Trans-4-hydroxy-2-hexenal is a neurotoxic product ofdocosahexaenoic (22:6; n-3) acid oxidation

Eric K. Long,* Tonya C. Murphy,* Laura J. Leiphon,* John Watt,� Jason D. Morrow,�Ginger L. Milne,� Jocelyn R. H. Howard� and Matthew J. Picklo Sr*,§

*Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, Grand Forks, North Dakota, USA

�Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, North Dakota, USA

�Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

§Department of Chemistry, University of North Dakota, Grand Forks, North Dakota, USA

Owing to the enrichment of polyunsaturated fatty acids(PUFA’s) in the CNS, lipid peroxidation is of particularimportance. The formation of lipid peroxidation products iselevated in multiple neurodegenerative diseases (e.g. Alz-heimer’s disease, Parkinson’s disease) and following acuteinjury (Yoritaka et al. 1996; Sayre et al. 1997; Reich et al.2001; Picklo et al. 2002). Peroxidation of PUFA’s leads tothe formation of cytotoxic lipid–aldehyde species (Esterbaueret al. 1990). Of these, 4-hydroxy-trans-2-nonenal (HNE),derived from the peroxidation of linoleic acid (18:2; n-6) andarachidonic acid (20:4; n-6) has gained the most attention asa neurotoxin (Keller et al. 1997, 1999; Kruman et al. 1997;Picklo et al. 2002).

Less appreciated, however, is that docosahexaenoic acid(DHA; 22:6; n-3) undergoes significant peroxidation in thebrain. DHA content in many brain regions is 30–50% higherthan that of arachidonic acid (Pawlosky et al. 2001; Salemet al. 2001; Lim et al. 2005). Elevated levels of the DHA-derived lipid peroxidation products F4-neuroprostanes

(F4-NPs) are observed in diseased regions of brain inpatients with Alzheimer’s disease, animals treated withkainic acid, and animals undergoing ethanol withdrawal(Reich et al. 2001; Montine et al. 2002; Musiek et al. 2004;Milne et al. 2006). These data suggest that DHA-derived

Received October 26, 2007; revised manuscript received November 26,2007; accepted November 26, 2007.Address correspondence and reprint requests to Matthew J. Picklo, Sr,

PhD, Department of Pharmacology, Physiology, and Therapeutics,University of North Dakota School of Medicine, 501 N, Columbia Rd,Grand Forks, ND 58203, USA. E-mail: mpicklo@medicine.nodak.eduAbbreviations used: ALDH5A, aldehyde dehydrogenase class 5A;

BSA, bovine serum albumin; CM-H2DCFDA, 5-(and-6)-chloro-methyl-2¢,7¢-dichlorodihydrofluorescein diacetate, acetyl ester; DHA,docosahexaenoic acid; F4-NP, F4-neuroprostane; HHE, trans-4-hydroxy-2-hexenal; HNE, trans-4-hydroxy-2-nonenal; KLH, keyhole limpethemocyanin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NOH, hydroxylamine; PBS, phosphate-buffered saline; PUFA,polyunsaturated fatty acid; ROS, reactive oxygen species; TBS, Tris-buffered saline; TBST, Tris-buffered saline Tween.

Abstract

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA)

is elevated in the CNS in patients with Alzheimer’s disease

and in animal models of seizure and ethanol withdrawal. One

product of DHA oxidation is trans-4-hydroxy-2-hexenal (HHE),

a six carbon analog of the n-6 fatty acid derived trans-4-

hydroxy-2-nonenal (HNE). In this work, we studied the

neurotoxic potential of HHE. HHE and HNE were toxic to

primary cultures of cerebral cortical neurons with LD50’s of

23 and 18 lmol/L, respectively. Toxicity was prevented by the

addition of thiol scavengers. HHE and HNE depleted neuronal

GSH content identically with depletion observed with

10 lmol/L of either compound. Using an antibody raised

against HHE–protein adducts, we show that HHE modified

specific proteins of 75, 50, and 45 kDa in concentration- and

time-dependent manners. The time-dependent formation of

HHE differed from that of F4-neuroprostanes following in vitro

DHA oxidation likely as a result of the different oxidation

pathways involved. Using purified mitochondrial aldehyde

dehydrogenase ALDH5A, we found that HHE was oxidized

6.5-fold less efficiently than HNE. Our data demonstrate that

HHE and HNE have similarities but also differences in their

neurotoxic mechanisms and metabolism.

Keywords: 4-hydroxyhexenal, 4-hydroxynonenal, docosa-

hexaenoic acid, glutathione, lipid peroxidation, neuropros-

tanes.

J. Neurochem. (2008) 105, 714–724.

d JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 714–724 doi: 10.1111/j.1471-4159.2007.05175.x

714 Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

lipid–aldehyde species may play a pathological role in CNSdisorders with significant components of oxidative damage.However, the toxicity of DHA-derived lipid–aldehydes inCNS systems is not well characterized.

Multiple aldehydes are derived from DHA peroxidation.Acrolein, neuroketals, 4-hydroxy-trans-2-cis-6-nonadienal,and 4-hydroxy-trans-2-hexenal (HHE) have been character-ized as DHA peroxidation products (Van Kuijk et al. 1990;Bernoud-Hubac et al. 2001; Kawai et al. 2007). Acrolein,also derived from arachidonate and linoleate, is highly toxicto neurons and levels of acrolein are elevated in Alzheimer’sdisease (Lovell et al. 2000, 2001). On the other hand, HHE(Fig. 1a) is reported to be a major product of DHAperoxidation in vitro (Van Kuijk et al. 1990). AlthoughHHE may be derived from other n-3 PUFA’s, in the brain,DHA is present at greater than 30-fold higher amounts thanall other n-3 PUFA’s combined (Pawlosky et al. 2001;Salem et al. 2001; Lim et al. 2005).

