Tài liệu Báo cáo khoa học: Aldehydes release zinc from proteins. A pathway from oxidative stress⁄lipid peroxidation to cellular functions of zinc pptx

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

from ketone bodies give rise to advanced glycation
end-products [2]. Concentrations of aldehydes also
increase with age and in diseases that are accompan-
ied by oxidative stress. Oxidative stress causes lipid
peroxidation and formation of aldehydes such as
malon(di)aldehyde, 4-hydroxynonenal (4-HNE), and
Keywords
acetaldehyde; acrolein; metallothionein;
oxidative stress; zinc
Correspondence
W. Maret, Division of Human Nutrition,
Preventive Medicine and Community
Health, The University of Texas Medical
Branch, 700 Harborside Drive, Galveston,
TX 77555, USA
Fax: +1 409 772 6287
Tel: +1 409 772 4661
E-mail:
(Received 2 May 2006, revised 14 July
2006, accepted 20 July 2006)
doi:10.1111/j.1742-4658.2006.05428.x
Oxidative stress, lipid peroxidation, hyperglycemia-induced glycations and
environmental exposures increase the cellular concentrations of aldehydes.
A novel aspect of the molecular actions of aldehydes, e.g. acetaldehyde and
acrolein, is their reaction with the cysteine ligands of zinc sites in proteins
and concomitant zinc release. Stoichiometric amounts of acrolein release
zinc from zinc–thiolate coordination sites in proteins such as metallothion-
ein and alcohol dehydrogenase. Aldehydes also release zinc intracellularly
in cultured human hepatoma (HepG2) cells and interfere with zinc-depend-
ent signaling processes such as gene expression and phosphorylation. Thus

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

hundred micromoles per liter [9], especially in indi-
viduals with a slow-metabolizing variant of mito-
chondrial aldehyde dehydrogenase. Accumulation of
acetaldehyde has been discussed in the pathology of
alcohol-induced tissue injury [10].
Cysteine is now recognized as a ligand in a large
number of zinc coordination sites. The cysteine ligands
are remarkably reactive towards oxidizing agents and
nucleophiles, both of which release zinc [11]. Even
minute amounts of released zinc are potent effectors of
cellular metabolism and signaling [12,13]. This study
addresses the reactivity of aldehydes with cysteine lig-
ands of zinc in proteins. Moreover, it demonstrates
that aldehydes release zinc from isolated proteins and
in cultured cells and that the released zinc affects phos-
phorylation signaling and gene expression.
Results
Aldehydes release zinc from zinc-binding proteins
The effect of aldehydes on the zinc-binding capacity of
zinc proteins was assayed by employing spectropho-
tometry and the chromophoric indicator 4-(2-pyridyl-
azo)-resorcinol (PAR) for zinc ions. Acrolein at
concentrations as low as 10 lm releases zinc from
metallothionein (MT) (Fig. 1). In this experiment, the
concentration of zinc MT isoform 2 (MT2) is 0.5 lm,
corresponding to 10 lm in thiols, as there are 20 cys-
teines in MT. Thus stoichiometric amounts of acrolein
with regard to the thiols in MT release zinc. The reac-
tion continues for 20 h until all seven zinc ions from
MT2 are released (Fig. 1). Zinc release is based on the

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

kinetics of zinc transfer from MT isoform 2 (MT2) (0.5 l
M)to
4-(2-pyridylazo)-resorcinol (PAR) (100 l
M) was monitored spectro-
photometrically in the absence and presence of 10 l
M acrolein in
20 m
M Tris ⁄ HCl (pH 7.4). The reaction was recorded immediately
after acrolein was added to the solution and recorded for 1200 min.
Zinc release is based on the reaction of MT with 10 l
M ebselen,
which releases all seven zinc ions from MT within 20 min [14],
because evaporation of liquid during the long time period of the
assay leads to a more concentrated sample, a higher absorbance
reading, and hence an apparent release of more zinc than is possi-
ble based on the initial concentration of 0.5 l
M MT2, when calcula-
ted on the basis of the extinction coefficient of PAR. Line 1: control
(no acrolein). Line 2: with acrolein.
Q. Hao and W. Maret Aldehydes and zinc metabolism
FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4301
(Fig. 3B). Incubation with acetaldehyde has virtually
no effect on its activity, whereas incubation with acro-
lein inhibits enzymatic activity, suggesting that acetal-
dehyde removes only the noncatalytic zinc and that
acrolein, an irreversible inhibitor [15], removes both
the noncatalytic and the catalytic zinc ions from the
enzyme.
Aldehydes react with the sulfhydryl groups of
metallothionein and thionein

