
diluted 1:2000 in “blotto” for 2 h at room temperature, we
visualized the signal using a chemiluminescence reagent (NEN,
Boston, MA, no. NEL100). For autoradiography the SDS-PAGE
gels were treated with an autoradiography enhancer (New
England Nuclear, MA, no. NEF 992) according to the manufac-
turer’s instructions, dried, and then exposed to film (Kodak
X-OMAT AR, Sigma, MO) for 3 weeks at - 80 °C. The films
from immunoblots and autoradiography were scanned with a
GS710 densitometer (Bio-Rad, Hercules, CA) and the signals
quantified using Quantity One Quantitation Software (Bio-Rad,
Hercules, CA).
Im m u n ocytoch em istr y. After HNE or HNE(Ac)3 exposure
Neuro 2A cells were immediately fixed in 4% paraformaldehyde
in phosphate buffered saline (PBS) for 30 min at room temper-
ature and then permeabilized with 1% Triton X-100 in PBS
containing 2% FBS for 20 min at room temperature. Microtu-
bules were visualized with an anti-â-tubulin antibody (Boe-
hringer Mannheim, Indianapolis, IN, no. 1111876) at 0.25 µg/
mL in PBS with 2% FBS overnight at 4 °C, followed by a FITC-
coupled anti-mouse IgG (Cappel, West Chester, PA, no. 55514)
at 3 µg/mL in PBS with 2% FBS for 1-2 h at room temperature.
To visualize HNE-modified proteins antibody no. 672 (specific
for HNE-protein adducts) (32) diluted 1:500 in PBS with 2%
FBS or anti-HNE Michael adducts antibody (Calbiochem, San
Diego, CA, no. 393205) diluted 1:200 in PBS with 2% FBS was
applied overnight at 4 °C. The secondary antibody, a FITC-
coupled affinity-purified goat anti-rabbit antibody (Cappel, West
Chester, PA no. 55662), was diluted 1:100 (final concentration
of 7.3 µg/mL active antibodies) in PBS with 2% FBS, and the
cells were incubated for 1.5 h at room temperature. We carefully
removed the glass slides with the cells from the 35 mm dishes
with a razor blade and mounted them in ProLong mounting
medium (Molecular Probes, Eugene, OR, no. P-7481). Immuno-
fluorescence was analyzed with a Zeiss Axiovert 135 microscope
(Carl Zeiss, INC., Thornwood, NY) using a Plan-Apochromat
100×/1.4 objective. For semiquantitative analysis of the micro-
tubule organization, microscope slides were scanned along a
fixed y-coordinate using an electronically controlled microscope
stage and the microtubule organization of 200-500 cells/slide
were categorized as either normal, mildly disrupted, or severely
disrupted. The analysis was done blinded to exposure conditions.
ANOVA, followed by repeated t-tests with Bonferroni correction
for multiple comparisons were used to compare the effects of
HNE and HNE(Ac)3. We acquired confocal images of cellular
fluorescence using a confocal laser-scanning microscope (Zeiss
LSM 410) equipped with a Plan-Apochromat 63×/1.4 objective.
F igu r e 1. Synthesis and hydrolysis of HNE(Ac)3. Chemical
structures of HNE, and HNE(Ac)3 and HNE(Ac)1, the triester
and monoester analogues of HNE are shown. HNE(Ac)3 was
synthesized by incubation of HNE with acetic anhydride in the
presence of an acid catalyst. Unlike its parent compound, this
triester analogue lacks an aldehyde group and therefore does
not share the electrophilicity of HNE. The initial product of
the lipase-catalyzed hydrolysis of HNE(Ac)3 is the monoester
HNE(Ac)1, which is then further cleaved into HNE.
