protective mechanism of metallothionein against copper–1,10-phenanthroline induced dna cleavage

Chemico-Biological Interactions 125 (2000) 221–232

Protective mechanism of metallothioneinagainst copper–1,10-phenanthroline induced

DNA cleavage

Jianhua Yang, R.N.S. Wong, M.S. Yang *Department of Biology, Hong Kong Baptist Uni6ersity, Kowloon Tong, Hong Kong

Received 30 August 1999; accepted 7 January 2000

Abstract

Metallothionein (MT) has been shown to protect DNA against cleavage induced by avariety of mutagenic agents. The mechanism has been attributed to its ability to eitherchelate transitional metals that participate in the Fenton reaction, or scavenge free radicalsby means of the abundant cystenyl residues of the proteins. In the present study, theprotective action of MT against DNA cleavage by the copper–1,10-phenanthroline[(OP)2Cu+] complex was studied in situ. At 0.1 mM, MT inhibited the (OP)2Cu+ inducedDNA cleavage by about 50% (IC50:0.1 mM). At 2.5 mM, the cleavage activity wascompletely inhibited. Similar to MT, cysteine can protect against DNA cleavage by(OP)2Cu+ (IC50 of approximately 3 mM), however, its action was 1500-fold less efficientthan MT. The combined action of MT and cysteine was additive. Reduced glutathione (1and 10 mM) did not protect the (OP)2Cu+ induced DNA cleavage. Sodium azide couldinhibit the cleavage only at high concentrations (IC40:25 mM). Spectrophotometric analy-sis showed that MT can inhibit the formation of the DNA[(OP)2Cu+] complex possibly bychelating Cu. It can also cause a dissociation of the complex after it was formed. In the latercase, the mechanism through which MT protects against the DNA cleavage might occurwhen MT fitted in closely with the complex, competing with the hydroxyl groups of thenucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.© 2000 Elsevier Science Ireland Ltd. All rights reserved.

www.elsevier.com/locate/chembiont

Abbre6iations: MT, metallothionein (MW�6100 Da); EDTA, ethylenediaminetetra-acetic acid; GSH,glutathione (MW�300 Da); scDNA, supercoiled DNA; ocDNA, open circular DNA; Cu, copper(atomic wt. 63.5); Cd, cadmium (atomic wt. 112.4); Zn, zinc (atomic wt. 91.2).

* Corresponding author. Tel.: +852-2339-7058; fax: +852-2336-1400.E-mail address: [email protected] (M.S. Yang)

0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (00 )00148 -4

J. Yang et al. / Chemico-Biological Interactions 125 (2000) 221–232222

Keywords: Metallothionein; Fenton reaction; Copper–1,10-phenanthroline [(OP)2Cu+] complex

1. Introduction

Metallothioneins (MT) is a group of low molecular weight, cytosolic-rich (30% oftotal amino acid composition), metal-binding protein with high affinity for variousmetals including Cd, Zn, Cu, Hg, Ag etc. [1]. MT is generally considered a cytosolicprotein. Recently, increasing evidence showed that the protein also resides in thenucleus of rapidly growing cells [2–4]. The exact function of MT in the nucleus isnot clear. MT was said to modulate the level of Zn in the nucleus which in turnregulate gene transcription [4–6]. It was also suggested that the protein might playan important role as an endogenous anti-mutagenic agent through protectionagainst a variety of oxidative damages [7,8].

The mode of action of MT in protection against DNA damage was studied. Itwas suggested that MT may act as chelator of transition metals such as Cu ions toterminate the Fenton-like reaction [9–11]. The cystenyl groups of the protein mayalso act by scavenging the free radicals generated [7,10,12]. However, in in vitrostudies, the efficiency of MT to protect against DNA cleavage from oxidativedamage was much higher than that of GSH [13]. The results suggested that the freeradical scavenging action of MT might be more complicate than through thecystenyl residues alone. In order to clarify the mode of action of MT, protectiveaction must be studied in a well-established model in which the mode of action ofDNA cleavage is understood.