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

does not react with TNB.
In order to determine whether the modification of
any of the eight lysines in MT by aldehydes would
Fig. 3. Aldehydes release zinc from alcohol dehydrogenase (ADH).
(A) The kinetics of zinc transfer from ADH (0.5 l
M, 12.8 unitsÆmL
)1
)
to 4-(2-pyridylazo)-resorcinol (PAR) (100 l
M) was monitored spectro-
photometrically in the absence and presence of aldehydes in
20 m
M Tris ⁄ HCl (pH 7.4). The ADH concentration is based on the
data provided by the manufacturer. The reaction was recorded for
20 min immediately after aldehydes were added to the solution.
Line 1: control (no aldehyde). Line 2: 1 m
M acetaldehyde. Line 3:
1m
M acrolein. (B) Effect of aldehydes on ADH activity. ADH
(0.15 units) was incubated with either 1 m
M acetaldehyde or 1 mM
acrolein for 20 min, the mixture was added to the buffer ⁄ substrate
mix, and the reaction was followed spectrophotometrically at
340 nm. d, control (no aldehyde preincubation); n, acetaldehyde;
m, acrolein.
Fig. 2. Zinc-releasing activities of different aldehydes. The amount
of zinc released from metallothionein isoform 2 (MT2) (0.5 l
M)by
aldehydes (1 m
M) was determined with 4-(2-pyridylazo)-resorcinol

(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%),

albeit statistically significant, a different approach was
employed to increase cellular acetaldehyde concentra-
tions. When cells were treated with 2 lm disulfiram to
inhibit aldehyde dehydrogenase and ethanol was
added, a significant release of zinc was detected, with
saturation of Zinquin reaching 22% (Fig. 5B). Ethanol
alone had a small but statistically significant effect,
while disulfiram alone lowered the amount of zinc
available to the probe due to its metal-chelating capa-
city [16].
Fig. 4. Effect of acetaldehyde on the thiol reactivity of metallothion-
ein isoform 2 (MT2). MT2 (1.2 l
M) was incubated without (line 1)
or with (line 2) acetaldehyde (1 m
M) for 30 min in 20 mM Tris ⁄ HCl
(pH 7.4), 5,5¢-dithiobis-2-nitrobenzoic acid (DTNB) was added to a
final concentration of 0.2 m
M, and the absorbance at 412 nm was
recorded. Line 1: control (no acetaldehyde). Line 2: 1 m
M acetalde-
hyde.
Fig. 5. Aldehydes increase the amount of available intracellular zinc
in HepG2 cells. (A) HepG2 cells (1 · 10
6
) were treated with acetal-
dehyde (1 m
M) or acrolein (10 lM) for 30 min. The cells were col-
lected and labeled with Zinquin ester. Fluorescence intensities
were recorded with excitation and emission wavelengths of 370
and 490 nm, respectively. (B) HepG2 cells (1 · 10

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-

ylpyrazole hydrochloride (4-MP)] and aldehyde dehy-
drogenase (disulfiram) were used in conjunction with
ethanol. 4-MP inhibits the conversion of ethanol to
acetaldehyde, lowering acetaldehyde concentrations,
whereas disulfiram inhibits the conversion of acetalde-
hyde to acetic acid, increasing the concentrations of
acetaldehyde. The concentration of MT in 4-MP ⁄ etha-
nol-treated cells does not change, whereas it increases
to 118 ngÆ(g cells)
)1
in disulfiram ⁄ ethanol-treated cells
(Fig. 6A). Treatment of HepG2 cells with 1 mm acetal-
dehyde increases the MT concentration two-fold.
These results clearly demonstrate that acetaldehyde
and not ethanol induces MT in HepG2 cells. Relatively
low concentrations of acrolein (10 lm) increase MT2
expression by 35% (Fig. 6B).
Aldehydes inhibit protein tyrosine phosphatase
activity in HepG2 cells through modulation of
intracellular zinc
To further investigate the effect of aldehydes on zinc-
mediated biological processes, the effects of acetalde-
hyde and acrolein on protein tyrosine phosphatase
(PTP) activity were investigated. The rationale for this
experiment is that intracellular zinc modulates PTP
activity [18]. Incubation of HepG2 cells with acetalde-
hyde or acrolein significantly inhibits PTP activity to
45% and 52% of the control, respectively (Fig. 7).
This inhibition could be caused by a reaction of
Fig. 6. Aldehydes increase the expression levels of metallothio-