Trans-4-hydroxy-2-hexenal has biological activity andsignaling properties (Je et al. 2004; Lee et al. 2004a,b;Choudhary et al. 2005; Lee and Wander 2005). HHE is apotent inducer of permeability transition in hepatic mito-chondria but not mitochondria isolated from brain (Kristalet al. 1996). In relation to neurodegenerative disease,elevated levels of HHE–histidine adducts are found inpatients with amyotrophic lateral sclerosis (Shibata et al.2004). In the present study, we tested the neurotoxicpotential of HHE in primary cultures of cerebral corticalneurons by examining multiple endpoints. Our datademonstrate that HHE and the n-6-derived HNE havesimilar LD50’s and both deplete GSH with similar potency.Through use of an anti-HHE–protein adduct antibody, wedemonstrate that multiple proteins are targets of HHEmodification and that adducts are localized to the nuclear/perinuclear region of neurons treated with HHE. Our dataindicate that the kinetics of HHE formation differ fromthat of F4-NP formation.

Materials and methods

ChemicalsTrans-4-hydroxy-2-hexenal was obtained from Cayman Chemical

(Ann Arbor, MI, USA). Purity and content of HHE was checked by

Fig. 1 Trans-4-hydroxy-2-hexenal (HHE) is toxic to primary cortical

neurons. (a) Structures of HHE, trans-4-hydroxy-2-nonenal (HNE),

and trans-2-hexenal. (b) Cultures of primary rat cortical neurons were

exposed to increasing concentrations of HHE, HNE, or trans-2-hexe-

nal for 24 h followed by determination of viability by the 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Note that HNE and HHE have a similar cytotoxic profiles whereas

trans-2-hexenal was less toxic. (c) Thiol scavengers protect against

HHE toxicity. Cortical neurons were exposed to HHE (50 lmol/L) in

the presence of potential cytoprotectants for 24 h followed by deter-

mination of cell viability by the MTT assay. Control cells received dil-

uent alone. For (c) data were compared using a one-way ANOVA with

Bonferroni’s post-test comparing protectants versus HHE alone.

*p < 0.05. For all experiments, data presented are the mean ± SD

(n = 3) for experiments performed on three separate days in triplicate.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724

HHE is a neurotoxin | 715

HPLC following purchase. HNE was synthesized in our laboratory

as previously described (Honzatko et al. 2005). NAD+, GSH, GSH-

ethyl ester, and trans-2-hexenal were purchased from Sigma-Aldrich

(St Louis, MO, USA). 5-(and-6)-chloromethyl-2¢,7¢-dichlorodi-hydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was

purchased from Invitrogen (Grand Island, NY, USA). DHA was

purchased from Nu-Chek-Prep, Inc. (Elysian, MN, USA). Hydrox-

ylamine (NOH) hydrochloride was purchased from Acros Organics

(Geel, Belgium). Bovine serum albumin (Fraction V, BSA) was

purchased from Fisher Scientific (Pittsburgh, PA, USA). Purified

recombinant human a-synuclein was the kind gift of Dr Julia M.

George (University of Illinois, Urbana, IL, USA) (Perrin et al. 2000,2001).

Neuron preparationExperimental protocols were in accordance with the NIH guidelines

for the use of live animals and were approved by the University of

North Dakota Institutional Animal Care and Use Committee. Timed-

pregnant rats (E18) were anesthetized with an i.p. injection of

ketamine (100 mg/kg + 0.1 mL) and xylazine (13 mg/kg) followed

by cervical dislocation. Rat pups were harvested and placed in ice-

cold dissecting media [0.002% of 0.5 mmol/L EDTA, 0.01% of

0.1 mmol/L EGTA, 1 mg/mL glucose in phosphate-buffered saline

(PBS), pH 7.4]. Brains were removed and placed in ice-cold

dissecting media. The cerebral cortex was dissected and the

meninges removed. Cortical pieces were minced in ice-cold

dissecting media and incubated in pre-warmed (37�C) 0.25%

trypsin for 15–20 min at 37�C. Trypsin was inactivated with fetal

bovine serum, and the tissue was added to 10 mL of plating media

(Neurobasal media, 10% L-glutamine, 1% penicillin/streptomycin,

10% B-27 supplement) and triturated to break up clumps. Neurons

were counted and plated on poly-d-lysine coated plates at a density

of 30 000 cells/cm2. Neurons were used on days 7–10. Immuno-

chemical staining of cells with anti-bIII tubulin (Sigma) demon-

strated that the cultures were 90–95% neurons. Neurons were grown

in a humidified incubator at 37�C under normal atmosphere

maintained with a final concentration of 5% CO2. Under these

conditions, O2 content is approximately 20%.

MTT activityCell viability was determined by the cellular reduction of 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its

formazan product (Mosmann 1983). For protection studies, cells

were briefly rinsed with PBS before addition of MTT to remove

thiol protectants from the media that interfered with the MTT assay.

Culture medium containing 5 mg/10 mL MTT was added to each

well. Cells were incubated for 45–60 min at 37�C. The MTT

solution was carefully aspirated and 1 N HCl : isopropanol (4 : 96

v/v) was added. The plate was placed on a rocking shaker for

30 min. The absorbance of the dissolved formazan reduction

product was quantified at 570 nm using a SpectraMax 384Plus

UV/VIS plate reader (Molecular Devices, Sunnyvale, CA, USA).