1,10-Phenanthroline (OP) is a potential mutagen. Under physiological conditions,OP, in the presence of Cu and a reducing agent such as ascorbic acid, mercap-toethanol or molecular oxygen, is able to induce the degradation of DNA by anoxidative mechanism [14–16,33]. The reaction involved the formation of (OP)2Cu+

complex which, in turn, interacted with the minor groove of DNA [16–18]. TheDNA[(OP)2Cu+] complex formed strongly absorbs light at 435 nm [19]. DNAstrand break occurred via proton abstraction from the deoxyribose C1% or C4%[18,20–24]. The DNA adducts formed were identified to be typical hydroxyl radicalinduced products similar to those produced upon exposure to radiation, hypoxan-thine/xanthine oxidase, or hydrogen peroxide in the presence of transition metalions [25]. Despite the detailed analysis, hydroxyl radical scavengers such as manni-tol and sodium formate could not inhibit DNA cleavage by the complex [25]. In thepresent study, experiments were performed to investigate the potential of MT toprotect against DNA cleavage by (OP)2Cu+. Furthermore, the use of this well-es-tablished system could, in turn, provide a means for studying the mode of action ofMT.

2. Materials and methods

Autoclaved double distilled water (Millipore) was used in all experiments.Tris–acetate, EDTA and boric acid were from Boehringer Mannheim DmbH,

J. Yang et al. / Chemico-Biological Interactions 125 (2000) 221–232 223

Fluka (Germany) and Huagongchang (Beijing, China) respectively. 1,10-Phenan-throline (MW 198.2), ascorbic acid (MW 176.1), HEPES, potassium chloride andall other reagents were from Sigma Chemical Co. (St. Louis, MO) except asindicated. DNA from herring sperm for the spectrum analyses was ordered fromServa Company.

2.1. Purity of MT

Cd/Zn-MT was a kind gift from Dr Liberatorie of DuPont Laboratory. Theprotein was prepared by the method described in [26] and is known to contain bothMT-I and MT-II isoforms. The purity of MT was analyzed by SDS-PAGE on a25% gel according to the recommended procedure by the manufacturer (PhastSystem, Pharmacia, USA). The amount of protein was determined by weight. Cdand Zn content in the protein were determined by graphite furnace atomicabsorption spectrometry (model 3110, Perkin Elmer, equipped with an HGA 600graphite furnace) at wavelengths of 228.8 and 213.9 nm, respectively.

2.2. Determination of DNA clea6age acti6ity

DNA cleavage assay was performed according to the method described in [27].Experiment was performed in a 20 ml NaH2PO4/Na2HPO4 buffer (pH 6.7) contain-ing 0.20 mg/ml supercoiled plasmid DNA (pBluescript II KS+ ), 12 mM CuCl2, 25mM 1,10-phenanthroline, 75 mM ascorbic acid. The reaction mixture was incubatedat 37oC. After incubating for 25 min, 1 ml of 0.1 M EDTA (final concentration 5mM) was added to terminate the reaction [28]. Two microliters of bromophenolblue was then added and the mixture was subjected to electrophoresis on 1.0%agarose gel at 80 V for 80 min. The gel was stained with ethidium bromide for 5min and photographed with a Polaroid MP4 land camera under illumination at 365nm. The DNA cleavage activity was measured by digitizing the photographic imageand quantified (QuantiScan from Biosoft). The ratio of open circular (oc) relativeto supercoiled (sc) density was determined. The degree of DNA cleavage activitywas expressed in terms of the percentage of conversion of the scDNA to ocDNAaccording to the following equation:

% DNA cleavage activity=[% of scDNA]control− [% of scDNA]sample

[% of scDNA]control

×100

2.3. Effect of different chemicals on (OP)2Cu+ induced DNA clea6age

Different concentrations of (1) Cd/Zn-MT (0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM);(2) cysteine (0. 0.5, 1, 2.5, 5 and 7.5 mM); (3) GSH (1 and 10 mM); (4) sodiumazaid (0, 1, 5, 15, 20, 25 mM); (5) CdCl2 (25 mM); (6) ZnCl2 (10 mM), orcombinations of (7) CdCl2 (25 mM) and ZnCl2 (10 mM) (a 5:2 raio similar to thatexpected for 5 mM MT); (8) Cd/Zn-MT (0.01, 0.05 and 0.1 mM) and cysteine (5mM), were added to the incubation mixture as described in the previous section.


The percentage of DNA cleavage was determined. Each study was carried out intriplicates. Statistical differences in DNA cleavage between the different treatmentswere analyzed by ANOVA. Significant difference between the treatments was set atPB0.05.