incubated HNE(Ac)3 in phosphate buffer in the absence
or presence of lipase and followed hydrolysis by gas
chromatography. We did not observe any hydrolysis of
HNE(Ac)3 in the absence of lipase during 2 h incubation
(Figure 2A). In the presence of lipase, HNE(Ac)3 was
initially hydrolyzed to 4-acetyloxy-2-nonenal (HNE[Ac]1),
the monoester of HNE, which was then further hydro-
lyzed to HNE (Figure 2B, see also Figure 1). After 3 min
of incubation, 11% of HNE(Ac)3 was converted into
HNE(Ac)1, after 15 min 100% of HNE(Ac)3 was hydro-
lyzed into HNE(Ac)1 (57%) and HNE (43%). After 60 min
incubation, the reaction mixture contained HNE(Ac)1
(28%) and HNE (72%). We observed only little additional
conversion of HNE(Ac)1 into HNE after the initial hour
of incubation (not shown). The results shown are repre-
sentative of several experiments performed. These ob-
servations show that HNE(Ac)3 does not undergo spon-
taneous hydrolysis within the time frame relevant for this
study and that lipase hydrolyzes HNE(Ac)3 initially into
HNE(Ac)1 and then into HNE (Figure 1).
The hydrolysis of HNE(Ac)3 by Neuro 2A cell lysates
was analyzed in the same way. We added HNE(Ac)3 to
Neuro 2A cell lysates in molar ratios [moles of HNE(Ac)3
per mg of cell protein] comparable to the ones used in
the experiments described below (equivalent to 100 µM)
and followed its hydrolysis by gas chromatography
(Figure 3). Hydrolysis of HNE(Ac)3 was rapid, such that
5.6% of HNE(Ac)3 was converted to HNE(Ac)1 upon
adding HNE(Ac)3 to the cell lysate. After 30 min, 88% of
HNE(Ac)3 was hydrolyzed into HNE(Ac)1 (86%) and HNE
(2%). After 60 min, the hydrolysis of HNE(Ac)3 was
complete with the conversion of 94.4% of HNE(Ac)3 into
HNE(Ac)1 (90%) and HNE (4.4%). Within the next 60
min, a small fraction of HNE(Ac)1 (2.8%) was hydrolyzed
to HNE, resulting in a total yield of 7.2% HNE. The sum
of the peak areas on the gas chromatography chromato-
grams [HNE(Ac)3, HNE(Ac)1, and HNE] stayed constant,
implicating that the amount of HNE(Ac)1 or HNE lost
through metabolism or adduction to cellular components
was too low to be detected with this method. Therefore,
Neuro 2A cells convert HNE(Ac)3 largely into HNE(Ac)1,
which differs from HNE only by having an acetyloxy-
instead of a hydroxyl-group on the C4 position (see Figure
1), and therefore shares the electrophilic character of
Resu lts
Hyd r olysis of HNE(Ac)3 by Lip a se a n d Neu r o 2A
Cell Lysa te. To analyze lipase-induced hydrolysis, we

An Intracellularly Activated HNE Analogue
Chem. Res. Toxicol., Vol. 15, No. 1, 2002 43
F igu r e 3. HNE(Ac)3 hydrolysis by Neuro 2A cell lysates.
HNE(Ac)3 was added to Neuro 2A cell lysate [600-800 nmol of
HNE(Ac)3/mg of cell protein] and incubated for 2 h. Aliquots
were removed at different times, extracted, and analyzed by
gas chromatography. The amount of HNE(Ac)3, HNE(Ac)1, and
HNE are plotted as a percentage of the total peak area (area
under peaks of all three compounds). The total peak area did
not change significantly over the time course of the experiment.
Values are means ( SEM; n ) 8 (some of the SEM-bars are
hidden by the symbols).
F igu r e 2. Lipase-catalyzed hydrolysis of HNE(Ac)3. HNE(Ac)3
in 20 mM phosphate buffer was incubated at 37 °C in the
absence (A) or presence (B) of lipase and hydrolysis followed by
gas chromatography. No hydrolysis was observed during 2 h of
incubation in the absence of lipase (A). In the presence of lipase
HNE(Ac)3 was quickly converted first to HNE(Ac)1 and then to
HNE.
HNE. When the ratio of HNE(Ac)3/mg of protein was
increased 2 or 4-fold, the hydrolysis of HNE(Ac)3 was
incomplete, such that within a 2 h time period only 60
or 27% of HNE(Ac)3 was hydrolyzed, respectively. Cell
lysates which had been boiled for 7 min before the assay
did not hydrolyze HNE(Ac)3 (data not shown).