GSH depletionNeurons were exposed to HHE or HNE at different concentrations

and time for characterization of GSH depletion. GSH content was

measured according to a method described by Dringen et al. (1999)with slight modifications. Following exposure, neurons were rinsed

with 1 mL PBS, lysed in 600 lL of 1% sulfosalicylic acid, scraped

from the plate and centrifuged 13 600 g, 15 min, 4�C. The

supernatant was used for GSH determination and pellets were

retained for protein quantification. GSH standards were prepared in

the same buffer as samples (1% sulfosalicylic acid); 10 lL of

supernatant or GSH standard was added to 90 lL of water in a 96-

well plate; 100 lL of reaction mixture (0.3 mmol/L 5,5¢-dithiobis-(2-nitrobenzoic acid), 1.0 mmol/L EDTA, 0.4 mmol/L NADPH,

1 U/mL GSH reductase in 0.1 mol/L sodium phosphate buffer, pH

7.5) was added. Samples were mixed and read immediately using a

SpectraMax 384Plus UV/VIS plate reader (Molecular Devices) at

405 nm for 2.5 min with readings at 15 s intervals. For protein

quantification, pellets were allowed to dry briefly followed by the

addition of 200 lL of 0.5 mol/L NaOH. The pellet was vortexed to

resuspend the protein. Protein content was determined using Protein

Assay Reagent (BioRad, Inc; Hercules, CA, USA).

Measurement of neuronal ROSNeurons were pre-treated with 7 lmol/L CM-H2DCFDA (Chi et al.2007). After 30 min, HHE (10, 20, 35, and 50 lmol/L) or H2O2

(250 lmol/L) as a positive control was added. Neurons were

incubated for an additional 3 h at 37�C. Fluorescence was measured

using SpectraMax Gemini XS plate fluorimeter (Molecular Devices)

with an excitation of 485 nm, emission of 540 nm and a cutoff of

515 nm. All procedures were performed under dim lighting to

preserve fluorescent signal.

Oxidation of DHADocosahexaenoic acid was oxidized using the method developed by

Kawai et al. (2006). DHA (10 mmol/L) was oxidized at 37�C using

50 lmol/L ammonium iron(II) sulfate hexahydrate and 1 mmol/L

ascorbic acid in sodium phosphate buffer at pH 7.4 and 6.5. Aliquots

(2 mL) were taken at 0, 2, and 6 h and added to 200 lL 10% 2,6-di-

tert-butyl-4-methylphenol (w/v). Samples were immediately deriv-

atized with pentafluorobenzyl NOH (see below) for subsequent

analysis.

Gas chromatographic-mass spectrometric analysis of HHEDocosahexaenoic acid oxidation samples and standards were

derivatized for 1 h by adding an equal amount of 50 mmol/L

O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (Alfa Aesar, Ward-

hill, MA, USA) in 50 mmol/L sodium acetate buffer, pH 5.0 (van

Kuijk et al. 1986). Benzaldehyde (50 lmol/L) was added as an

internal standard prior to derivatization. The pH of the final reaction

was approximately 6.0. After 1 h, the reaction was acidified using

three drops of concentrated sulfuric acid to precipitate excess

O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine. Samples were

extracted twice into methylene chloride, dried over sodium sulfate,

evaporated under nitrogen and raised in the appropriate volume of

methylene chloride. Samples and standards were placed in auto-

sampler vial inserts. The trimethylsilyl derivative of the 4-hydroxy

group was formed by diluting the sample twofold in N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (99 : 1;

Supelco-Sigma) and allowed to derivatize for 12 h at 22�C.For the analysis of HHE content, gas chromatography with

negative-ion chemical ionization mass spectrometry was used. The

gas chromatographic-mass spectrometric system consisted of a

Trace GC, a PolarisQ ion trap mass spectrometer, and an AS3000

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

716 | E. K. Long et al.

autosampler (Thermo-Finnigan Corp.). All analyses employed a

1 lL splitless injection. The initial temperature was held at 50�Cfor 1 min followed by a 20�C per minute increase to 320�C, whichwas held for 10 min. Total ion current was measured from 6 to

24 min. Methane was used as a reagent gas at a flow rate of 1.5 mL/

min. HHE was quantified using the characteristic m/z 241 fragment

and benzaldehyde was quantified using the characteristic m/z 281

fragment.

HPLC analysis of HNE and HHEWhen appropriate, HNE and HHE content were determined by

HPLC using a Shimadzu HPLC system, an ODS2 (4.6 mm ·250 mm) column and 220 nm detection (Murphy et al. 2003a,b).The solvent consisted of 30% acetonitrile : 70% water (v/v) in

isocratic mode.

Analysis of F4-neuroprostanes

F4-neuroprostane content was quantified using gas chromatography

with negative-ion chemical ionization mass spectrometry with

selected ion monitoring utilizing deuterated 17-F4-NeuroP

(1.19 ng/sample) as the internal standard (Musiek et al. 2004).