2.4. Determination of (OP)2Cu+ and DNA interaction by spectrophotometricmethod

A characteristic feature of the interaction between Cu, OP, ascorbic acid andDNA is the formation of a complex with strong absorption spectra at 435 nm [19].Spectral titration was performed to monitor the formation of the complex. Thetitration was carried out under anaerobic condition according to that specifiedpreviously (160 mM DNA, 50 mM OP, 20 mM CuCl2 and 2 mM ascorbic acid [19]),except that instead of argon, the mixture was flushed with nitrogen. The effect ofMT on the formation of complex can be monitored on the spectrophoto-meter (Hitachi U-2000). MT was added either at the same time when Cu, OP andascorbic acid were added to DNA, or 20 min after Cu, OP and ascorbic acid wereadded. In the later case, the DNA[(OP)2Cu+] complex would have already beenformed.

2.5. Identification of the Cu containing fraction after MT and DNA[(OP)2Cu+]interaction

To study whether metal exchange occurred between MT the DNA[(OP)2Cu+] inthe reaction system, the DNA[(OP)2Cu+] was separated on a Sephadex G-75column immediately after addition of MT. The column was eluted with 20 mMTris–HCl pH 7.5. DNA content in each of the 5-ml fractions collectedwas monitored by absorbance at 260 nm. Copper was determined by graphitefurnace atomic absorption spectrometry, and MT by differential pulse polarogra-phy [29].

3. Results

3.1. Purity of MT used

Fig. 1 shows the SDS-PAGE profile of Cd/Zn-MT used in the experiment. Theprotein consists of two bands believed to be MT-I and MT-II as specified in [26].The molar ratio of Cd and Zn in the MT sample was 5:2.

3.2. DNA clea6age by (OP)2Cu+

Fig. 2 shows the DNA cleavage activity of the (OP)2Cu+ complex formed fromvarious mixtures of CuCl2, OP and ascorbic acid. The combination of 25 mMOP+12 mM CuCl2+75 mM ascorbic acid (lane 4), resulted in a complete conver-


sion of scDNA to ocDNA. This combination was selected for use in all subsequentexperiments.

3.3. MT protects against (OP)2Cu+ induced DNA clea6age

Fig. 3 shows MT inhibited the DNA cleavage activity by the (OP)2Cu+ complex.The degree of inhibition was concentration-dependent. At about 0.1 mM, Cd/Zn-MT significantly reduced (PB0.05) DNA cleavage by about 50%. The DNAcleavage activity of the (OP)2Cu+ complex was almost completely inhibited by MTat concentrations above 2.5 mM. Fig. 4 shows that while 0.1 mM MT caused a 50%reduction in DNA cleavage induced by CuOP, addition of Cd and Zn alone or in

Fig. 1. SDS-PAGE profile of Cd/Zn-MT used.

Fig. 2. Conversion of DNA from supercoiled form (sc) to open circular form (oc) by addition ofdifferent concentrations of 1,10-phenanthroline (OP), CuCl2 and ascorbic acid (asc). Lane 1, plasmidDNA (control); lane 2, +25 mM OP+2.5 mM CuCl2+25 mM asc; lane 3, +25 mM OP+5.0 mMCuCl2+50 mM asc; Lane 4, +25 mM OP+12.0 mM CuCl2+75 mM asc; lane 5, +50 mM OP+12.0mM CuCl2+25 mM asc; lane 6,+50 mM OP+25.0 mM CuCl2+50 mM asc; lane 7, +50 mM OP+50.0mM CuCl2+75 mM asc; lane 8, +75 mM OP+12.5 mM CuCl2+25 mM asc; lane 9, +75 mMOP+25.0 mM CuCl2+50 mM asc.


Fig. 3. Protective effect of Cd/Zn-MT on DNA strand break induced by [(OP)2Cu+] complex. Reactionmixture contains DNA, and conditions specified in lane 4 of Fig. 1.

Fig. 4. Effect of Cd*, Zn* and GSH on the [(OP)2Cu+] induced DNA cleavage (* the concentrationsused were equivalent to that in 5 mM Cd/Zn-MT).

a 5:2 ratio were unable to protect DNA cleavage. The results suggested that theprotective action was not due to the metals in the protein.

