Metallothionein protects DNA from copper-induced but not iron-induced cleavage in vitro

ELSEVIER Chemico-Biological Interactions 96 (1995) 143- I55

Metallothionein protects DNA from copper-induced but not iron-induced cleavage

in vitro

Lu Cai”, James Koropatnicka3b3c, M. George Cherian*”

“Department of Pathology, Health Sciences Centre, University of Western Ontario, London, Ontario, N6A SCl, Canada

bDepartment of Oncology, The University of Western Onfario, London, Ontario, Canada CDeparrment of Microbiology, The University of Western Ontario, London, Ontario, Canada

Received 18 May 1994; revision received 14 September 1994; accepted 27 September 1994

Abstract

Iron and copper ions mediate generation of reactive oxygen radicals from 0, and H,O, by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce double-strand breaks in DNA in vitro in the presence of H,O, and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-binding protein metallothionein (MT) to protect DNA from damage. Strand cleavage was measured by loss of fluorescence after binding to ethidium bromide and by increased mobility of DNA in agarose. The results show that Cu(II), Fe(U) and Fe(II1) all can induce damage to calf thymus DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the concentration of H202. On the other hand, DNA fragmentation was significant only in the presence of high concentrations of Fe(I1) or Fe(II1). Addition of Zn-MT to the reaction mixture prior to addition of Cu(I1) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not effective in protecting DNA from Cu-induced damage, as compared to Zn-MT. The formation of Cu(1) from Cu(I1) in the presence of hydrogen peroxide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT may protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.

Keywords: Metallothionein; Oxidative DNA damage; Copper; Iron; DNA damage protection

* Corresponding author, Tel.: +I 519 6612030; Fax: +I 519 6613370

0009-2797/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0009-2797(94)03585-V

144 L. Cai el ul. / Chemico-Biologicul Interactions 96 (1995) 143-155

1. Introduction

Superoxide radical (02 :) and hydrogen peroxide (Hz02) are generated by normal cellular metabolism [l]. Excessive levels of these species in vivo may be harmful: for example, oxidative DNA damage has been suggested to be critical in aging, mutagenesis and carcinogenesis [2]. However, neither superoxide (02 :) nor HzOz appears to produce DNA strand breakage [3] or modification of DNA bases [4] under physiological conditions. Most of the toxicity of oxygen and hydrogen peroxide in vivo is thought to arise from metal ion-catalyzed production of highly reactive hydroxyl radical (OH ’ ) by the Fenton reaction [3,5,6]. The metal ions could be bound to DNA or chromatin in vivo, or oxidative stress could liberate them from intracellular storage sites with subsequent binding to DNA [7]. DNA is, in fact, damaged in the presence of reactive oxygen radical-producing systems (such as hydrogen peroxide in the presence of iron [4,8,9]). Cu(I1) ions can also participate in formation of OH * through the Fenton reaction and induce DNA damage [5,8]. Furthermore, Cu(I1) appears to be potentially more reactive in mediating oxygen radical-induced cytotoxicity and genotoxicity than Fe ions (8,10-141. Cu(I1) may also enhance the effects of other oxidising agents, including ionizing radiation [l&16] and UV radiation [17]. Therefore, an understanding of the mechanism of iron- or copper-induced DNA damage will be important in developing strategies to prevent these harmful effects.

Metallothioneins (MTs) are small (< 10 kDa) proteins containing a high propor- tion (approximately 30%) of cysteine residues and have been suggested to play a role in both essential metal homeostasis and resistance to heavy metal toxicity [18]. Furthermore, a number of biological functions have been suggested for MT [ 19,201, including an antioxidant role [2 1,221. Because of its high cysteine content, MT may bind and inactivate a variety of radicals, including hydroxyl and organic radicals induced by metals [23], radicals induced by irradiation [24,25] and radicals induced by the xanthine-xanthine oxidase reaction in vivo [26].

