Aldehydes release zinc from proteins. A pathway from
oxidative stress
⁄
lipid peroxidation to cellular functions
of zinc
Qiang Hao and Wolfgang Maret
Departments of Preventive Medicine & Community Health and Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA
The aldehyde group is the most reactive among the
functional groups of biomolecules. It is involved in
Schiff base formation in the chemistry of pyridoxal
phosphate-catalyzed reactions, and in vision photo-
receptors, where retinal reacts with the e-amino group
of a specific lysine in rhodopsin. There are many
sources of endogenous aldehydes. For instance, glycer-
aldehyde 3-phosphate is an intermediate in glycolysis.
Thiohemiacetal ⁄ thioester intermediates between glycer-
aldehyde 3-phosphate and the sulfhydryl group of the
active site cysteine are formed during turnover of
glyceraldehyde 3-phosphate dehydrogenase, demon-
strating that aldehydes also react with the sulfhydryl
group of cysteine. Several enzymes control the levels
of aldehydes by oxidation or reduction, thus avoiding
unspecific reactions of endogenous aldehydes and
detoxifying xenobiotic aldehydes. In many degenerat-
ive diseases, the concentrations of aldehydes increase,
and their reactivity becomes a liability. In diabetes,
for example, prolonged elevation of blood glucose, an
aldose, leads to nonenzymatic glycations such as the
addition of glucose to the a-amino groups of the
b-chains of hemoglobin [1]. In yet other glycation reac-
tions, a-hydroxy-aldehydes or oxy-aldehydes formed

both acetaldehyde and acrolein induce the expression of metallothionein
and modulate protein tyrosine phosphatase activity in a zinc-dependent
way. Since minute changes in the availability of cellular zinc have potent
effects, zinc release is a mechanism of amplification that may account for
many of the biological effects of aldehydes. The zinc-releasing activity of
aldehydes establishes relationships among cellular zinc, the functions of
endogenous and xenobiotic aldehydes, and redox stress, with implications
for pathobiochemical and toxicologic mechanisms.
Abbreviations
ADH, alcohol dehydrogenase; DNP, 2,4-dinitrophenyl; DTNB, 5,5¢-dithiobis-2-nitrobenzoic acid; 4-HNE, 4-hydroxynonenal; MCA,
(7-methoxycoumarin-4-yl)-acetyl; 4-MP, 4-methylpyrazole hydrochloride; MRE, metal response element; MT, metallothionein; MT2,
metallothionein isoform 2; MTF-1, metal response element-binding transcription factor-1; PAR, 4-(2-pyridylazo)-resorcinol; PTP, protein
tyrosine phosphatase; TCEP, tris(2-carboxyethyl)-phosphine; TPEN, N ,N,N¢,N¢-tetrakis(2-pyridylmethyl)-ethylenediamine.
4300 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS
acrolein [3,4]. Aldehydes from the environment can
exacerbate the burden of exposure. Endogenous alde-
hydes that increase during these and other episodes of
exposure include: formaldehyde, used as a preservative
but also found in cigarette smoke and burning veget-
ation; acrolein, found in cigarette smoke, herbicides,
and acrylics, and produced during fossil fuel combus-
tion, during petrochemical processing, and when over-
heating cooking oil; and methylglyoxal, a metabolite
formed during acetone detoxification [5,6]. Endogen-
ously generated or inhaled aldehydes are involved in
cardiovascular disease, atherosclerosis, vascular com-
plications of diabetes [7] and respiratory diseases [8].
Another prominent example is acetaldehyde, the meta-
bolic product of ethanol from alcoholic beverages.
Excess acetaldehyde can accumulate to levels of a few