Production of anti-HHE–protein adduct antiserumMariculture keyhole limpet hemocyanin (KLH; Pierce Chemical,

Rockford, IL, USA) was diluted with water to 10 mg/mL and

dialyzed versus 0.1 mol/L sodium phosphate (pH 8.0) with

0.15 mol/L NaCl overnight at 4�C. HHE was added at a final

concentration of 13 mmol/L (1.3 lmol/mg KLH) and incubated for

6 h at 37�C. After 6 h an aliquot was removed for determination of

protein-bound carbonyls using dinitrophenylhydrazine (Levine

et al. 1990). Protein-bound carbonyl content following HHE

incubation was 0.43 lmol/mg KLH. To the remainder of the

reaction, NOHÆHCl (Acros Organics) was added to a final

concentration of 20 mmol/L and incubated at 37�C for 30 min to

derivatize any free aldehydes of HHE–protein Michael adducts to an

oxime. This process prevents cyclic hemiacetal formation bet-

ween the 4-hydroxy group of HHE and the aldehyde. NaBH4

(100 mmol/L final concentration) was then added to reduce any

remaining HHE and potentially to reduce oximes and imines and the

reaction was incubated 1 h at 37�C. NOH and NaBH4 were removed

by dialysis twice versus a 1000-fold excess of 0.1 mol/L sodium

phosphate (pH 7.2) with 0.15 mol/L NaCl. The HHE–KLH

derivative was sent to Chemicon, Inc. (Temecula, CA, USA) for

immunization of three rabbits to produce polyclonal rabbit antisera.

Resulting antisera were screened by immunoblot and ELISA for

specificity (see specific figure legends).

Immunodetection of HHE–protein and HNE-protein adductsFor immunoblot analysis of HHE– or HNE–protein adducts,

neurons were plated in either 25 or 75 cm2 plates. Neurons were

exposed to HHE or HNE as described in the specific figure legends.

After incubation, the medium was aspirated and the neurons washed

with cold PBS and harvested in 3 mL of PBS. For detection of

HHE–modified proteins, cells were pelleted by centrifugation, and

the resulting pellet was resuspended in 0.5 mL of ice-cold buffer

(20 mmol/L sodium phosphate, 1 mmol/L diethylenetriaminepenta-

acetic acid, 50 lmol/L 2,6-di-tert-butyl-4-methylphenol, 0.5% v/v

Triton X-100, 2 mmol/L EDTA) with 25 mmol/L NOH and

mammalian protease inhibitor cocktail (Sigma), according to

manufacturers instructions. The lysate was sonicated for three, ten

second bursts with a Sonic Dismembrator Model 100 (Fisher

Scientific) at power level 3. The samples were then incubated on ice

for 30 min for NOH derivatization of free aldehydes. For detection

of HNE-modified proteins, lysis buffer without NOH was used and

following sonication the samples were incubated with a final

concentration of 10 mmol/L NaBH4 (Neely et al. 1999).Proteins were separated by sodium dodecyl sulfate–polyacryl-

amide gel electrophoresis using 10% polyacrylamide gels and

transferred to an Immobilon Transfer Membrane (Millipore, Inc.,

Bedford, MA, USA) for 55 min at 90 V. The blot was blocked with

3% (w/v) non-fat dry milk in Tris-buffered saline (TBS) for 2 h at

22�C. Blots were incubated overnight at 4�C with either anti-HHE

antiserum (1 : 4000) or anti-HNE antibody (1 : 2000; Calbiochem,

San Diego, CA, USA) in blocking solution containing 0.2% Tween

20 (TBST). The rabbit anti-HNE–protein adduct antibody recog-

nizes adducts reduced with NaBH4. The blot was washed four times

at 10 min each with TBST and incubated with a goat anti-rabbit

conjugated to horse radish peroxidase (1 : 5000; Promega, Madison,

WI, USA) for 2 h at 22�C. After washing three times at 10 min each

with TBST and three times for 10 min each with TBS, the blot was

developed using Pierce Detection Reagent and Pierce CL-Xposure

film (Pierce Biotechnology, Inc.).

Immunolocalization of anti-HHE protein adducts was performed

on neurons plated onto poly-D-lysine coated coverslips. Neurons

were plated and used as described above except that they were plated

onto poly-D-lysine coated coverslips set into six-well plates. Neurons

were treated for 6 h with or without HHE followed by washing with

PBS and fixation for 25 min at 22�C with 4% (v/v) formaldehyde

diluted in PBS. Following fixation the coverslips were washed with

PBS three times. For NOH derivatization, coverslips were treated

with 25 mmol/L NOH in 0.1 mol/L sodium phosphate (pH 7.4) for

30 min at 22�C. Following derivatization, coverslips were rinsed

three times with TBS. Permeabilization and quenching of endo-

genous horseradish peroxidase activity was accomplished by

incubating coverslips with 0.1% Triton X-100/0.3% H2O2 in TBS

for 30 min at 22�C. Following three rinses with TBS, coverslips

were blocked with 3% non-fat dry milk (w/v) in TBS for 2 h at 22�C.Primary anti-HHE–protein adduct antiserum (R68) was diluted

1 : 2000 in 3% non-fat dry milk (w/v) in TBST and incubated 4�Covernight. Coverslips were rinsed three times with TBST, incubated

for 2 h at 22�C with goat anti-rabbit conjugated to horse radish

peroxidase (1 : 5000; Promega) diluted in 3% non-fat dry milk (w/v)

in TBST. Coverslips were washed three times with TBST. Coverslips

were developed using the DAB peroxidase substrate kit (Vector

Laboratories, Inc., Burlingame, CA, USA) and placed onto slides

using Vectashield Hardset mounting medium with DAPI (Vector

Laboratories, Inc.). Following immunocytochemical preparation, all

slides were viewed and photographed using an Olympus BX-51

fluorescent microscope with attached Olympus DP-71 color camera

with dedicated software. Areas of interest were digitized and

prepared for page-reproduction using Adobe Photoshop (v.6.0).

Preparation and activity of recombinant rat aldehydedehydrogenase class 5ARecombinant rat aldehyde dehydrogenase class 5A (ALDH5A)

possessing an N-terminal poly-histidine tag was prepared as

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724

HHE is a neurotoxin | 717

described previously (Brichac et al. 2007). Assays were performed

in sodium phosphate buffer (40 mmol/L, pH 7.4) at 37�C with

1 mmol/L NAD+ as cofactor. ALDH activity was measured as the

increase in NADH formation at A340 nm following the addition of the

aldehyde substrate.

