aldehydes release zinc from proteins. a pathway from oxidative stress/lipid peroxidation to cellular...

Aldehydes release zinc from proteins. A pathway fromoxidative stress ⁄lipid peroxidation to cellular functionsof zincQiang 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: [email protected]

(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 lM) to

4-(2-pyridylazo)-resorcinol (PAR) (100 lM) was monitored spectro-

photometrically in the absence and presence of 10 lM acrolein in

20 mM 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 lM 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 lM 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 lM, 12.8 unitsÆmL)1)

to 4-(2-pyridylazo)-resorcinol (PAR) (100 lM) was monitored spectro-

photometrically in the absence and presence of aldehydes in

20 mM 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 mM acetaldehyde. Line 3:

1 mM acrolein. (B) Effect of aldehydes on ADH activity. ADH

(0.15 units) was incubated with either 1 mM 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 lM) by

aldehydes (1 mM) was determined with 4-(2-pyridylazo)-resorcinol

(PAR) after 30 min. Ebselen (10 lM) 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


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 lM) was incubated without (line 1)

or with (line 2) acetaldehyde (1 mM) 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 mM, and the absorbance at 412 nm was

recorded. Line 1: control (no acetaldehyde). Line 2: 1 mM acetalde-

hyde.

Fig. 5. Aldehydes increase the amount of available intracellular zinc

in HepG2 cells. (A) HepG2 cells (1 · 106) were treated with acetal-

dehyde (1 mM) 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 · 106) were treated

with 2 lM disulfiram for 1 h to inhibit aldehyde dehydrogenases.

After addition of 5 mM 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.



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 mM), 4-methylpyrazole

hydrochloride (4-MP) ⁄ ethanol (5 lM ⁄ 5 mM), disulfiram ⁄ ethanol

(5 lM ⁄ 5 mM) or acetaldehyde (1 mM) were incubated with

2 · 106 HepG2 cells for 12 h. (B) Acrolein (10 lM) was incubated

with 2 · 106 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 mM) or acrolein (10 lM) was incubated with 2 · 106 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 lM 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.



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–SCys)

coordination environments in proteins

Zn–SCys 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–SCys 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–SCys 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



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–SCys bonds in ADH and concomit-

ant zinc release underscores the significance of these

reactions for compromising the functions of other pro-

teins with Zn–SCys 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

(A220 ¼ 48 000 m)1Æcm)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-



tion for 4 h at 4000 g using Centricon� 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 (A220 ¼ 159 000 m)1Æcm)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 (A343 ¼ 7600

m)1Æcm)1) with a Beckman-Coulter DU� 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. Zn7-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 (A500 ¼ 65 000 m)1Æcm)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.

A 1 mm 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) in 20 mm 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



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 mgÆmL)1 gentamicin sulfate.

Cells were maintained at 5% CO2 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 · 106 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

5 mm, 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-BCATM 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 · 106 cells were

incubated with the dye as described above, washed three

times with Dulbecco’s NaCl ⁄Pi, and detached in 3 mL of

NaCl ⁄Pi, 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

(Fmax). The minimum fluorescence (Fmin) 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 ) Fmin) ⁄ (Fmax ) Fmin)] · 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 Fmax.

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 · 106) 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 CdCl2 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 lL of

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 HNO3. Cadmium concen-

trations in the supernatants were determined by atomic

absorption spectrophotometry (Perkin-Elmer model 5100).

MT concentrations were calculated based on an MT ⁄Cdstoichiometry of 1 : 7.

PTP assay

PTP activity in HepG2 cells was determined with a tyro-

sine-phosphorylated oligopeptide MCA-Gly-Asp-Ala-Glu-

Tyr(PO3H2)-Ala-Ala-Lys(DNP)-Arg-NH2 (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 �C in 20 mm

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-



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.

References

1 Robertson RP (2004) Chronic oxidative stress as a cen-

tral mechanism for glucose toxicity in pancreatic islet

beta cells in diabetes. J Biol Chem 279, 42351–42354.