reaction of MT with 10 lm ebelsen, which releases all
seven zinc ions from MT within 20 min [14]. At a con-
centration of 1 mm acrolein, all seven zinc ions are
released within 6 h.
The zinc-releasing activity of other aldehydes was
determined with the same assay (Fig. 2). Because some
aldehydes are much less reactive than acrolein, the
measurements were performed at aldehyde concentra-
tions of 1 mm (Fig. 2). Among the aldehydes tested,
acrolein is the most reactive aldehyde, followed by
butyraldehyde, propionaldehyde, acetaldehyde, benzal-
dehyde, and glyceraldehyde. 4-HNE releases only 5%
of zinc from MT, while malondialdehyde releases only
3%. At physiologic pH, malondialdehyde exists as the
enolate, which is much less reactive than its enol form
at acidic pH (b-hydroxyacrolein).
The following investigations focus on the effects of
acetaldehyde and acrolein because of the relevance of
these aldehydes for the biological effects of ingested
ethanol and lipid peroxidation, respectively.
In order to explore whether or not acetaldehyde
releases zinc from other zinc–sulfur coordination envi-
ronments, its reaction with the zinc enzyme yeast alco-
hol dehydrogenase (ADH) in the absence of coenzyme
was followed with the PAR assay. Acetaldehyde
(1 mm) also releases zinc from this enzyme (Fig. 3A,
line 2). Acrolein (1 mm) releases significantly more zinc
than acetaldehyde (Fig. 3A, line 3). The activity of the
enzyme is affected differently by the two aldehydes
Fig. 1. Acrolein releases zinc from metallothionein (MT). The

A thiol assay with 5,5¢-dithiobis-2-nitrobenzoic acid
(DTNB, Ellman’s reagent) was employed to explore
the reactions of MT2 with acetaldehyde (Fig. 4). When
the ratio between MT2 and DTNB is 1 : 200, the reac-
tion reaches a plateau after 2 h (Fig. 4, line 1), at
which point all of the 20 sulfhydryl groups in MT are
titrated with DTNB. Preincubation of MT2 with acet-
aldehyde for 30 min changes the sulfhydryl reactivity
of MT2 significantly. Only 67% of the thiols now
react, indicating that the remaining 33% are modified
with acetaldehyde and can no longer react with DTNB
(Fig. 4, line 2). Under these conditions, 2.1 zinc ions
are released from MT. The reaction of the apoprotein
thionein (1.2 lm) with DTNB (200 lm) is rapid and
complete in less than 10 min. Acetaldehyde (1 mm)
quenches the reactivity of the 20 thiols in thionein, as
the absorbance does not change when DTNB is added.
To determine whether or not acetaldehyde also reacts
directly with 2-nitro-5-theobenzoic acid, the product of
the reaction of DTNB with thiols, the excess of acetal-
dehyde in the above reaction mixture was removed
enzymatically with yeast ADH [1 unitÆmL
)1
(one unit
converts 1 micromole ethanol per min at pH 8.8,
25 °C)] and NADH (2 mm) before DTNB was added.
As virtually the same absorbance reading was recor-
ded, save for a small increase due to the sulfhydryls in
ADH, the experiment demonstrates that acetaldehyde
reacts directly with the sulfhydryl groups of MT and

(PAR) after 30 min. Ebselen (10 l
M) was used as a positive control
because it releases all seven zinc ions from MT within 20 min. Data
are presented as means ± SD of triplicate determinations.
Aldehydes and zinc metabolism Q. Hao and W. Maret
4302 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS
contribute to zinc release, the E-amino groups of
lysines in MT2 were carbamoylated with potassium
cyanate and the modified protein was assayed for zinc
release as described above. Acetaldehyde releases
almost the same amount of zinc from the modified
protein (90%), clearly indicating that the reaction of
lysines in MT with aldehydes has little, if any, effect
on zinc release and that the predominant mechanism
of zinc release is the modification of the cysteine lig-
ands of zinc.
Aldehydes increase the concentration of
available cellular zinc
Cultured human hepatocellular carcinoma (HepG2)
cells were used to examine whether or not aldehydes
release zinc intracellularly. HepG2 cells were incubated
with acetaldehyde (1 mm) or acrolein (10 lm) for
30 min, and Zinquin ester was added to introduce a
fluorescent chelating agent into the cell for measure-
ment of intracellular zinc. HepG2 cells without any
treatment have a fluorescence signal that corresponds
to 15.4% saturation of Zinquin with zinc (Fig. 5A).
Treatment of cells with acrolein (10 lm) increases the
saturation to 22%. Because the effect of acetaldehyde
(1 mm) on zinc saturation of Zinquin is small (17%),