3.4. SH groups are responsible for protection against DNA clea6age

Fig. 5 shows that cysteine can reduce DNA cleavage activity by the (OP)2Cu+

complex. However, the concentration of cysteine needed to inhibit the reaction by50% was approximately 3 mM. Taking into account that 1 M of MT contains 20 M


of cysteines, the efficacy of MT in inhibiting DNA cleavage is 1500-fold higher thanthat of cysteine. The combined effect of MT and cysteine on the DNA cleavageactivity of the the (OP)2Cu+ complex was additive (Fig. 6). Glutathione atconcentrations of 1 and 10 mM did not protect against the the (OP)2Cu+ complexinduced DNA cleavage activity. The result demonstrated that the protective actionof Cd/Zn-MT was due to the presence of SH groups. Furthermore, the SH groupson MT is more efficient than if it were in the form of cysteine or glutathione.

Fig. 5. Protective effect of cysteine on DNA cleavage induced by the [(OP)2Cu+] complex.

Fig. 6. Combined effect of cysteine and Cd/Zn-MT on the [(OP)2Cu+] induced DNA cleavage.


Fig. 7. Effect of sodium azide on the [(OP)2Cu+] induced DNA cleavage.

3.5. Protecti6e action is not through direct free radical sca6enger

The protective action of Cd/Zn-MT against DNA cleavage by the (OP)2Cu+

complex had been suggested to be due to free radical generation. However, inprevious studies, free radical scavengers, such as mannitol, were unable to preventDNA cleavage [25]. Similarly, Fig. 7 shows that high concentration (25 mM) ofsodium azide, which is also a known free radical scavenger, was only able to reducethe DNA cleavage activity by 40%. These results concurs with previous studieswhich demonstrated that the mechanism of (OP)2Cu+ complex induced DNAcleavage is not simply by scavenging free radicals.

3.6. The possible protecti6e mechanism of MT against (OP)2Cu+ induced DNAclea6age

The interaction between Cu++, OP, ascorbic acid and DNA resulted in theformation of DNA. [(OP)2Cu+] complex which has a characteristic absorbency at435 nm [19]. Fig. 8 shows the kinetic of the complex formation. Upon addition ofall reactants, the complex was formed within 5 min and was stable for over 20 min.When MT was added prior to addition of ascorbic acid, the formation of the(OP)2Cu+ complex was prevented (Fig. 9). As the affinity of different metals to MTfollows the order ZnBCdBCu [1], it is possible that the MT could easily bind Cuand prevent the complex formation. However, when MT was added 20 min afterthe formation of the complex, the peak absorbance also disappeared. This can onlybe possible if MT could reach into the DNA[(OP)2Cu+] complex causing adissociation of the complex.


Fig. 10 shows the Sephadex G-75 chromatographic profile of the chemicalmixture after MT was added to the DNA[(OP)2Cu+] complex. Cu ions wereseparated into two distinct fractions, one associated with DNA and the other withMT. The molar ratio of Cu to MT was approximately 11:1, very close to thetheoretical ratio of 12 [1]. The result provided evidence to demonstrate that metalexchange occurred and MT could chelate Cu after the DNA[(OP)2Cu+]complex was formed. The results of these experiments showed that the mostprobable way for MT to protect against [(OP)2Cu+] induced DNA cleavage is tochelate Cu. The protein could not only bind Cu prior to the formation of theDNA[(OP)2Cu+] complex, it could also removed Cu from the complex after it wasformed.

Fig. 8. Formation of the DNA[(OP)2Cu+] complex as detected at wavelength of 435 nm. Reaction wasperformed under anaerobic condition in a mixture specified in [19].

Fig. 9. Effect of Cd/Zn-MT (2mM) added to the mixture at 20 minute after the initiation ofDNA[(OP)2Cu+] complex formation.


Fig. 10. Separation of the DNA[(OP)2Cu+] complex on a Sephdex G-75 column after Cd/Zn-MT* wasadded to the mixture(as in Fig. 8). Cu content in each fraction was measured by graphite furnace atomicabsorption spectrometry, and MT by differential pulse polarography (* Cd/Zn-MT was added 20 minafter the initiation of DNA[(OP)2Cu+] complex).

4. Discussion

The molecular structure of MT demonstrated that the protein could eitherscavenge free radicals or bind to specific metals such as Cu. Both of these functionsmight be involved in its ability to protect against chemically induced DNAcleavage. Using the well-established model of [(OP)2Cu+] induced DNA damage,the mode of action of MT in this specific system indicated that the protective actionof MT is superior to other compounds that simply scavenge free radicals e.g.cysteine, GSH and sodium azide. The most likely possibility is through chelatingCu, thus, terminating the Fenton-like reaction. Also, the interaction can even occurthrough a stereospecific interaction between MT and the DNA[(OP)2Cu+] complexafter the complex was formed. A stereospecific interaction between MT and othermolecules had been demonstrated in a study on the mechanism of Zn exchangebetween MT and the Gal4 protein [30]. Metal transfer between proteins occurredthrough ligand exchange in which the two proteins interact through close contact,placing the metal at appropriate sites for transfer to take place. Thus, themetal-binding characteristic of MT in cells was not only a means of protectingcellular organelles against metal toxicity, but also agianst processes which eventu-ally led to free radical generation.