Statistical analysesData analysis was performed using one-way and two-way ANOVA as

appropriate with Prism 4.0 software (GraphPad, Inc., San Diego,

CA, USA). Data are presented as the mean ± SD.

Results

Our initial experiments were designed to test the neurotoxicpotential of HHE. For comparison, we also tested the toxicityof the nine-carbon HNE and the non-hydroxylated analog,trans-2-hexenal (Fig. 1a). As shown in Fig. 1, HHE andHNE had similar LD50’s of 23 lmol/L for HHE and18 lmol/L for HNE (Fig. 1b). trans-2-Hexenal was lesspotent than HHE and HNE indicating that the C4-hydroxylmoiety enhances a,b-unsaturated aldehyde toxicity. Protec-tion against HHE toxicity was effected by co-incubation withthe thiol-based scavengers N-acetyl cysteine, GSH, and GSHethyl ester (Fig. 1c). The antioxidant a-tocopherol was noteffective, suggesting that secondary, toxic lipid peroxidationevents were not occurring.

Glutathione depletion is a common toxic effector of a,b-unsaturated aldehydes (Neely et al. 2005; Arakawa et al.2006; Malone and Hernandez 2007). HHE significantlydepleted GSH content in a concentration-dependent manner(Fig. 2a). A similar concentration-dependent GSH depletionwas observed for HNE. The data in Fig. 2(b and c) show thatdepletion of GSH occurred within 15 min of HHE (20 lmol/L) exposure. While our GSH detection method monitorsGSH as well as GSSG, likely, HHE reacts only with GSH.This suggests that the amount of free GSH depletion weobserved is an underestimation in the loss of the totalneuronal GSH pool.

Because HHE depleted GSH levels, we predicted thatHHE would cause subsequent accumulation of reactiveoxygen species (ROS). We tested this hypothesis throughmeasuring the oxidation of the ROS-sensitive dye, CM-H2DCFDA (Chi et al. 2007). Exposure of neurons toincreasing concentrations of HHE led to an increase inROS content (Fig. 3). However, significant increases in DCFfluorescence were not observed until concentrations of HHEexceeded levels needed to deplete GSH content.

Our next series of experiments were designed to studythe formation of HHE–protein adducts. Since the proteinadducts of a,b-unsaturated aldehydes are immunogenic, wedeveloped antibodies against HHE–protein adducts. Immu-nogen was prepared by incubating KLH with HHE. Toincrease the specificity of the antibodies, we incubated theresulting HHE–KLH with NOH such that the aldehyde

component of any HHE–Michael adducts would beconverted to an oxime derivative. The antiserum fromone rabbit (R68) recognized HHE–protein adducts thatneeded to be derivatized with NOH but not NaBH4

(Fig. 4a) indicating that the antiserum recognized HHE–protein Michael adducts converted to the oxime derivativewith NOH (Fig. 4b). The R68 antiserum was specific forHHE–protein adducts but not those against HNE or

Fig. 2 Trans-4-hydroxy-2-hexenal (HHE) exposure depletes neuronal

GSH. (a) Cortical neurons were treated with increasing concentrations

of HHE or trans-4-hydroxy-2-nonenal (HNE) for 6 h before washing

and harvesting for GSH content. (b and c) Measurement of GSH loss

at increasing timepoints from 15 min to 6 h demonstrate that HHE

(20 lmol/L) caused rapid depletion of neuronal GSH. Neurons were

treated with HHE (20 lmol/L) and were harvested at increasing times

for determination of GSH content. Data presented are the mean ± SD

(n = 3) for three separate experiments.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

718 | E. K. Long et al.

trans-2-hexenal modified protein as determined by ELISAand immunoblot (Fig. 4c and d). The antiserum from onerabbit (R67) was reactive towards HHE-modified andHNE–modified proteins derivatized with NOH (not shown).As shown by ELISA using primary neurons treated withHHE, the NOH derivatization step was necessary forantibody recognition in whole cells (Fig. 4e).

The R68 antiserum was used to determine the localizationof HHE–protein adducts following HHE treatment. Neuronswere treated with HHE (0, 20, and 50 lmol/L) for six hoursfollowed by washing and fixing. For most but not allneurons, staining was heaviest in the nuclear/perinuclear area(Fig. 5a–c). For comparison, neurons were also stained forbIII tubulin as a cytoplasmic protein (Fig. 5d). No staining oronly faint immunoreactivity was observed in cells receivingHHE but not derivatized with NOH (Fig. 5e) and controlcells without HHE treatment (Fig. 5f). This localization isdifferent from the cytoplasmic localization noted for HNE–protein adducts in diseased regions of brain of patients withAlzheimer’s disease (Montine et al. 1997, 1998).

We then used the R68 antiserum to assess the multi-plicity of HHE–modified proteins accruing after HHEexposure and the time-course of adduct formation. Neuronswere exposed for 6 h to increasing concentrations of HHE(0–50 lmol/L). Immunoblot analysis of the neuron lysatesshows that HHE adducts accumulated in a dose-dependentmanner and that specific proteins of approximately 75, 50,and 45 kDa were targeted (Fig. 6a). A clear elevation inHHE–protein adduct formation was noted after exposure to5 lmol/L HHE. To define a time course of adductformation, neurons were exposed to 20 lmol/L HHEfollowed by harvesting at increasing timepoints. Immuno-

blot analysis demonstrated that these proteins were adductedfollowing a 30 min exposure to 20 lmol/L HHE (Fig. 6b).