2 Beisswenger PJ, Howell SK, Nelson RG, Mauer M &

Szwergold BS (2003) Alpha-oxoaldehyde metabolism

and diabetic complications. Biochem Soc Trans 31,

1358–1363.

3 Esterbauer H, Schaur RJ & Zollner H (1991) Chemistry

and biochemistry of 4-hydroxynonenal, malonaldehyde

and related aldehydes. Free Radic Biol Med 11, 81–128.

4 Kehrer JP & Biswal SS (2000) The molecular effects of

acrolein. Toxicol Sci 57, 6–15.

5 Dost FN (1991) Acute toxicology of components of

vegetation smoke. Rev Environ Contam Toxicol 119, 1–46.

6 Thornalley PJ (1996) Pharmacology of methylglyoxal:

formation, modification of proteins and nucleic acids,

and enzymatic detoxification ) a role in pathogenesis

and antiproliferative chemotherapy. Gen Pharmacol 27,

565–573.

7 Uchida K (2000) Role of reactive aldehyde in cardiovas-

cular diseases. Free Radic Biol Med 28, 1685–1696.

8 Leikauf GD (1992) Mechanisms of aldehyde-induced

bronchial reactivity: role of airway epithelium. Res Rep

Health Eff Institute 49, 1–35.

9 Johnsen J, Stowell A & Morland J (1992) Clinical

responses in relation to blood acetaldehyde levels. Phar-

macol Toxicol 70, 41–45.

10 Eriksson CJ (2001) The role of acetaldehyde in the

actions of alcohol (update 2000). Alcohol Clin Exp Res

25, 15S–32S.

11 Maret W (2004) Zinc and sulfur: a critical biological

partnership. Biochemistry 43, 3301–3309.

12 Beyersmann D & Haase H (2001) Functions of zinc in

signaling, proliferation and differentiation of mamma-

lian cells. Biometals 14, 331–341.

13 Frederickson CJ, Koh JY & Bush AI (2005) The neuro-

biology of zinc in health and disease. Nat Rev Neurosci

6, 449–462.

14 Jacob C, Maret W & Vallee BL (1998) Ebselen, a sele-

nium-containing redox drug, releases zinc from metal-

lothionein. Biochem Biophys Res Commun 248, 569–573.

15 Rando RR (1974) Allyl alcohol-induced irreversible

inhibition of yeast alcohol dehydrogenase. Biochem

Pharmacol 23, 2328–2331.

16 Shiah SG, Kao YR, Wu FYH & Wu CW (2003) Inhibi-

tion of invasion and angiogenesis by zinc-chelating

agent disulfiram. Mol Pharmacol 64, 1076–1084.

17 Kagi JHR (2001) Overview of metallothionein. Methods

Enzymol 205, 613–626.

18 Haase H & Maret W (2003) Intracellular zinc fluctua-

tions modulate protein tyrosine phosphatase activity in

insulin ⁄ insulin-like growth factor-1 signaling. Exp Cell

Res 291, 289–298.

19 Maret W (1994) Oxidative metal release from metallo-

thionein via zinc–thiol ⁄ disulfide interchange. Proc Natl

Acad Sci USA 91, 237–241.

20 Chen Y & Maret W (2001) Catalytic selenols couple the

redox cycles of metallothionein and glutathione. Eur J

Biochem 268, 3346–3353.

21 Chen Y, Irie Y, Keung WM & Maret W (2002) S-nitro-

sothiols react preferentially with zinc thiolate clusters of

metallothionein III through transnitrosation. Biochemis-

try 41, 8360–8367.

22 Deetz JS, Luehr CA & Vallee BL (1984) Human liver

alcohol dehydrogenase isozymes: reaction of aldehydes

and ketones. Biochemistry 23, 6822–6828.

23 Toninello A, Pietrangeli P, De Marchi U, Salvi M &

Mondovi B (2006) Amine oxidases in apoptosis and

cancer. Biochim Biophys Acta 1765, 1–13.

24 Tirumulai R, Rajesh Kuram T, Mai KH & Biswal S

(2002) Acrolein causes transcriptional induction of

phase II genes by activation of Nrf2 in human lung type

II epithelial (A549) cells. Toxicol Lett 132, 27–36.