F igu r e 4. In vitro protein modification by HNE(Ac)3 is lipase
dependent. HNE adduction to BSA incubated with HNE (lanes
1 and 2) or HNE(Ac)3 (lanes 3 and 4) in the presence (+) or
absence (-) of lipase was analyzed. (A) BSA was incubated with
HNE or HNE(Ac)3 and HNE adduction to BSA analyzed by
immunoblotting using an antibody specific for HNE-protein
adducts. (B) BSA was incubated with [3H]HNE or [3H]HNE-
(Ac)3 and protein modification analyzed by 3H-autoradiography.
In Vitr o Mod ifica tion of P r otein by HNE(Ac)3
Dep en d s on th e P r esen ce of Lip a se. In vitro reactiv-
ity of HNE(Ac)3 was tested and compared to HNE. To
mimic exposure conditions used in cell culture experi-
ments described below, HNE(Ac)3 and HNE were incu-
bated with BSA in cell culture medium (N2 medium) for
1-2 h at 37 °C in the presence or absence of lipase. Our
immunoblot analysis using an antibody specific for
HNE-protein adduct demonstrates that, while incuba-
tion of BSA (1.6 mg/mL) with HNE (4.5 mM) resulted in
adduction of this aldehyde to BSA regardless of the
presence of lipase, no BSA adducts were observed in
samples incubated with HNE(Ac)3 (4.5 mM) in the
absence of lipase. When lipase was present, HNE-BSA
adducts were detectable (Figure 4A). This shows that
HNE(Ac)3 does not adduct to protein, is not spontane-
ously hydrolyzed in solution to HNE, and that the
formation of HNE-BSA adducts depends on the hydroly-
sis of HNE(Ac)3 by lipase. To detect protein adducts not
recognized by the anti-HNE antibody, we incubated BSA
with [3H]HNE(Ac)3 and [3H]HNE and analyzed BSA
adduction by autoradiography. While HNE-adduction to
BSA was clearly detectable in the absence or presence
of lipase, binding of radioactivity to BSA incubated with
[3H]HNE(Ac)3 was dependent on lipase (Figure 4B).
Distr ibu tion of HNE(Ac)3 a n d Its P r od u cts in to
th e Cellu la r Lip id a n d P r otein P ools. We next
compared the distribution of radioactivity from [3H]HNE
and [3H]HNE(Ac)3 into cellular protein and lipid fractions

44 Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Neely et al.
F igu r e 5. Comparison of HNE modified proteins in Neuro 2A
cells exposed to HNE and HNE(Ac)3. (A) Neuro 2A cells were
exposed to vehicle only (lane 1), 25 µM HNE (lane 2), 250 µM
HNE(Ac)3 (lane 3), or 500 µM HNE(Ac)3 (lane 4) for 1 h. Protein
modification of the cell lysates was analyzed by immunoblotting
using an anti-HNE-protein adduct antibody as described in the
materials and methods section. Molecular weight markers (kDa)
are shown on the left. Arrowheads indicate two proteins that
were modified by HNE, but not detectably modified by HNE-
(Ac)3. A protein modified preferentially in cells exposed to HNE-
(Ac)3 is indicated by an arrow. No protein modification was
observed in cells exposed to vehicle only (lane 1). Three and a
half times as much cell lysate was applied from cells exposed
to HNE(Ac)3 (lanes 3 and 4) than HNE treated cells (lane 2).
(B) Neuro 2A cells were exposed to [3H]HNE (50 µM, lane 1;
100 µM lane 2) or [3H]HNE(Ac)3 (250 µM, lane 3; 500 µM, lane
4) for 1 h and protein modifications analyzed by autoradiogra-
phy. Molecular weight markers (kDa) are shown on the left.
Equal amounts of protein were applied to all lanes.
F igu r e 6. Subcellular localization of HNE-protein adducts in
Neuro 2A cells exposed to HNE and HNE(Ac)3. Neuro 2a cells
were exposed to 25 µM HNE (A) or 250 µM HNE(Ac)3 (B) for 1
h. Immunofluorescence was performed using an antibody spe-
cific for HNE-protein adducts as described in the Materials and
Methods and analyzed by confocal laser scanning microscopy.