6
) were treated
with 2 l
M disulfiram for 1 h to inhibit aldehyde dehydrogenases.
After addition of 5 m
M ethanol to the medium and incubation for
another hour, cells were collected and cellular zinc was measured
as described above. Data are presented as means ± SD of triplicate
determinations. Fluorescence changes are insignificant when etha-
nol is added to the cells. Disulfiram decreases the fluorescence
intensity slightly (see text). The asterisk indicates significance at
P < 0.05.
Q. Hao and W. Maret Aldehydes and zinc metabolism
FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4303
Aldehydes induce expression of metallothionein
in HepG2 cells
A cadmium-binding assay was used to examine the
expression levels of MT in HepG2 cells after aldehyde
treatment. The experiment is based on the hypothesis
that released zinc induces the expression of MT.
The MT concentration in control HepG2 cells is
75.4 ± 7.6 ngÆ(g cells)
)1
(Fig. 6). Treating the cells
with ethanol, a known inducer of MT [17], for 12 h
increases the concentration of MT to 101 ngÆ(g cells)
)1
.
To examine whether ethanol or its metabolic product
acetaldehyde induces MT, inhibitors of ADH [4-meth-

nein (MT) in HepG2 cells. (A) Ethanol (5 m
M), 4-methylpyrazole
hydrochloride (4-MP) ⁄ ethanol (5 l
M ⁄ 5mM), disulfiram ⁄ ethanol
(5 l
M ⁄ 5mM) or acetaldehyde (1 mM) were incubated with
2 · 10
6
HepG2 cells for 12 h. (B) Acrolein (10 lM) was incubated
with 2 · 10
6
HepG2 cells for 12 h. Control or treated cells were
collected, washed, and homogenized. MT concentrations were
determined with a cadmium-binding assay. Data are presented as
means ± SD of triplicate determinations. The asterisk indicates sig-
nificance at P < 0.05. No significant difference was found for
4-MP ⁄ ethanol treatment.
Fig. 7. Aldehydes inhibit protein tyrosine phosphatase (PTP) activity
in HepG2 cells through a zinc-mediated mechanism. Acetaldehdye
(1 m
M) or acrolein (10 lM) was incubated with 2 · 10
6
HepG2 cells
for 12 h. Control or treated cells were collected, washed, and
homogenized. PTP activity was measured with a fluorescent phos-
photyrosine peptide. An aliquot of the homogenized cells was incu-
bated with 5 l
M N,N,N¢,N¢-tetrakis(2-pyridylmethyl)-ethylenediamine
(TPEN) for 30 min before measurement of PTP activity. –, without
TPEN; +, with TPEN. Emission wavelength 395 nm, excitation

activity relationship for the limited number of alde-
hydes tested here cannot be given, as many factors
other than steric factors determine the reactivity. In
aqueous solutions, aldehydes undergo side reactions
that compete with the reactivity under investigation.
Examples are slow oxidation to the corresponding
acid, aldol condensation of short-chain aldehydes and
hydration of alkyl aldehydes to gem-diols [22]. There-
fore, it is critical to prepare fresh stock solutions from
the anhydrous aldehyde immediately before the experi-
ment. In addition, the two aldehydes discussed,
acrolein and acetaldehyde, react differently with sulf-
hydryls. Acetaldehyde reacts via the aldehyde group,
whereas acrolein, an a,b-unsaturated aldehyde, forms a
Michael adduct. The zinc-releasing activity of alde-
hydes has implications for toxicologic and patho-
biochemical mechanisms.
Acrolein
Concentrations of cellular aldehydes increase during
environmental and nutritional exposures, as well as in
various diseases with oxidative stress that increases
lipid peroxidation. Malondialdehyde, 4-HNE and acro-
lein are the major aldehyde products of lipid peroxida-
tion. Acrolein is also formed from spermine and
spermidine by amine oxidases [23]. In the brain of Alz-
heimer’s disease victims, the concentrations of acrolein
and 4-HNE increase 7–8-fold [24–26]. For refer-
ence, basal values in hippocampus are 0.3 and
0.265 nmolÆ(mg protein)
)1