25 Markesbery WR & Lovell MA (1998) Four-hydroxyno-

nenal, a product of lipid peroxidation, is increased in the

brain in Alzheimer’s disease. Neurobiol Aging 19, 33–36.

26 Lovell MA, Xie CS & Markesbery WR (2001) Acrolein

is increased in Alzheimer’s disease brain and is toxic to

primary hippocampal cultures. Neurobiol Aging 22, 187–

194.

27 Bartfay WJ, Hou D, Lehotay DC, Luo X, Bartfay E,

Backx PH & Liu PP (2000) Cytotoxic aldehyde genera-

tion in heart following acute iron-loading. J Trace Elem

Med Biol 14, 14–20.

28 Dinkova-Kostova AT, Holtzclaw WD & Wakabayashi

N (2005) Keap1, the sensor for electrophiles and oxi-

dants that regulates the phase 2 response, is a zinc

metalloprotein. Biochemistry 44, 6889–6899.

29 Helander A, Lowenmo C & Johansson M (1993) Distri-

bution of acetaldehyde in human blood: effects of etha-

nol and treatment with disulfiram. Alcohol Alcohol 28,

461–468.

30 Isse T, Oyama T, Kitagawa K, Matsuno K, Matsumoto

A, Yoshida A, Nakayama K, Nakayama K & Kawa-

moto T (2002) Diminished alcohol preference in trans-

genic mice lacking aldehyde dehydrogenase activity.

Pharmacogenetics 12, 621–626.

31 Keung WM, Lazo O, Kunze L & Vallee BL (1995)

Daidzin suppresses ethanol consumption by Syrian

golden hamsters without blocking acetaldehyde metabo-

lism. Proc Natl Acad Sci USA 92, 8990–8993.



32 Poschl G & Seitz HK (2004) Alcohol and cancer. Alco-

hol Alcohol 39, 155–165.

33 Palmiter RD & Findley SD (1995) Cloning and func-

tional characterization of a mammalian zinc transporter

that confers resistance to zinc. EMBO J 14, 639–649.

34 Ayaz M & Turan B (2006) Selenium prevents diabetes-

induced alterations in [Zn2+]i and metallothionein level

or rat heart via restoration of cell redox cycle. Am J

Physiol Heart Circ Physiol 290, H1071–H1080.

35 Atar D, Backx PH, Appel MM, Gao WD & Marban E

(1995) Excitation–transcription coupling mediated by

zinc influx through voltage-dependent calcium channels.

J Biol Chem 270, 2473–2477.

36 Maret W, Jacob C, Vallee BL & Fischer EH (1999)

Inhibitory sites in enzymes: zinc removal and reactivation

by thionein. Proc Natl Acad Sci USA 96, 1936–1940.

37 Ye B, Maret W & Vallee BL (2001) Zinc metallothio-

nein imported into liver mitochondria modulates

respiration. Proc Natl Acad Sci USA 98, 2317–2322.

38 Nagy LA (2004) Molecular aspects of alcohol metabo-

lism: transcription factors involved in early ethanol-

induced liver injury. Annu Rev Nutr 24, 55–78.

39 Aroor AR & Shukla SD (2004) MAP kinase signaling

in diverse effects of ethanol. Life Sci 74, 2339–2364.

40 Atkinson KJ & Rao RK (2001) Role of protein tyrosine

phosphorylation in acetaldehyde-induced disruption of

epithelial tight junctions. Am J Physiol Gastrointest

Liver Physiol 280, G1280–G1288.

41 Rao RK, Seth A & Sheth P (2004) Recent advances in

alcoholic liver disease I. Role of intestinal permeability

and endotoxemia in alcoholic liver disease. Am J Physiol

Gastrointest Liver Physiol 286, G881–G884.

42 Roman J, Gimenez A, Lluis JM, Gasso M, Rubio M,

Caballeria J, Pares A, Rodes J & Fernandez-Checa JC

(2000) Enhanced DNA binding and activation of tran-

scription factors NF-kappa B and AP-1 by acetaldehyde

in HEPG2 cells. J Biol Chem 275, 14684–14690.