The present study offered a model for studying the mechanism of MT to protectagainst [(OP)2Cu+] induced DNA damage. The protective action of MT againstother toxic chemicals might be different. A number of studies examined theprotective action of MT against DNA damage by Fe+++ [10,11] and Fe+++

complex [31,32]. As MT is not a good chelator for ferric salt, it was demonstrated


that MT was unable to protect DNA damage by free iron [11,32]. But MT was ableto protect DNA against damage by ferric salt in the presence of either EDTA [31]or nitrilotriacetic acid [32]. While the effectiveness of MT against [(OP)2Cu+]induced DNA damage was 1500-fold higher than that with cysteine and still higherwith GSH, the effectiveness of MT on Fe-complex induced DNA damage was only5-fold better than GSH [32], It is likely that MT simply acted as a free radicalscavenger in this system.

Acknowledgements

The study was supported by the Faculty Research Grant (FRG/95-96/II-22) ofthe Hong Kong Baptist University.

References

[1] J.H.R. Kagi, Y. Kojima, Chemistry and biochemistry of metallothionein, Exp. Suppl. 52 (1987)25–61.

[2] D.M. Templeton, D. Banerjee, M.G. Cherian, Metallothionein synthesis and localization in relationto metal storage in rat liver during gestation, Can. J. Biochem. Cell Biol. 63 (1985) 16–22.

[3] N. Nartey, M.G. Cherian, D. Banerjee, Immunohistochemical localization of metallothionein inhuman thyroid tumors, Am. J. Pathol. 129 (1987) 177–182.

[4] C. Hogstrand, N.J. Gassman, B. Popova, C.M. Wood, P.J. Walsh, The physiology of massive zincaccumulation in the liver of female squirrelfish and its relationship to reporduction, J. Exp. Biol.199 (1996) 2543–2554.

[5] D.H. Hamer, Metallothionein, Ann. Rev. Biochem. 55 (1986) 913–951.[6] J. Zheng, R. Heuchel, W. Schaffner, J.H.R. Kagi, Thionein (apometallothionein) can modulate

DNA binding and transcription activation by zinc finger containing factor Sp1, FEBS Lett. 279(1991) 310–312.

[7] L.S. Chubatsu, R. Meneghini, Metallothionein protects DNA from oxidative damage, Biochem. J.291 (1993) 193–198.

[8] M.G. Cherian, The significance of the nuclear and cytoplasmic location of metallothioneine inhuman lliver and tumor cells, Environ. Health Perspect. 102 (1994) 131–133.

[9] M. Good, M. Vasak, Iron(II)-substituted metallothionein: evidence for the existence of iron–thio-late clusters, Biochemistry 25 (1986) 8353–8356.

[10] L. Cai, J. Koropatnick, M.G. Cherian, Protection from metal-induced DNA damage by metalloth-ionein in an in vitro system, in: B. Sarkar (Ed.), Genetic Response to Metals, Marcel Dekker, Inc,New York, Basel, Hong Kong, 1994, pp. 101–119.

[11] L. Cai, J. Koropatnick, M.G. Cherian, Metallohtionein protects DNA from copper-induced, butnot iron-induced cleavage in vitro, Chem. Biol. Interact. 96 (1995) 143–155.

[12] E.I. Goncharova, T.G. Rossman, The antimutagenic effects of metallothionein may involve freeradical scavenging, in: B. Sarkar (Ed.), Genetic Response to Metals, Marcel Dekker, New York,1995, pp. 87–100.

[13] T. Miura, S. Muraoka, T. Ogiso, Antrioxidant activity of metallothionein compared with reducedglutathione, Life Sci. 60 (1997) 301–309.

[14] D.S. Sigman, D.R. Graham, V. D’Aurora, A.M. Stern, Oxygen-dependent cleavage of DNA by the1,10-phenanthroline-cuprous complexinhibition of escherichia coli DNA polymerase I, J. Biol.Chem. 254 (1979) 12269–12272.


