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 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: womaret@utmb.edu (Received 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 metallothionein and alcohol dehydrogenase Aldehydes also release zinc intracellularly in cultured human hepatoma (HepG2) cells and interfere with zinc-dependent 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 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 photoreceptors, where retinal reacts with the e-amino group of a specific lysine in rhodopsin There are many sources of endogenous aldehydes For instance, glyceraldehyde 3-phosphate is an intermediate in glycolysis Thiohemiacetal ⁄ thioester intermediates between glyceraldehyde 3-phosphate and the sulfhydryl group of the active site cysteine are formed during turnover of glyceraldehyde 3-phosphate dehydrogenase, demonstrating 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 degenerative 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 reactions, 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 accompanied by oxidative stress Oxidative stress causes lipid peroxidation and formation of aldehydes such as malon(di)aldehyde, 4-hydroxynonenal (4-HNE), and 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,NÂ,NÂ-tetrakis(2-pyridylmethyl)-ethylenediamine 4300 FEBS Journal 273 (2006) 43004310 ê 2006 The Authors Journal compilation ª 2006 FEBS Q Hao and W Maret acrolein [3,4] Aldehydes from the environment can exacerbate the burden of exposure Endogenous aldehydes that increase during these and other episodes of exposure include: formaldehyde, used as a preservative but also found in cigarette smoke and burning vegetation; acrolein, found in cigarette smoke, herbicides, and acrylics, and produced during fossil fuel combustion, during petrochemical processing, and when overheating cooking oil; and methylglyoxal, a metabolite formed during acetone detoxification [5,6] Endogenously generated or inhaled aldehydes are involved in cardiovascular disease, atherosclerosis, vascular complications of diabetes [7] and respiratory diseases [8] Another prominent example is acetaldehyde, the metabolic product of ethanol from alcoholic beverages Excess acetaldehyde can accumulate to levels of a few hundred micromoles per liter [9], especially in individuals with a slow-metabolizing variant of mitochondrial 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 ligands of zinc in proteins Moreover, it demonstrates that aldehydes release zinc from isolated proteins and in cultured cells and that the released zinc affects phosphorylation 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 spectrophotometry and the chromophoric indicator 4-(2-pyridylazo)-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 (MT2) is 0.5 lm, corresponding to 10 lm in thiols, as there are 20 cysteines in MT Thus stoichiometric amounts of acrolein with regard to the thiols in MT release zinc The reaction 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 [14] At a concentration of mm acrolein, all seven zinc ions are released within h Aldehydes and zinc metabolism Fig Acrolein releases zinc from metallothionein (MT) The kinetics of zinc transfer from MT isoform (MT2) (0.5 lM) to 4-(2-pyridylazo)-resorcinol (PAR) (100 lM) was monitored spectrophotometrically 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 Zinc release is based on the reaction of MT with 10 lM ebselen, which releases all seven zinc ions from MT within 20 [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 possible based on the initial concentration of 0.5 lM MT2, when calculated on the basis of the extinction coefficient of PAR Line 1: control (no acrolein) Line 2: with acrolein 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 concentrations of mm (Fig 2) Among the aldehydes tested, acrolein is the most reactive aldehyde, followed by butyraldehyde, propionaldehyde, acetaldehyde, benzaldehyde, 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 environments, its reaction with the zinc enzyme yeast alcohol 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 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4301 Aldehydes and zinc metabolism Q Hao and W Maret Fig Zinc-releasing activities of different aldehydes The amount of zinc released from metallothionein isoform (MT2) (0.5 lM) by aldehydes (1 mM) was determined with 4-(2-pyridylazo)-resorcinol (PAR) after 30 Ebselen (10 lM) was used as a positive control because it releases all seven zinc ions from MT within 20 Data are presented as means ± SD of triplicate determinations (Fig 3B) Incubation with acetaldehyde has virtually no effect on its activity, whereas incubation with acrolein inhibits enzymatic activity, suggesting that acetaldehyde 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 : 200, the reaction reaches a plateau after h (Fig 4, line 1), at which point all of the 20 sulfhydryl groups in MT are titrated with DTNB Preincubation of MT2 with acetaldehyde for 30 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 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 4302 Fig Aldehydes release zinc from alcohol dehydrogenase (ADH) (A) The kinetics of zinc transfer from ADH (0.5 lM, 12.8 unitsỈmL)1) to 4-(2-pyridylazo)-resorcinol (PAR) (100 lM) was monitored