43 Butcher HL, Kennette WA, Collins O, Zalups RK &

Koropatnick J (2004) Metallothionein mediates the level

and activity of nuclear factor kappa B in murine fibro-

blasts. J Pharmacol Exp Ther 310, 589–598.

44 Bracken WM & Klaassen CD (1987) Induction of hepa-

tic metallothionein by alcohols: evidence for an indirect

mechanism. Toxicol Appl Pharmacol 87, 257–263.

45 Zhou Z, Sun X & Kang YJ (2002) Metallothionein pro-

tection against alcoholic liver injury through inhibition

of oxidative stress. Exp Biol Med 227, 214–222.

46 Kang YJ (1999) The antioxidant function of metallo-

thionein in the heart. Proc Soc Exp Biol Med 222, 263–

273.

47 Zhou Z, Wang L, Song Z, Saari JT, McClain CJ &

Kang YJ (2005) Zinc supplementation prevents alco-

holic liver injury in mice through attenuation of oxida-

tive stress. Am J Pathol 166, 1681–1690.

48 Krezoski SK, Villalobos J, Shaw CF III & Petering DH

(1988) Kinetic lability of zinc bound to metallothionein

in Ehrlich cells. Biochem J 255, 483–491.

49 Choudhary S, Xiao T, Srivastava S, Zhang W, Chan

LL, Vergara LA, Van Kuijk FJGM & Ansari NH

(2005) Toxicity and detoxification of lipid-derived alde-

hydes in cultured retinal pigmented epithelial cells. Toxi-

col Appl Pharmacol 204, 122–134.

50 Yang Y, Maret W & Vallee BL (2001) Differential

fluorescence labeling of cysteinyl clusters uncovers high

tissue levels of thionein. Proc Natl Acad Sci USA 98,

5556–5559.

51 Sagher D, Brunell D, Hejtmancik JF, Kantorow M,

Brot N & Weissbach H (2006) Thionein can serve as a

reducing agent for the methionine sulfoxide reductases.

Proc Natl Acad Sci USA 103, 8656–8661.

52 Carbone DL, Doorn JA & Petersen DR (2004)

4-Hydroxynonenal regulates 26S proteasomal degrada-

tion of alcohol dehydrogenase. Free Radic Biol Med 37,

1430–1439.

53 Hao Q & Maret W (2005) Imbalance between pro-oxi-

dant and pro-antioxidant functions of zinc in disease.

J Alzheimer’s Dis 8, 161–170.

54 Maret W & Sandstead HH (2006) Zinc requirements

and the risks and benefits of zinc supplementation.

J Trace Elem Med Biol 20, 3–18.

55 Oteiza PI, Clegg MS, Zago MP & Keen CL (2000) Zinc

deficiency induces oxidative stress and AP-1 activation

in 3T3 cells. Free Radic Biol Med 28, 1091–1099.

56 Vasak M (1991) Metal removal and substitution in

vertebrate and invertebrate metallothioneins. Methods

Enzymol 205, 452–458.

57 Pedersen AO & Jacobsen J (1980) Reactivity of the thiol

group in human and bovine albumin at pH 3)9, asmeasured by exchange with 2,2¢-dithiodipyridine. Eur JBiochem 106, 291–295.

58 Hunt JB, Neece SH & Ginsburg A (1985) The use of

4-(2-pyridylazo)resorcinol in studies of zinc release from

Escherichia coli aspartate transcarbamoylase. Anal Bio-

chem 146, 150–157.

59 Maret W (2002) Optical methods for measuring zinc

binding and release, zinc coordination environments in

zinc finger proteins, and redox sensitivity and activity of

zinc-bound thiols. Methods Enzymol 348, 230–237.

60 Zeng J (1991) Lysine modification of metallothionein by

carbamylation and guanidination. Methods Enzymol

205, 433–437.

61 Eaton DL & Cherian MG (1991) Determination of

metallothionein in tissues by cadmium-hemoglobin affi-

nity assay. Methods Enzymol 205, 83–88.



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