Although HNE and HHE had similar cytotoxic and GSHdepletion characteristics, it is possible that HHE and HNEpossess different cellular protein adduction targets or differ-ent reactivity towards the same target. To test this possibility,neurons were exposed to either HNE or HHE (20 lmol/L)for 6 h followed by harvesting for immunoblot analysis. Thedata in Fig. 7a show that although similar sized proteins wereadducted by HHE and HNE, the apparent amounts of thoseproteins varied, suggesting that there exist different reactiv-ities towards the same proteins. The extent that HNE and

(a) (b)

(c)

(d) (e)

Fig. 4 Characterization of anti-trans-4-hydroxy-2-hexenal (HHE)–

protein adduct antiserum. (a) The antiserum (R68) recognizes HHE–

protein adducts derivatized with hydroxylamine (NOH). Following

binding of the bovine serum albumin (BSA) or HHE–modified BSA to

the ELISA plate selected wells were treated with NOH or NOH fol-

lowed by NaBH4. The data in (a) indicate that the antiserum recog-

nized an HHE–Michael protein adduct derivatized to an oxime with

NOH as drawn in (b). (c) The R68 antiserum has high specificity for

BSA–HHE adducts by immunoblot compared with HNE–BSA or trans-

2-hexenal–BSA adducts. Proteins were treated with NOH prior to

electrophoresis; 20 ng of each modified protein was loaded in their

respective lanes. (d) The R68 antiserum recognizes the HHE–BSA

adducts by ELISA versus other aldehyde derivatives. Samples were

derivatized with NOH prior to blocking. (e) The R68 antibody recog-

nizes HHE adducts on fixed neurons treated with HHE. Note that NOH

treatment was necessary for antibody recognition. Neurons were

treated with HHE (50 lmol/L) for 6 h, washed with PBS, and fixed with

formalin. Wells were treated with or without NOH prior to incubation

with the R68 antibody.

Fig. 3 Trans-4-hydroxy-2-hexenal (HHE) exposure elevates neuronal

reactive oxygen species (ROS) production. Neurons were pre-incu-

bated for 30 min with the ROS sensitive dye DCFDA (7 lmol/L)

followed by exposure to HHE at increasing concentrations for 3 h.

Exposure to hydrogen peroxide (250 lmol/L) increased DCF fluores-

cence by 284 ± 65% above control (not shown). Data are the

mean ± SD (n = 6) for experiments performed on two separate days

in triplicate. Data were compared using a one-way ANOVA with Bon-

ferroni’s post-test comparing cells receiving HHE versus control

(0 lmol/L HHE) cells, *p < 0.05.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724

HHE is a neurotoxin | 719

HHE had different reactivities with the same protein wasassessed by incubating HNE or HHE (50 lmol/L individu-ally) with BSA (50 lmol/L) or recombinant humana-synuclein (50 lmol/L) and measuring the loss of HHEand HNE as an indicator of protein alkylation. Because aprevious report demonstrated that HNE-modified a-synucleinwas neurotoxic a comparison of HHE and HNE modificationof a-synuclein was performed (Qin et al. 2007). HNEalkylated BSA at a faster rate than HHE; however wedetected no loss of either HNE or HHE when incubated witha-synuclein (Fig. 7b). The lack of HNE loss during incuba-tion with a-synuclein may be attributed in part to the 1 hincubation period we performed versus the overnight incu-bation described by Qin et al. (2007). There was no loss ofthese alkenals when BSA was omitted, indicating that non-specific loss did not occur.

To further clarify the disposition of HHE, we furtherexamined HHE formation resulting from DHA oxidation.HHE formation occurred in a time-dependent manner. Wetested the extent to which the amount of HHE generatedwas dependent upon the pH of the incubation using pH 7.4and 6.5 to mimic control and the acidotic condition thatoccurs in ischemia (Nemoto and Frinak 1981; Paschenet al. 1987; Hoffman et al. 1998). No significant differencein HHE generation was observed when comparing the pHof the reaction (Fig. 8a). As F4-NP formation results from

DHA oxidation, the same samples also were analyzed forF4-NP content. F4-NP formation increased with time;however, the marked increase in HHE content at 6 h wasnot mirrored by a large increase in F4-NP content(Fig. 8b). Similar to HHE formation, there was no effectof pH.

Mechanisms exist for the detoxification of 4-hydroxy-alkenals like HHE and HNE. In isolated brain mitochondria,detoxification of HNE is mostly through oxidation to trans-4-hydroxy-2-nonenoic acid by ALDH5A. We examinedwhether HHE was an efficient substrate for ALDH5Acompared with HNE (Fig. 9). HHE had a 13-fold higherKm (388 ± 40 lmol/L) than HNE (28.5 ± 2.6 lmol/L).However, the Vmax of ALDH5A for HHE was about twofoldhigher (3.52 ± 0.2 lmol/min/mg; kcat = 12.89 s)1) com-pared with HNE (1.7 ± 0.1 lmol/min/mg; kcat = 6.22 s)1).A comparison of kcat/Km shows that ALDH5A is 6.5-foldmore efficient towards utilization of HNE.

Discussion

Lipid peroxidation of n-3 PUFA’s occurs under basalconditions and is elevated in disease states. Multiple studieshave shown that CNS levels of the DHA oxidation productsF4-NP’s are elevated in Alzheimer’s disease, ethanol with-drawal, and inflammation. In this work, we examined the

(a) (b)

(c) (d)

(e) (f)

Fig. 5 Trans-4-hydroxy-2-hexenal (HHE)–

protein adducts are localized to the nuclear

region of neurons. Cultures were treated

with 0, 20, and 50 lmol/L HHE for 6 h prior

to washing and fixing with formalin. (a and

b) Neurons treated with HHE (20 lmol/L).