spectrophotometrically 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 immediately after aldehydes were added to the solution Line 1: control (no aldehyde) Line 2: mM acetaldehyde Line 3: mM acrolein (B) Effect of aldehydes on ADH activity ADH (0.15 units) was incubated with either mM acetaldehyde or 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 the reaction of DTNB with thiols, the excess of acetaldehyde in the above reaction mixture was removed enzymatically with yeast ADH [1 unitỈmL)1 (one unit converts micromole ethanol per at pH 8.8, 25 °C)] and NADH (2 mm) before DTNB was added As virtually the same absorbance reading was recorded, 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 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Hao and W Maret Aldehydes and zinc metabolism Fig Effect of acetaldehyde on the thiol reactivity of metallothionein isoform (MT2) MT2 (1.2 lM) was incubated without (line 1) or with (line 2) acetaldehyde (1 mM) for 30 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: mM acetaldehyde 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 ligands 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 measurement 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 concentrations When cells were treated with 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, Fig Aldehydes increase the amount of available intracellular zinc in HepG2 cells (A) HepG2 cells (1 · 106) were treated with acetaldehyde (1 mM) or acrolein (10 lM) for 30 The cells were collected 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 lM disulfiram for h to inhibit aldehyde dehydrogenases After addition of 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 ethanol is added to the cells Disulfiram decreases the fluorescence intensity slightly (see text) The asterisk indicates significance at P < 0.05 while disulfiram alone lowered the amount of zinc available to the probe due to its metal-chelating capacity [16] FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4303 Aldehydes and zinc metabolism Q Hao and W Maret 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 ngỈ(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 ngỈ(g cells))1 To examine whether ethanol or its metabolic product acetaldehyde induces MT, inhibitors of ADH [4-methylpyrazole hydrochloride (4-MP)] and aldehyde dehydrogenase (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 acetaldehyde to acetic acid, increasing the concentrations of acetaldehyde The concentration of MT in 4-MP ⁄ ethanol-treated cells does not change, whereas it increases to 118 ngỈ(g cells))1 in disulfiram ⁄ ethanol-treated cells (Fig 6A) Treatment of HepG2 cells with mm acetaldehyde 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 zincmediated biological processes, the effects of acetaldehyde 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 acetaldehyde 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 Aldehydes increase the expression levels of metallothionein (MT) in HepG2 cells (A) Ethanol (5 mM), 4-methylpyrazole hydrochloride (4-MP) ⁄ ethanol (5 lM ⁄ mM), disulfiram ⁄ ethanol (5 lM ⁄ mM) or acetaldehyde (1 mM) were incubated with · 106 HepG2 cells for 12 h (B) Acrolein (10 lM) was incubated with · 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 significance at P < 0.05 No significant difference was found for 4-MP ⁄ ethanol treatment 4304 Fig 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 · 106 HepG2 cells for 12 h Control or treated cells were collected, washed, and homogenized PTP activity was measured with a fluorescent phosphotyrosine peptide An aliquot of the homogenized cells was incubated with lM N,N,N¢,N¢-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) for 30 before measurement of PTP activity –, without TPEN; +, with TPEN Emission wavelength 395 nm, excitation wavelength 328 nm Data are presented as means ± SD of triplicate determinations The asterisk indicates significance at P < 0.05 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Hao and W Maret 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 Oxidation 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 glutathione disulfide, nitric oxide and reducible seleniumcontaining compounds release zinc from proteins with Zn–SCys sites [19–21] Based on the above results, aldehydes 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 aldehydes 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] Therefore, it is critical to prepare fresh stock solutions from the anhydrous aldehyde immediately before the experiment In addition, the two aldehydes discussed, acrolein and acetaldehyde, react differently with sulfhydryls Acetaldehyde reacts via the aldehyde group, whereas acrolein, an a,b-unsaturated aldehyde, forms a Michael adduct The zinc-releasing activity of aldehydes has implications for toxicologic and pathobiochemical 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 acrolein are the major aldehyde products of lipid peroxidation Acrolein is also formed from spermine and spermidine by amine oxidases [23] In the brain of Alzheimer’s disease victims, the concentrations of acrolein and 4-HNE increase 7–8-fold [24–26] For reference, basal values in hippocampus are 0.3 and Aldehydes and zinc metabolism 0.265 nmolỈ(mg protein))1, respectively In acute iron loading ⁄ toxicosis, cytotoxic aldehydes increase through lipid peroxidation, which is initiated by Fenton chemistry-generated free radicals [27] In diabetes, there are pathways for the increased formation of a-ketoaldehydes such as glyoxal and methylglyoxal from glyceraldehyde 3-phosphate Autoxidation of a-hydroxyaldehydes to a-ketoaldehydes generates hydrogen peroxide, 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 protein Keap1, a zinc metalloprotein with Zn–SCys coordination 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 concomitant zinc release provide direct experimental support 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 protein-bound [29] However, acetaldehyde concentrations are significantly higher when alcoholic beverages are consumed, in individuals with an inactive mitochondrial aldehyde dehydrogenase or in alcoholic patients under treatment with disulfiram or other alcoholsensitizing drugs In animals treated with aldehyde dehydrogenase inhibitors and ethanol, blood acetaldehyde can reach concentrations of almost 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 ensuing 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 magnitude 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 cellular zinc have profound biological effects Thus, an FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4305 Aldehydes and zinc metabolism Q Hao and W Maret increase from 520 to 870 pm ‘free’ zinc is characteristic for a transition between normal and diabetic cardiomyocytes [34] Changes from picomolar to low nanomolar concentrations of zinc affect gene expression in cardiomyocytes [35] Similarly, low nanomolar concentrations of zinc inhibit phosphorylation signaling, metabolic enzymes, and mitochondrial respiration [18,36,37] Because a very potent zinc signal is generated, aldehyde-induced zinc release from proteins is significant for even relatively small increases of aldehyde 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 Acetaldehyde 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 interaction of aldehydes with protein sulfhydryls, an indirect action of aldehydes via binding of released zinc to protein 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 supplementation activates metal response element (MRE)-binding transcription factor-1 (MTF-1) and transcription of the apoprotein thionein, which also reacts with aldehydes Indeed, addition of a hexapeptide that contains three of the 20 cysteines of thionein suppresses the formation of protein–hydroxynonenal adducts in retinal pigmented epithelial cells [49] Most cells have concentrations 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 4306 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 acetaldehyde with the Zn–SCys bonds in ADH and concomitant zinc release underscores the significance of these reactions for compromising the functions of other proteins 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 conditional zinc deficiency will increase cellular damage by aldehydes Zinc deficiency elicits oxidative stress [55], thus increasing lipid peroxidation and aldehyde concentrations, releasing more zinc from proteins, and initiating a vicious cycle that will exacerbate zinc deficiency 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 (dithiothreitol) from Calbiochem (San Diego, CA), and Zinquin ester from Molecular Probes (Eugene, OR) All other chemicals 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], mg of MT2 was dissolved in 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 with HCl, and centrifuged at 10 000 g for (Eppendorf centrifuge model 5415C, Hamburg, Germany) to remove any precipitate The clear supernatant was then loaded onto a Sephadex 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 ẳ 48 000 m)1ặcm)1) and assay of thiols A ten-fold molar excess of zinc sulfate was added to the nitrogen gaspurged solution of thionein, and the pH value was adjusted to 8.6 by slowly adding nitrogen gas-purged m Tris base The sample was concentrated to about mL by centrifuga- FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Hao and W Maret tion for 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 mLỈh)1 MT fractions were pooled after measuring the concentration of protein (A220 ¼ 159 000 m)1Ỉcm)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 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 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 minimize 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 absorbance at 220 nm and by assaying its thiols with 2,2¢-dithiodipyridine (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 mgặL)1 2,2Â-dithiodipyridine [57] and taking absorbance readings (A343 ẳ 7600 m)1ặcm)1) with a Beckman-Coulter DUÒ 800 UV–visible spectrophotometer (Fullerton, CA) PAR metal transfer assay Metallochromic indicators provide a rapid means of investigating 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 mm stock solution in 20 mm Tris ⁄ HCl, pH 7.4) were incubated with or without aldehydes and the absorbance change was followed (A500 ẳ 65 000 m)1ặcm)1) Aldehyde stock solutions (100 mm) were prepared immediately 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 ‘malondialdehyde’ Evaporation of acetaldehyde during measurements was minimized by sealing the cuvettes with