Direct addition of MT inhibited Fe-EDTA-induced DNA damage in an aqueous in vitro system [27]. On the other hand, Cd/Zn-MT has been shown to damage supercoiled plasmid DNA through the generation of free radicals [28]. The effect of MT on the ability of free radicals to damage DNA is, therefore, controversial. How- ever, the cellular compartmentalization of MT within nuclei during development [29] and in some human tumours [30] and the importance of metals in chromatin structure [31] and gene regulation [32] suggest a close association of MT and DNA in biological systems, although no direct binding of MT to DNA has been demonstrated. We investigated the protective effect of MT in DNA damage using a free radical-generating system in which DNA was cleaved in vitro in the presence of activated oxygen species. These free radicals were generated from hydrogen peroxide in the presence of ascorbic acid by a Fenton reaction, which was critically dependent on the presence of metals.

2. Materials and methods

2.1. Chemicals L-Ascorbic acid (sodium salt), bathocuproinedisulfonic acid (disodium salt;

L. Cai et al. / Chemico-Biological Interactions 96 (1995) 143-155 145

BCS), cupric chloride dihydrate, deoxyribonucleic acid (sodium salt from calf thymus), EDTA (disodium salt), ethidium bromide, albumin (Fraction V, from bovine plasma) and histone (from calf thymus), were all obtained from Sigma Chemical Co., (St. Louis, MO). Hydrogen peroxide, K2HP04 and KH2P04 were from Fisher Corp. (Misissauga, Ontario). All reagents were analytical grade. Solutions were prepared in sterile deionized water. Stock phosphate buffer solutions were treated with chelex resin (Bio-Rad) by a published method [12,33] to remove metal ions. Metallothionein was isolated from rabbit liver as described earlier [34] and was elec- trophoretically pure Zn-MT.

2.2. Reaction conditions and measurement of DNA oxidation The ethidium bromide binding (EBB) assay [35] based on the formation of a fluor-

escent complex between double-strand DNA and ethidium bromide was used to measure DNA damage. Exposure to free radicals, generated by metal ion/ascorbate/peroxide damaged DNA and inhibited the binding of EB to DNA with decreas- ed fluorescence. Several forms of DNA damage (including strand scission, base oxidation and base liberation) are believed to contribute to the loss of fluorescence [12,35]. Hence, the assay detects a broad range of different DNA lesions. A 2 ml standard reaction mixture contained 20 mM Chelex-treated phosphate buffer (pH 7.0), 66.4 c(g DNA/ml, 50 PM CuC12, 2 mM H202 and 2 mM sodium ascorbate. To measure the effect of MT on DNA damage and/or Cu(1) formation, varying amounts of rabbit liver Zn-MT (lo- 1500 &ml) were added to the reaction mixture after addition of CuClz and before addition of H202. Reactions were carried out at 30°C for 30 min and terminated by addition of a stock solution of EDTA to a final concentration of 10 mM. Ten ~1 of a 1 mM EB solution were added and fluorescence was measured using a Turner Fluorometer (excitation at 510 nm and emission at 590 nm). Since the enhancement in the fluorescence of EB was dose-dependent following interaction with DNA, it was a good measure of the integrity of the DNA. In controls, 100% fluorescence was assessed in a solution containing all reagents (including DNA) except H202. EDTA was added before the CuC12 and ascorbate in 100% fluorescence controls. Zero fluorescence was assessed in a solution identical to the 100% reference solution except DNA. The loss in fluorescence was used as a measure of DNA damage.

2.3. DNA gel electrophoresis Calf thymus DNA samples were prepared as for fluorescence analysis (with the

omission of added ethidium bromide). Thirty ~1 (including 10 pg of DNA plus 3 11 loading buffer 10.25% bromophenol blue, 0.25% xylene cyan01 FF, 30% glycerol in water]) were loaded onto agarose gels (1 .O%) in TAE buffer (40 mM Tris-acetate, 1 mM EDTA) and subjected to electrophoresis in the presence of EB (0.5 &ml in TAE) for 2-3 h. Photographs were taken on a UV (312 nm) transilluminator.