Following fixation coverslips were treated

with NOH. (a) is a low magnification of the

stained cultures with an increased magnifi-

cation shown in (b). Note the heavy locali-

zation to nuclei (N) versus cytoplasmic (c)

staining observed in cultures treated

with 50 lmol/L HHE in (c). (d) Untreated

neurons were stained for bIII-tubulin to

show cytoplasmic (c) staining with lighter

nuclei (N). Neurons in (e) were treated with

HHE (50 lmol/L) but not derivatized with

NOH and in (f) were not treated with HHE,

demonstrating the specificity of staining.

Figures shown were from the same cul-

tures, treated and stained in parallel and

under the same conditions. The scale bar

indicates 50 lm.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

720 | E. K. Long et al.

neurotoxic potential of trans-4-hydroxy-2-hexenal (HHE), aproduct of oxidative damage to DHA. Our results demon-strate that HHE was toxic to primary cultures of rat cerebralcortical neurons, depleted neuronal GSH levels and elevatedneuronal ROS content. HHE protein adduction occurred inconcentration- and time-dependent manner. Our resultsindicate that the kinetics of HHE formation from DHAoxidation differ from that of F4-NPs.

Most studies examining the neurotoxicity of aldehydesproduced from lipid peroxidation have focused on theproduct HNE, generated from the n-6 PUFA’s arachidonicacid and linoleic acid. Yet, comparisons of the disposition ofHNE and HHE in neuronal systems have not beenperformed. Our studies demonstrated no difference in thecytotoxic potency (LD50 for HNE of 18 lmol/L and LD50 forHHE of 23 lmol/L) as measured by loss of MTT activity orby GSH depletion. This is in contrast to the lower potency ofHHE (LD50 of 60 lmol/L) versus HNE (LD50 of 40 lmol/L)towards inducing cell death in ARPE-19 retinal pigmentedepithelial cells (Choudhary et al. 2005). Similar to ourresults, the LD50 of HHE for YPEN-1 rat prostate cells areapproximately 20 lmol/L (Lee et al. 2004b) . In both these

other cell types, apoptosis was evident (Lee et al. 2004b;Choudhary et al. 2005). Our cytotoxicity data with trans-2-hexenal indicate that the neurotoxic potential of HHE andHNE in this system is governed by the 4-hydroxyalkenalmotif independent of the two carbon and five carbon tails incontrast to the data obtained using ARPE-19 cells (Choudh-ary et al. 2005).

Similar to studies examining protection against HNE-mediated toxicity, thiol scavengers such as N-acetyl cysteine,GSH, and GSH analogs provided protection against HHEtoxicity (Kruman et al. 1997; Neely et al. 2000; Arakawaet al. 2006).

The parallel toxicity data of HHE andHNE are in contrast tothe difference between the activities of these aldehydes withknown detoxification systems. Previous data by Hubatschet al. (1998) found that that HHE is a poor substrate (31-foldless active) versus HNE for conjugation by glutathione-S-transferases A4-4. Our data show that HHE is a poorersubstrate (�6.5 fold) than HNE for the mitochondrialALDH5A. On the other hand, HHE and HNE are detoxified

(a)

(b)

Fig. 7 Trans-4-hydroxy-2-hexenal (HHE) has adduction characteris-

tics different than trans-4-hydroxy-2-nonenal (HNE). (a) Two indepen-

dent neuron cultures were treated with either HHE (20 lmol/L) or HNE

(20 lmol/L) for 6 h before harvesting for immunoblot detection of

protein adducts. The black arrows demonstrate protein bands of similar

masses. The gray arrow denotes a difference in immunoreactive

protein banding between the two treatments. (b) HHE and HNE show

protein-dependent adduction. HNE or HHE, 50 lmol/L each, were

incubated with bovine serum albumin (BSA) (50 lmol/L) or human

recombinant a-synuclein (a-Syn) in 50 mmol/L phosphate buffer at pH

7.4 for increasing times followed by determination of remaining HHE

or HNE as an indicator of protein adduction. Incubations were termi-

nated by the addition of 1/10 volume of 6% phosphoric acid.

HHE (µmol/L)

20 µmol/L HHETime (h)

0.5 1 3 6

0150

(a)

(b)

100755037

25

10

10075

50

37

25

10

5 10 20 50BSA-HHE

BSA-HHE

0 HHE

Fig. 6 Trans-4-hydroxy-2-hexenal (HHE) forms adducts with selected

neuronal proteins. (a) Adducts are formed in a concentration-depen-

dent manner. Neuronal cultures were treated with increasing con-

centrations of HHE for 6 h. Major immunoreactive bands are found at

approximately 75, 50, and 45 kDa. (b) Adduct formation occurs in a

time-dependent manner. Neurons were treated with 20 lmol/L HHE

and harvested at increasing times. Immunoreactive proteins of the

same mass were found in unexposed cells suggesting the presence of

HHE–protein adducts under basal conditions.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724

HHE is a neurotoxin | 721

by aldose reductase with similar efficiency (He et al. 1998).Thus, the two carbon tail of HHE has a significant effect uponits detoxification versus HNE.