Parafilm A 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 mm glyceraldehyde with PAR was subtracted The experiments were repeated at least three times Aldehydes (1 mm) were also Aldehydes and zinc metabolism 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 formaldehyde 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 spectrophotometrically at 412 nm (25 °C) The number of sulfhydryls modified by acetaldehyde was determined by incubating MT or thionein with acetaldehyde for 30 min, removing the excess of aldehyde with unitỈmL)1 of yeast ADH, 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 established protocol [60] Briefly, mg of MT2 was concentrated with Centricon centrifugal microconcentrators (MWCO 3000; Millipore), and diluted with 0.5 m sodium borate buffer (pH 9.2) to a final concentration of 10 mgỈmL)1, and solid potassium cyanate was added to a final concentration of 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 · cm) Protein concentrations were determined spectrophotometrically at 220 nm Yeast ADH assay ADH activity was determined with acetaldehyde as substrate The assay was performed in 0.1 m Tris ⁄ HCl (pH 8.0), 0.67 mm NADH, 100 mm KCl, 10 mm 2-mercaptoethanol, 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 lL) with an equal volume of either mm acetaldehyde or mm acrolein and incubating for 20 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 Collection, Manassas, VA) were cultured in DMEM containing FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS 4307 Aldehydes and zinc metabolism Q Hao and W Maret 4.5 gỈL)1 glucose, supplemented with 10% (v ⁄ v) FBS (defined; Hyclone, Salt Lake City, UT), 0.12 mgỈmL)1 streptomycin sulfate, and 0.1 mgỈmL)1 gentamicin sulfate Cells were maintained at 5% CO2 and 37 °C in a humidified atmosphere All other cell culture products were purchased 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 concentrations of mm and 10 lm, respectively, and incubated for 30 Additionally, cells were incubated for h with tetraethylthiuram disulfide (disulfiram), an aldehyde dehydrogenase inhibitor, at a final concentration of lm, ethanol was added to each well to a final concentration of 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 concentration 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 (Rockford, 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, · 106 cells were incubated with the dye as described above, washed three times with Dulbecco’s NaCl ⁄ Pi, and detached in 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 acquisition and processing electronics from ISS (Champaign, IL) Fluorescence intensities are the averages of three measurements The working range for measurements of fluorescence intensity was determined by adding zinc and the ionophore pyrithione (20 lm final concentrations for both) The measured 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 14 000 g (Eppendorf centrifuge model 5415C) was mixed with the same volume of a CdCl2 solution (2 lgỈmL)1), and incubated at 25 °C for 10 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 The samples were then placed on ice for min, and centrifuged at 14 000 g for (Eppendorf centrifuge model 5415C); another aliquot of 100 lL of 2% hemoglobin solution was then added to the supernatant, 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 concentrations in the supernatants were determined by atomic absorption spectrophotometry (Perkin-Elmer model 5100) MT concentrations were calculated based on an MT ⁄ Cd stoichiometry of : PTP assay PTP activity in HepG2 cells was determined with a tyrosine-phosphorylated oligopeptide MCA-Gly-Asp-Ala-GluTyr(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 mm Tris-(2-carboxyethyl)-phosphine (Molecular Probes) and lm substrate in a total volume of mL After 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 by adding 10 lL of chymotrypsin ⁄ 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 monitored at 328 ⁄ 395 nm, with slit widths of 1.5 nm (excitation) and 10 nm (emission), using an SLM-8000 spectrofluorimeter Background fluorescence was determined 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 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-Elvehjem homogenizer with at least 20 strokes Microscopic inspection verified that 90% of the cells were broken The supernatant (200 lL) obtained after centrifugation at 4308 Acknowledgements We thank Dr V M Sadagopa Ramanujam (The University of Texas Medical 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modification of metallothionein by carbamylation and guanidination Methods Enzymol 205, 433–437 Eaton DL & Cherian MG (1991) Determination of metallothionein in tissues by cadmium-hemoglobin affinity assay Methods Enzymol 205, 83–88 FEBS Journal 273 (2006) 4300–4310 ª 2006 The Authors Journal compilation ª 2006 FEBS ... ligands 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. .. that elevated levels of aldehydes affect zinc metabolism and that zinc release and ensuing binding of zinc to other proteins is one aspect of the molecular actions of aldehydes that are generated... lipid peroxidation and metabolism of ethanol Zinc signals generated by aldehydes The concentrations of ‘free’ zinc are orders of magnitude smaller than those of total cellular zinc, which is a few