[15] D.S. Sigman, Nuclease activity of 1,10-phenanthroline–copper ion, Acc. Chem. Res. 19 (1986)180–186.

[16] J.M. Veal, R.L. Rill, Sequence specificity of DNA cleavage by bis-(1,10-phenanthroline) copper(I),Biochemistry 27 (1988) 1822–1827.

[17] A. Spassky, D.S. Sigman, Nuclease activity of 1,10-phenanthroline–copper ion. Comformationalanalysis and footprinting of the lac Operon, Biochemistry 24 (1985) 8050–8056.

[18] M. Kuwabara, C. Yoon, T. Goyne, T. Thederahn, D. Sigman, Nuclease activity of 1,10-phenan-throline–copper ion: reaction with CGCGAATTCGCG and its complexs with netropsin andEcoRI, Biochemistry 25 (1986) 7401–7408.

[19] J.M. Veal, R.L. Rill, Noncovalent DNA binding of bis(1,10-phenanthroline) copper (I) and relatedcompounds, Biochemistry 30 (1991) 1132–1140.

[20] S. Goldstein, G. Czapski, Mechanisms of the reaction of some copper complexes in the presence ofDNA with O2

− �, H2O2 and molecular oxygen, J. Am. Chem. Soc. 108 (1986) 2244–2250.

[21] L. Pope, K. Reich, D. Graham, D. Sigma, Products of DNA cleavage by the 1,10-phenanthroline–copper complex — inhibitor of escherichia coli DNA polymerase I, J. Biol. Chem. 257 (1982)12121–12128.

[22] G.R. Johnson, N.B. Nazhat, Kinetics and mechanism of the reaction of the bis(1,10-phenanthro-line) copper(I) ion with hydrogen peroxide in aqueous solution, J. Am. Chem. Soc. 109 (1987)1990–1994.

[23] S. Goldstein, G. Czapski, H. Cohen, D. Meyerstein, Formation and decomposition of transientcomplexes with a copper–carbon d-bond in the reaction of copper (I) phenanthroline with aliphaticfree radicals. A pulse-radiolysis study, Inorg. Chem. 27 (1988) 4130–4135.

[24] T.B. Thederahn, M.D. Kuwabara, T.A. Larsen, D.S. Sigman, Nuclease activity of 1,10-phenanthro-line–copper: kinetic mechanism, J. Am. Chem. Soc. 111 (1989) 4941–4946.

[25] M. Dizdaroglu, O.I. Aruoma, B. Halliwell, Modification of bases in DNA by copper ion–1,10-phenanthroline complexes, Biochemistry 29 (1990) 8447–8451.

[26] R.D. Comeau, K.W. McDonald, G.L. Tolman, M. Vasak, F.A. Liberatore, Gram scale purificationand preparation of rabbit liver zinc metallothionein, Prep. Biochem. 22 (1992) 151–164.

[27] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning; A laboratory manual, second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989.

[28] J.M.C. Gutteridge, B. Halliwell, The role of superoxide and hydroxyl radicals in the degradation ofDNA and deoxyribose induced by a copper-phenanthroline complex, Biochem. Pharmacol. 31(1982) 2801–2805.

[29] J.H. Yang, Y.G. Cao, M.S. Yang, Determination of metallothionein content in hepatoma cells bydifferential pulse polarography, Chem. Biol. Interact. 115 (1998) 109–116.

[30] W. Maret, K.S. Larsen, B.L. Vallee, Coordinatiion dynamics of biological zinc ‘clusters’ inmetallothioneins and in the DNA-binding domain of the transcription factor Gal4, Proc. Natl.Acad. Sci. USA 94 (1997) 2233–2237.

[31] J. Abel, N. de Reuiter, Inhibition of hydroxyl-radical-generated DNA degradation by metalloth-ioneine, Toxicol. Lett. 47 (1989) 1910196.

[32] L. Cai, G. Tsiapalis, M.G. Cherian, Protective role of zinc-metallothonein on DNA damage in vitroby ferric nitrilotriacetate (Fe-NTS) and ferric salts, Chem. Biol. Interact. 115 (1998) 141–151.

[33] J.M. Veal, K. Merchant, R.L. Rill, The influence of reducing agent and 1,10-phenanthrolineconcentration on DNA cleavage by phenanthroline+copper, Nucl. Acids Res. 19 (1991) 3383–3388.

.

protective mechanism of metallothionein against copper–1,10-phenanthroline induced dna cleavage

Documents