Protein adduction is a major mechanism by which agentslike HHE, HNE, and acrolein can exert neurotoxicity.Although levels of HHE–protein adducts are elevated inamyotrophic lateral sclerosis (Shibata et al. 2004), our resultsare the first to demonstrate that HHE modifies specificproteins rather than modifying proteinaceous nucleophilicgroups non-discriminantly. Three major proteins weretargeted by HHE adduction in concentration- and time-dependent manners. Two proteins of approximately of 75 and45 kDa were primary targets at 5 lmol/L HHE and10 lmol/L HHE whereas a protein of approximately50 kDa became labeled more prominently at 20 lmol/LHHE. The finding that immunoreactive proteins of the samemasses were observed in untreated neurons, albeit lessintensely compared with HHE-treated neurons, suggests thatHHE–modified proteins exist within neurons under these

conditions. The selective modification of these proteins candepend upon multiple factors including the amount ofexpression of the protein and the interaction of HHE towardsone particular protein motif versus another. More detailedproteomic analysis is needed to clarify these issues.

Our experiments directly comparing HHE modification toHNE modification show that HNE modified three majorproteins of similar masses. Potentially these are the sameproteins although this needs to be confirmed using subsequentproteomic techniques. Comparison of banding intensity sug-gests that the HNE targeted these proteins in differing amountscompared with HHE. We cannot exclude however that theseproteins were adducted to the same extent possibly at differentsites or that different types of adducts (e.g.Michael adducts vs.Schiff bases) were formedwith differing or no reactivities withthe antibodies. These data, along with those demonstratingdivergent reactivities towards BSA and detoxification en-zymes, indicate that HHE has biochemical properties that areunique and others that are similar to HNE.

The mechanisms involving HHE formation from DHAhave not been explored. Based upon the mechanismsproposed for HNE, we propose that HHE formation is theresult of the extraction of the bis-allylic hydrogens of C18and C15 and subsequent peroxide formation at C20 and C16of DHA (Schneider et al. 2001, 2004). In vitro DHAoxidation studies indicate that the C20 and C16 hydroper-oxides combined are the most abundant hydroperoxidesformed (VanRollins and Murphy 1984). Whether the C20and C16 hydroperoxides of DHA are the most abundant onesformed in vivo under pathological or control conditions arenot known.

Fig. 8 Trans-4-hydroxy-2-hexenal (HHE) formation does not mirror

F4-neuroprostane (F4-NP) formation from docosahexaenoic acid

(DHA) oxidation. (a) DHA (10 mmol/L) was oxidized in the presence of

Fe2+ (50 lmol/L)/ascorbate (1 mmol/L) at either pH 6.5 or 7.4. Sam-

ples were taken at the timepoints shown and analyzed by gas chro-

matography with negative-ion chemical ionization mass spectrometry

(GC-NICI/MS) for HHE and F4-NP content. Data presented are the

mean ± SD, n = 3. Two-way ANOVA revealed no significant effect of pH

upon HHE or F4-NP production although the time of incubation was a

significant factor for both products.

Fig. 9 Trans-4-hydroxy-2-hexenal (HHE) is a less efficient substrate

for aldehyde dehydrogenase class 5A (ALDH5A) detoxification versus

trans-4-hydroxy-2-nonenal (HNE). HHE and HNE were compared as

substrates for recombinant rat ALDH5A. NADH formation (Abs

340 nm) was used as a measure of activity. Non-linear regression

analysis utilizing the Michaelis–Menten equation was used to deter-

mine Km and Vmax. Assays contained 3 lg of purified enzyme in a

0.3 mL final volume. The calculated Km’s were 388 ± 40 lmol/L for

HHE and 28.5 ± 2.6 lmol/L for HNE. The calculated Vmax’s were

3.52 ± 0.2 lmol/min/mg for HHE and 1.7 ± 0.1 lmol/min/mg for HNE.

Data are the mean ± SD for assays performed in triplicate.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

722 | E. K. Long et al.

Our data demonstrate differences in the rates of formation ofHHE versus F4-NPs. Oxidation of DHA produced similarlevels of HHE and F4-NPs after 2 h, but far greater amounts ofHHE than F4-NPs after 6 h. The increase in HHE from 2 to 6 his consistent with a transition from the initiation to thepropagation phase of DHA oxidation (van Kuijk et al. 1995).However, the amounts of F4-NP formed after 6 h are notconsistent with a transition to the propagation phase, as levelsdo not appear to have increased exponentially. The mecha-nisms underlying the differences in formation are complex. F4-NPs are only a fraction of the total class of NP that are formedfrom DHA oxidation (Roberts et al. 1998). Furthermore, F4-NPs result from reduction of the bicyclic endoperoxide whoserearrangement can result in alternative products such asneuroketals (Roberts et al. 1999; Bernoud-Hubac et al. 2001).In contrast, formation of HHE from DHA likely occurs in afashion similar to formation of HNE from n-6 fatty acids andwould not require multiple rearrangements (Schneider et al.2001, 2004). As their kinetics of formation vary significantly,HHE and F4-NPs cannot be used interchangeably as biomar-kers of the magnitude of oxidative damage to DHA.

In summary, our data indicate that HHE is a neurotoxicaldehyde generated from DHA oxidation. Further study isneeded to determine the extent to which HHE levels may beelevated in disease conditions with increased F4-NP concen-trations like Alzheimer’s disease (Reich et al. 2001). Thesecurrent data along with those examining the neurotoxicity ofacrolein and HNE indicate that lipid peroxidation spawns theformation of multiple neurotoxic species with similar anddivergent effects (Keller et al. 1997, 1999; Kruman et al.1997; Lovell et al. 2000).

Acknowledgements

The authors gratefully acknowledge NIH grants P20 RR17699-01

COBRE (MJP) from the NCRR, AA15145-01 (MJP) GM15431

(JDM), ES131215 (JDM), ES000267 (JDM), and DK48831 (JDM).

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Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 714–724� 2008 The Authors

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