No staining was observed in cells incubated with vehicle only.
Arrows point to cellular nuclei.
of Neuro 2A cells. After 1 h exposure to 250 µM [3H]HNE,
2.4 ( 0.5% of the total radioactivity applied to the
cultures was associated with cells. Of this cell-associated
radioactivity 93.5 ( 0.8% (2.2% of total label) remained
with the protein fraction, while 6.5 ( 0.8% (0.2% of total
label) was associated with the lipid fraction. In cells
incubated with 250 µM [3H]HNE(Ac)3 for 1 h, 1.7 ( 0.3%
of the added radioactivity was associated with the cells
of which 79.9 ( 2.1% (1.4% of total label) was partitioned
into the protein fraction and 20.1 ( 2.1% (0.3% of total
label) into the lipid fraction. Thus, the total amount of
cell-associated label was not significantly different after
[3H]HNE or [3H]HNE(Ac)3 treatment. However, a statis-
tically significantly larger fraction of the radioactive label
was associated with the proteins in cells incubated with
[3H]HNE than in cells exposed to [3H]HNE(Ac)3 (p e
0.001).
arrow) was preferentially modified in cells treated with
HNE(Ac)3.
In addition to these qualitative differences, we also
observed a quantitative difference in protein modifica-
tion. To achieve protein modification detectable by im-
munoblot analysis, exposure of cells to higher concen-
trations of HNE(Ac)3 than HNE were necessary. Thus, a
20-fold higher concentration of HNE(Ac)3 (500 µM)
resulted in less detectable protein modification than what
was observed after HNE (25 µM) treatment of the cells
(Figure 5A, compare lane 2 and 4). This difference in
signal intensity could be due to the specificity of the anti-
HNE-protein adduct antibody used in the immunoblot
analysis, which may not recognize a protein-adduct
formed by HNE(Ac)1. We therefore performed autorad-
iography of Neuro 2A cells exposed to tritiated HNE or
HNE(Ac)3 (Figure 5B). Total radioactivity associated with
the proteins resolved by SDS-PAGE was 3-4-fold less
in extracts of cells incubated with [3H]HNE(Ac)3 than in
cells incubated with [3H]HNE, a difference much less
dramatic than the one observed on immunoblots. As
observed on immunoblots, the patterns of proteins
modified in Neuro 2A cells exposed to [3H]HNE and
HNE(Ac)3 F or m s HNE-P r otein Ad d u cts in Neu r o
2A Cells. Exposure of Neuro 2A cells to HNE(Ac)3
resulted in protein modifications detectable with antibod-
ies specific for HNE-protein adducts (Figure 5A). This
confirms intracellular conversion of HNE(Ac)3 to HNE.
Although the patterns of HNE-adducted proteins in cells
exposed to HNE and HNE(Ac)3 were similar, they were
not identical. Two proteins, one with MW > 212 kDa
(Figure 5A, single arrowhead) and another with a 43.8
kDa < MW < 86 kDa (Figure 5A, double arrowhead) were
adducted by HNE, but not detectably modified by HNE-
(Ac)3. Another protein with MW < 43.8 kDa (Figure 5A,

An Intracellularly Activated HNE Analogue
Chem. Res. Toxicol., Vol. 15, No. 1, 2002 45
F igu r e 7. HNE and HNE(Ac)3 disrupt the microtubule network. Neuro 2A cells were exposed to HNE or HNE(Ac)3 for 1 h and
their microtubule organization analyzed by immunocytochemistry using an anti-â-tubulin antibody. In control cells the microtubule
network is dense with microtubules radiating out to the periphery of the cells (A). HNE at 25 µM causes a severe loss of the microtubule
network (B). HNE(Ac)3 applied at 25 µM induces a more moderate change, with many cells showing loss of mainly the peripheral
microtubules (C). 50 µM HNE(Ac)3 leads to a near complete loss of the microtubule network in the majority of the cells (D). Bar 20
) µm. (E) Neuro 2A cells were exposed to HNE or HNE(Ac)3 and the degree of microtubule disruption quantified as described in the
materials and methods section. Values are means ( SEM; n ) 5 (control); n ) 4 (25 µM HNE); n ) 4 (25 µM HNE[Ac]3); n ) 3 (50
µM HNE[Ac]3.