drial aldehyde dehydrogenase or in alcoholic patients
under treatment with disulfiram or other alcohol-
sensitizing drugs. In animals treated with aldehyde
dehydrogenase inhibitors and ethanol, blood acetalde-
hyde can reach concentrations of almost 1 mm [30,31].
Acetaldehyde is discussed as a mediator of tissue
injury in alcoholic liver disease and myopathies, in the
etiology of cancer of the respiratory and digestive
tracts, and in other diseases [10,32].
In summary, the reactivity of aldehydes with zinc
proteins demonstrates that elevated levels of aldehydes
affect zinc metabolism and that zinc release and ensu-
ing binding of zinc to other proteins is one aspect of
the molecular actions of aldehydes that are generated
during lipid peroxidation and metabolism of ethanol.
Zinc signals generated by aldehydes
The concentrations of ‘free’ zinc are orders of magni-
tude smaller than those of total cellular zinc, which is
a few hundred micromoles per liter [33]. Very small
but significant changes in the availability of cellu-
lar zinc have profound biological effects. Thus, an
Q. Hao and W. Maret Aldehydes and zinc metabolism
FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4305
increase from 520 to 870 pm ‘free’ zinc is characteristic
for a transition between normal and diabetic cardio-
myocytes [34]. Changes from picomolar to low nano-
molar concentrations of zinc affect gene expression in
cardiomyocytes [35]. Similarly, low nanomolar concen-
trations of zinc inhibit phosphorylation signaling,
metabolic enzymes, and mitochondrial respiration

Micromolar cellular concentrations of MT [48] make it
a significant source of aldehyde-released zinc. Zinc
released in the cell or zinc provided by supplementa-
tion activates metal response element (MRE)-binding
transcription factor-1 (MTF-1) and transcription of
the apoprotein thionein, which also reacts with alde-
hydes. Indeed, addition of a hexapeptide that contains
three of the 20 cysteines of thionein suppresses the for-
mation of protein–hydroxynonenal adducts in retinal
pigmented epithelial cells [49]. Most cells have concen-
trations of thionein commensurate with those of MT
[50]. Reactions of aldehydes with cellular thiols such as
thionein and glutathione will affect the cellular redox
balance and the capacity to scavenge reactive species.
Thionein, with its 20 thiols, is an efficient reducing
agent [20] and can serve as a cofactor for methionine
sulfoxide reductase, an enzyme that protects tissue
against oxidative injury [51]. The reaction of acetalde-
hyde with the Zn–S
Cys
bonds in ADH and concomit-
ant zinc release underscores the significance of these
reactions for compromising the functions of other pro-
teins with Zn–S
Cys
sites, such as ‘zinc fingers’. 4-HNE
modifies the cysteine ligands in liver ADH, leading to
ubiquitinylation and proteasomal degradation [52].
However, whether the released zinc is cytoprotective
or cytotoxic depends on the concentrations of released

collected and quantified based on both absorbance readings
(A
220
¼ 48 000 m
)1
Æcm
)1
) and assay of thiols. A ten-fold
molar excess of zinc sulfate was added to the nitrogen gas-
purged solution of thionein, and the pH value was adjusted
to 8.6 by slowly adding nitrogen gas-purged 1 m Tris base.
The sample was concentrated to about 2 mL by centrifuga-
Aldehydes and zinc metabolism Q. Hao and W. Maret
4306 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS
tion for 4 h at 4000 g using CentriconÒ centrifugal filter
devices (MWCO 3000) (Millipore, Bedford, MA), loaded
onto a Sephadex G-50 column (1 · 120 cm), and eluted
with 20 mm Tris ⁄ HCl (pH 7.4) at a flow rate of 10 mLÆh
)1
.
MT fractions were pooled after measuring the concentra-
tion of protein (A
220
¼ 159 000 m
)1
Æcm
)1
) and thiols and
determining zinc by atomic absorption spectrophotometry
(Perkin-Elmer model 5100, Wellesley, MA).

indicator. Binding of zinc ions changes its absorbance at
500 nm. Zn
7
-MT2 or yeast ADH (0.5 lm) and PAR
(100 lm from a 1 mm stock solution in 20 mm Tris ⁄ HCl,
pH 7.4) were incubated with or without aldehydes and the
absorbance change was followed (A
500
¼ 65 000 m
)1
Æcm
)1
).
Aldehyde stock solutions (100 mm) were prepared immedi-
ately before use. Owing to the toxicity of some aldehydes,
all stock solutions were prepared in a fume hood. A stock
solution of 4-HNE was prepared from the compound
stored at ) 80 °C and used immediately. Malonaldehyde
tetrabutylammonium salt was used as a source of ‘malondi-
aldehyde’. Evaporation of acetaldehyde during measure-
ments was minimized by sealing the cuvettes with Parafilm.
A1mm solution of dl-glyceraldehyde (Sigma) in 20 mm
Tris ⁄ HCl (pH 7.4) was found to contain 20 lm zinc. Thus
the absorbance change after incubation of 1 mm glyceralde-
hyde with PAR was subtracted. The experiments were
repeated at least three times. Aldehydes (1 mm) were also
mixed with PAR (100 lm) in the absence of MT, and the
absorbance at 500 nm was recorded. With the exception of
formaldehyde, none of the aldehydes affects the absorbance
of PAR. The data for the reaction of MT with formalde-