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

wavelength 328 nm. Data are presented as means ± SD of tripli-
cate determinations. The asterisk indicates significance at P < 0.05.
Aldehydes and zinc metabolism Q. Hao and W. Maret
4304 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS
acetaldehyde with the catalytic cysteine of PTP, zinc
inhibition of PTP, or both. After addition of the
zinc-chelating agent N,N,N¢,N¢-tetrakis(2-pyridylmethyl)-
ethylenediamine (TPEN), PTP activity in both control
and aldehyde-treated cells increases, indicating that
aldehydes affect PTP activity in part through zinc
release and zinc inhibition of PTP.
Discussion
Aldehydes affect zinc–sulfur (Zn–S
Cys
)
coordination environments in proteins
Zn–S
Cys
sites in proteins are remarkably reactive. Oxi-
dation of the sulfur ligands and concomitant zinc
release establishes multiple pathways for redox control
of zinc metabolism and dynamic regulation of protein
structure and function [11]. Oxidants such as glutathi-
one disulfide, nitric oxide and reducible selenium-
containing compounds release zinc from proteins with
Zn–S
Cys
sites [19–21]. Based on the above results, alde-
hydes can now be added to the growing list of agents
that affect the cellular functions of zinc. A structure–

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

, respectively. In acute iron
loading ⁄ toxicosis, cytotoxic aldehydes increase through
lipid peroxidation, which is initiated by Fenton chem-
istry-generated free radicals [27]. In diabetes, there are
pathways for the increased formation of a-keto-
aldehydes such as glyoxal and methylglyoxal from
glyceraldehyde 3-phosphate. Autoxidation of a-hydroxy-
aldehydes to a-ketoaldehydes generates hydrogen per-
oxide, which contributes to oxidative stress and lipid
peroxidation in the disease [1].
Acrolein induces transcription of phase II genes by
activating the transcription factor Nrf2 [24]. Nrf2
translocates to the nucleus when released from the pro-
tein Keap1, a zinc metalloprotein with Zn–S
Cys
co-
ordination and the sensor for electrophiles such
as aldehydes. A proposed mechanism of activation
involves a reaction of electrophiles with the cysteine
ligands of Keap1, followed by zinc release [28]. The
reactions of aldehydes with MT and ADH and con-
comitant zinc release provide direct experimental sup-
port for such a mechanism.
Acetaldehyde
Under normal conditions, aldehyde dehydrogenases
maintain acetaldehyde at relatively low levels, e.g.
below 0.2 lm for plasma acetaldehyde that is not pro-
tein-bound [29]. However, acetaldehyde concentrations
are significantly higher when alcoholic beverages are
consumed, in individuals with an inactive mitochon-

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

[18,36,37]. Because a very potent zinc signal is gener-
ated, aldehyde-induced zinc release from proteins is
significant for even relatively small increases of alde-
hyde concentrations. Hence, the actions of zinc may
explain at least some of the regulatory functions of
ethanol and its metabolite acetaldehyde in cellular
signaling, where molecular mechanisms remain largely
unknown [38,39]. There is a striking similarity between
the effects of acetaldehyde and those of zinc. Acetalde-
hyde inhibits PTP 1B in Caco-2 cells and increases
protein tyrosine phosphorylation, much as zinc does in
other cell lines [18,40,41]. Also, acetaldehyde affects
the nuclear factor- jB pathway in a way similar to zinc
or MT [42,43]. Indeed, in addition to a direct interac-
tion of aldehydes with protein sulfhydryls, an indirect
action of aldehydes via binding of released zinc to pro-
tein sulfhydryls is evident from the effects of released
zinc on gene expression (Fig. 6) and phosphorylation
signaling (Fig. 7). Short-chain alcohols induce thionein
through an indirect mechanism [44]. It is now apparent
that the induction occurs through zinc that is released
by aldehydes formed from the corresponding alcohols
during metabolism.
Protective functions of zinc and MT against
ethanol toxicity
Both zinc and MT protect the liver and the heart
against the toxic effects of ethanol [45–47]. The above
results suggest that a critical aspect of the protective
function of MT is the scavenging of the acetaldehyde
formed from ethanol and concomitant zinc release.