[3H]HNE(Ac)3 are similar, but not identical. The most
obvious difference again was a protein of MW > 212 kDa
that was modified by HNE, but not detectably by HNE-
(Ac)3. Whether or not that band represents the same high
molecular weight protein observed to be preferentially
modified by HNE by immunoblotting (Figure 5A) is not
known at this time.
We analyzed the subcellular distribution of HNE-
protein adducts by immunocytochemistry using an an-
tibody specific for HNE-Michael protein adducts and
observed a striking difference in the staining pattern in
cells treated with HNE and HNE(Ac)3. Exposure to HNE
for 1 h resulted in HNE-protein adducts in the plasma
membrane as well as cytoplasm, but the nucleus was
spared (Figure 6A, arrow). In contrast, cells incubated
with HNE(Ac)3 showed clear nuclear immunoreactivity
in addition to cytoplasmic and plasma membrane immu-
nofluorescence (Figure 6B, arrow). As observed in the
immunoblot analysis study described above, higher con-
centrations of HNE(Ac)3 than HNE were required to
obtain a signal detectable with the anti-HNE-protein
adduct antibody.
E ffect s of H NE (Ac)3 on Cellu la r Micr ot u b u le
Or ga n iza tion . HNE causes disruption of microtubules
in Neuro 2A cells at low concentrations and after short
exposure times (33). We exposed Neuro 2A cells to HNE-
(Ac)3 or HNE for 1 h and characterized the microtubule
network as normal (Figure 7A), mildly disrupted (Figure
7C), or severely damaged (Figure 7, panels B and D). A
majority of control cells (83.9 ( 8.5%) displayed a normal
dense network of microtubules radiating out to the
periphery of the cell (Figure 7, panels A and E); the
remaining 14.7 ( 8.9% showed moderate changes in the
microtubule distribution. After 1 h exposure to 25 µM
HNE(Ac)3, 36.6 ( 11.7% of Neuro 2A cells showed severe
and 63.2 ( 11.5% moderate microtubule disruption with
loss mainly from the cellular periphery (Figure 7, panels
C and E) (0.2% of the cells had normal microtubule
network). Prolonging the time of exposure to 25 µM HNE-
(Ac)3 did not increase the percentage of cells with severe
disruption of the microtubule network (data not shown).
At 50 µM HNE(Ac)3 89.6 ( 0.6% of the cells had lost all
or almost all of their microtubules (Figure 7D, E), with
the remaining 10.4 ( 0.6% cells displaying a moderate
disruption of the microtubule network. This is compa-
rable to the effect of 25 µM HNE which results in 87.7 (
2.9% showing severe and 12.3 ( 2.9% moderate distur-
bance of the microtubules (Figure 7, panels B and E).
Thus, HNE(Ac)3 causes disruption of microtubules with
approximately 2-fold less efficacy than HNE.
Discu ssion
The goal of this study was to develop a tool that would
allow the comparison of cellular effects caused by endog-
enously produced and externally applied HNE. We
designed an HNE analogue that itself is unreactive by
converting the aldehyde into a diacetate. Once inside the
cell, enzyme-catalyzed hydrolysis of the ester groups (34)
was expected to release the aldehyde group. At this point,
we have no knowledge of the cellular site(s) and enzyme-
(s) responsible for the hydrolysis of HNE(Ac)3. Compari-
son of the sites of HNE delivery resulting from HNE(Ac)3
metabolism and HNE production after intracellular
generation of reactive oxygen species would be complex,
since the site of HNE production by the latter pathway
likely depends on the type of oxidative insult as well as
cell type. Indeed, the pattern of HNE-adducted proteins
is different in synaptosomes exposed to FeSO4 or Aâ (35).