ADH activity was determined with acetaldehyde as sub-
strate. The assay was performed in 0.1 m Tris ⁄ HCl
(pH 8.0), 0.67 mm NADH, 100 mm KCl, 10 mm 2-mercap-
toethanol, 2 mm acetaldehyde and 0.0007% (w ⁄ v) BSA.
The reaction was monitored by measuring the decrease in
NADH absorbance at 340 nm after initiation of the reaction
by addition of enzyme (0.15 units). The effects of aldehydes
on ADH activity were examined by mixing ADH (0.15 units
in 5 lL) with an equal volume of either 2 mm acetaldehyde
or 2 mm acrolein and incubating for 20 min. An aliquot was
then added to the assay solution to initiate the reaction.
Aldehydes introduced into the assay in this way increase the
total aldehyde concentration by less than 1%.
Tissue culture
HepG2 cells (#HB-8065, American Type Culture Collec-
tion, Manassas, VA) were cultured in DMEM containing
Q. Hao and W. Maret Aldehydes and zinc metabolism
FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4307
4.5 gÆL
)1
glucose, supplemented with 10% (v ⁄ v) FBS
(defined; Hyclone, Salt Lake City, UT), 0.12 mgÆmL
)1
streptomycin sulfate, and 0.1 mg ÆmL
)1
gentamicin sulfate.
Cells were maintained at 5% CO
2
and 37 °C in a humid-
ified atmosphere. All other cell culture products were pur-

i
, and the fluorescence intensity (F) was measured
at 370 nm (excitation) and 490 nm (emission) with an
SLM-8000 spectrofluorimeter equipped with data acquisi-
tion and processing electronics from ISS (Champaign, IL).
Fluorescence intensities are the averages of three measure-
ments. The working range for measurements of fluorescence
intensity was determined by adding zinc and the ionophore
pyrithione (20 lm final concentrations for both). The meas-
ured value corresponds to the maximum fluorescence
(F
max
). The minimum fluorescence (F
min
) was obtained
from a reading in the presence of the zinc-chelating agent
TPEN (100 lm). The percentage of saturation was then
calculated from [(F ) F
min
) ⁄ (F
max
) F
min
)] · 100. Addition
of 20 lm zinc alone increased fluorescence slightly. This
fluorescence increase is quenched with cell-impermeable
EDTA, and is therefore due to zinc binding to residual,
extracellular Zinquin. This fluorescence was subtracted
from F
max

MT concentrations were calculated based on an MT ⁄ Cd
stoichiometry of 1 : 7.
PTP assay
PTP activity in HepG2 cells was determined with a tyro-
sine-phosphorylated oligopeptide MCA-Gly-Asp-Ala-Glu-
Tyr(PO
3
H
2
)-Ala-Ala-Lys(DNP)-Arg-NH
2
(Calbiochem, La
Jolla, CA) [18]. In this peptide, the DNP group quenches
the fluorescence of the (7-methoxycoumarin-4-yl)-acetyl
(MCA) group. Assays were performed at 37 °Cin20mm
Hepes ⁄ NaOH (pH 7.5) containing 1 mm Tris-(2-carboxy-
ethyl)-phosphine (Molecular Probes) and 1 lm substrate in
a total volume of 1 mL. After 5 min of equilibration of
substrate with buffer, the reaction was initiated by adding
an aliquot containing 10 mg of total protein from the
extract of the control or aldehyde-treated cells (sample from
determination of MT concentration). The reaction was
quenched after 15 min by adding 10 lL of chymotryp-
sin ⁄ sodium orthovanadate to final concentrations of 0.05%
(w ⁄ v) and 0.1 mm, respectively. Chymotrypsin cleaves only
the peptide that is dephosphorylated by PTPs. Cleavage
disrupts fluorescence resonance energy transfer, thereby
increasing MCA fluorescence. MCA fluorescence was mon-
itored at 328 ⁄ 395 nm, with slit widths of 1.5 nm (excita-
tion) and 10 nm (emission), using an SLM-8000

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