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

zinc, as zinc has both pro-antioxidant and pro-oxidant
functions [53]. If concerns for safety can be overcome
[54], zinc supplementation could be an efficient way of
inducing MT ⁄ thionein for protection against toxic
aldehydes. On the other hand, nutritional or condi-
tional zinc deficiency will increase cellular damage by
aldehydes. Zinc deficiency elicits oxidative stress [55],
thus increasing lipid peroxidation and aldehyde con-
centrations, releasing more zinc from proteins, and
initiating a vicious cycle that will exacerbate zinc defi-
ciency and increase the toxicity of aldehydes.
Experimental procedures
Materials
4-HNE was obtained from Biomol (Plymouth Meeting,
PA), Sephadex G-25 and G-50 from Amersham Biosciences
(GE Healthcare, Piscataway, NJ), Cleland’s reagent (dithio-
threitol) from Calbiochem (San Diego, CA), and Zinquin
ester from Molecular Probes (Eugene, OR). All other chem-
icals were from Sigma (St Louis, MO).
Reconstitution of MT2 with zinc
Commercial rabbit MT2 (Sigma) contains both cadmium
and zinc. To prepare zinc MT2 [56], 5 mg of MT2 was dis-
solved in 1 mL of 20 mm Tris ⁄ HCl (pH 7.4) containing
50 mg dithiothreitol, and incubated at 25 °C for 24 h. After
incubation, the sample was adjusted to pH 1 with HCl, and
centrifuged at 10 000 g for 5 min (Eppendorf centrifuge
model 5415C, Hamburg, Germany) to remove any precipi-
tate. The clear supernatant was then loaded onto a Sepha-
dex G-25 column (1 · 120 cm), which was equilibrated and
eluted with 10 mm HCl. Fractions containing thionein were

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).

Preparation of thionein from MT2
Zinc MT2 (0.5 mg) was incubated in 1 mL of 20 mm
Tris ⁄ HCl (pH 7.4) containing 0.1 m dithiothreitol overnight
at 25 °C. The reaction mixture was adjusted to pH 2 with
HCl, and thionein was separated from excess dithiothreitol
and zinc ions by gel filtration on a Sephadex G-25 column
(1 · 30 cm) equilibrated with 10 mm HCl at 25 °C. To min-
imize the oxidation of thionein, the elution buffer (20 mm
Tris ⁄ HCl, pH 7.4) was purged with nitrogen gas. Thionein
was located in the fractions by measurement of its absorb-
ance at 220 nm and by assaying its thiols with 2,2¢-dithiodi-
pyridine (see below). Thionein was either used immediately
or stored at liquid nitrogen temperatures.
Thiol assay
The concentration of thiols in MT was determined by
incubating the protein with 100 mgÆL
)1
2,2¢-dithiodipyridine
[57] and taking absorbance readings (A
343
¼ 7600
m
)1
Æcm
)1
) with a Beckman-Coulter DUÒ 800 UV–visible
spectrophotometer (Fullerton, CA).
PAR metal transfer assay
Metallochromic indicators provide a rapid means of investi-
gating metal–protein equilibria [58,59]. PAR is such an