Analyzing the dynamics of HNE(Ac)3 hydrolysis, we
observed that Neuro 2A cell lysates hydrolyze 94.5% of
HNE(Ac)3 into HNE(Ac)1 (87.3%) and HNE (7.2%) within

46 Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Neely et al.
a 2 h period relevant for this study. To avoid intracellular
formation of HNE(Ac)1, we attempted to synthesize 1,1-
bis(acetyloxy)-2(E)-nonene [HNE(Ac)2], a diester of HNE
which retains the hydroxyl group on the C4 position of
the aldehyde and would therefore be hydrolyzed solely
into HNE. However, our attempts to synthesize HNE-
(Ac)2 were not successful. HNE(Ac)1 differs from HNE
solely by having an acetyloxy- instead of a hydroxyl-group
on the C4 position. HNE(Ac)1 and HNE are both R,â-
unsaturated aldehydes and therefore are electrophilic
molecules with comparable reactivity toward nucleophilic
side chains.
ing plasma membrane, cytoplasm, and nucleus. Using an
antibody specific for HNE-adducted proteins, we have
presented evidence that the degree of protein modifica-
tion in at least one subcellular compartment, the nucleus,
is different in cells exposed to exogenously added HNE
and cells producing endogenous HNE. In addition, the
pattern of HNE-adducted proteins although similar, was
not identical in Neuro 2A cells exposed to HNE and HNE-
(Ac)3. This new tool will allow us to focus on the relevant
targets and cellular functions affected by HNE, one of
the major and most reactive products formed during lipid
peroxidation (17).
While performing immunoblot and immunocytochemi-
cal analysis using an antibody specific for HNE-protein
adducts, we observed that the signal resulting from
protein modifications of Neuro 2A cells exposed to HNE-
(Ac)3 was substantially less intense than the one observed
in cells exposed to HNE. This is likely a result of the
specificity of the antibodies used and not due to a
difference in reactivity of HNE(Ac)1 and HNE. The anti-
HNE-protein adduct antibodies used require a 4-hy-
droxyl group on the C4 position of the aldehyde (32, 36-
38). These antibodies, therefore, only recognize HNE-
adducted proteins, and adducts formed by HNE(Ac)1 are
not likely to be recognized efficiently on immunoblots or
by immunocytochemistry. This explanation is substanti-
ated by our observations that (1) the difference in protein-
associated radioactivity is less than 2-fold in cells exposed
to [3H]HNE(Ac)3 (1.4%) and cells exposed to [3H]HNE
(2.2%) and (2) HNE(Ac)3 is only two times less efficient
than HNE in disrupting cellular microtubules.
The proteins modified in Neuro 2A cells exposed to
HNE or HNE(Ac)3 were similar, but not identical. At
least two proteins were preferentially modified by HNE,
and one was preferentially modified by HNE(Ac)3. As
discussed above, the proteins revealed on the immuno-
blots represent only HNE-adducted proteins, while the
proteins on the autoradiographs reveal HNE- and HNE-
(Ac)1-adducted proteins. The nature of these proteins and
the relevance of their modification to biological effects
are not known at present. A similar observation was
made by Keller and colleagues who found that the
pattern of HNE-modified proteins was different in each
of three synaptosome preparations exposed to externally
applied HNE, FeSO4, or Aâ (35). In addition, using these
antibodies specific for HNE-adducted proteins for immu-
nocytochemistry, we observed more nuclear staining in
Neuro 2A cells exposed to HNE(Ac)3 than in cells treated
with HNE. These observations support the hypothesis
that the compartmentalization of proteins in relation to
the site of HNE generation may be important with
respect to the protein species that are modified. Other
investigators have demonstrated that exogenously added
HNE reacts first with plasma membrane proteins (39,
40), whereas HNE formed endogenously in cells or
organelles exposed to oxidative stress is present in high
concentrations in intracellular membranes (21, 24).
In conclusion, in this study we developed a tool that
allows us to form intracellularly HNE. We, therefore,
have for the first time the possibility to observe the effects
of intracellularly formed HNE in the absence of the
plethora of other products and processes that occur in
cells undergoing oxidative stress. This molecule allows
us to test the appropriateness of extracellular HNE
exposure to study effects of this aldehyde on functions
associated with different cellular compartments, includ-
Ack n ow led gm en t. This work was supported by NIH
Grants AG00774, ES10196, and AG16835 to Dr. Thomas
J . Montine.
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