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-

hyde were corrected for the absorbance changes in the
absence of MT.
Thiol reactivities of MT and thionein
The reactivity of thiols in MT and thionein was determined
with DTNB under pseudo-first-order rate conditions. The
reaction between MT or thionein (1.2 lm) and DTNB
(200 lm)in20mm Tris ⁄ HCl (pH 7.4) was followed spec-
trophotometrically at 412 nm (25 °C). The number of sulf-
hydryls modified by acetaldehyde was determined by
incubating MT or thionein with acetaldehyde for 30 min,
removing the excess of aldehyde with 1 unitÆmL
)1
of yeast
ADH, 2 mm NADH and 100 mm potassium chloride, and
then assaying the protein with DTNB.
Modification of lysine residues in MT
Lysine residues in MT were modified according to an estab-
lished protocol [60]. Briefly, 1 mg of MT2 was concentrated
with Centricon centrifugal microconcentrators (MWCO
3000; Millipore), and diluted with 0.5 m sodium borate buf-
fer (pH 9.2) to a final concentration of 10 mgÆmL
)1
, and
solid potassium cyanate was added to a final concentration
of 1 m. The reaction mixture was incubated at 37 °C for
24 h. Excess potassium cyanate was then removed by gel
filtration on a Sephadex G-25 column (0.2 · 8 cm). Protein
concentrations were determined spectrophotometrically at
220 nm.
Yeast ADH assay

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-

chased from Gibco (Invitrogen, Carlsbad, CA).
Determination of available cellular zinc
HepG2 cells (1 · 10
6
cells per well) were seeded in 12-well
plates and grown for 24 h. Freshly prepared acetaldehyde
and acrolein were added to the medium to final concentra-
tions of 1 mm and 10 lm, respectively, and incubated for
30 min. Additionally, cells were incubated for 1 h with
tetraethylthiuram disulfide (disulfiram), an aldehyde dehy-
drogenase inhibitor, at a final concentration of 2 lm, eth-
anol was added to each well to a final concentration of
5mm, and the cells incubated for an additional hour [18].
The fluorescence probe Zinquin ethyl ester (dissolved in
dimethyl sulfoxide) was added to the cells to a final concen-
tration of 25 lm. The measurements were normalized by
measuring the total protein concentration of each sample
with a Micro-BCA
TM
protein assay kit from Pierce (Rock-
ford, IL). The protein concentration of control cells without
disulfiram or ethanol was taken as 100%. To determine the
extent of saturation of Zinquin with zinc, 1 · 10
6
cells were
incubated with the dye as described above, washed three
times with Dulbecco’s NaCl ⁄ P
i
, and detached in 3 mL of
NaCl ⁄ P

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

.
Determination of MT in HepG2 cells
The total amount of MT in HepG2 cells was determined
with a cadmium-binding assay [61] with modifications.
HepG2 cells (2 · 10
6
) were homogenized in a Potter-Elveh-
jem homogenizer with at least 20 strokes. Microsco-
pic inspection verified that 90% of the cells were broken.
The supernatant (200 lL) obtained after centrifugation at
14 000 g (Eppendorf centrifuge model 5415C) was mixed
with the same volume of a CdCl
2
solution (2 lgÆmL
)1
), and
incubated at 25 °C for 10 min. One hundred microliters of
bovine hemoglobin solution (2%, w ⁄ v) was added to the
tubes, and the sample was mixed and heated in a boiling
water bath for 2 min. The samples were then placed on ice
for 5 min, and centrifuged at 14 000 g for 2 min (Eppen-
dorf centrifuge model 5415C); another aliquot of 100 lLof
2% hemoglobin solution was then added to the superna-
tant, and heating, cooling and centrifugation were repeated.
Finally, a 500 lL aliquot of the supernatant was removed
and diluted with 3.5 mL of 0.1 m HNO
3
. Cadmium concen-
trations in the supernatants were determined by atomic
absorption spectrophotometry (Perkin-Elmer model 5100).

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

spectrofluorimeter. Background fluorescence was deter-
mined in the absence of cell extract and was subtracted.
Statistical analysis
Values are given as means ± SD and analyzed by Student’s
t-test. Significance was assessed at the P<0.05 level.
Acknowledgements
We thank Dr. V. M. Sadagopa Ramanujam (The
University of Texas Medical Branch, Galveston, TX)
for help with metal analyses by atomic absorption
spectrophotometry (supported by the Human Nutri-
Aldehydes and zinc metabolism Q. Hao and W. Maret
4308 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS
tion Research Facility) and Professor Richard Glass
(University of Arizona, Tucson, AZ) for helpful dis-
cussions. This work was supported by NIH Grant
GM 065388 to WM.
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