2.4. Formation of Cu(I) Generation of Cu(1) from Cu(I1) in the presence of hydrogen peroxide and ascor-

bate was determined using bathocuproinedisulfonic acid (BCS), according to the method of Li and Trush [lo]. Varying concentrations of Cu(I1) (as CuCl$ and BCS were added to H202 and sodium ascorbate in PBS (pH 7.0) at 30°C to achieve a

146 L. Cai et al. / Chemico-Biological Interactions 96 (1995) 143-155

final BCS concentration of 0.3 mM in a total volume of 2 ml. The concentration of stable BCS Cu(I) complex formed was measured by absorption at 480 nm, in a Beckman DU-9 spectrophotometer.

2.5. Statistical analysis Data were analysed according to the two-tailed Student r-test. The results of at

least 3 measurements were presented as mean f S.D..

3. Results

3.1. Free radical-induced DNA damage in the presence of Fe and Cu Cu(II), Fe(I1) and Fe(II1) salts mediated different amounts of DNA damage in the

presence of hydrogen peroxide and ascorbic acid (Fig. 1). A concentration of 50 PM Cu(I1) reduced EB/DNA fluorescence by more than 95%, while equal molar concentrations of Fe(I1) and Fe(II1) reduced fluorescence by only about 30%. Thus, the ability of copper and iron ions to mediate free radical-induced DNA damage was in the order- Cu(I1) >> Fe(I1) i= Fe(III), similar to previous reports.

1 x loo-

a,

a 2 75-

4 50-

2 0 25-

O- m a

Fig. I. Comparison of DNA damage induced by copper and iron salts in the presence of H,O, and ascor-

bate. In the absence of metals, DNA-EB gave a measurable amount of fluorescence (control), which is

defined as 100%. Cu(II), Fe(II) and Fe(II1) (50 pM in the presence of 2 mM H,O, and 2 mM sodium

ascorbate) incubated with DNA for 30 min caused loss of EB induced DNA fluorescence. The degree of

fluorescence loss is expressed as % of control and indicates the extent of DNA damage (see Materials and

methods). Asterisks indicate a significant difference compared to DNA unexposed to metal salts

(*P < 0.05, ** P < 0.01).


Iron. Fe(I1) mediated DNA damage can be increased by increasing both metal and hydrogen peroxide concentrations. Relatively high Fe(I1) levels (125 PM) and hydrogen peroxide levels (4 mM) were required to induce a 50% loss in fluorescence (data not shown).

Copper. Cu(I1) was a more effective mediator of DNA damage in the presence of hydrogen peroxide than Fe(I1) or Fe(III), causing a 50% loss in fluorescence at less than 5 FM Cu(I1) in the presence of 2 mM hydrogen peroxide (Fig. 2). Maximum loss (95%) of fluorescence in 2.0 mM hydrogen peroxide occurred at 10 PM copper chloride; in 0.2 mM hydrogen peroxide, maximum DNA damage (70%) was induced by 50 PM copper chloride. The generation of Cu(1) from Cu(I1) may be the active ion mediating free radical production. In our experimental conditions, the amount of metal available for Cu(1) formation (and not hydrogen peroxide concentration) was rate-limiting, with Cu(1) formation reaching a plateau at approximately 75 PM CuClz (data not shown). Thus, Cu(I1) and Fe(I1) or Fe(II1) differed in their ability to generate free radicals from H202, and Cu(I1) was the most effective.

3.2. Protective effects of MT in DNA damage induced by copper and iron Zinc-bound MT (Zn-MT) inhibited the ability of Cu(I1) to induce DNA damage

in the presence of hydrogen peroxide (2 mM) and sodium ascorbate (2 mM). The data are presented in Fig. 3 (A,B). The inhibitory effect of Zn-MT on radical- induced DNA damage was dose-dependent in the presence of hydrogen peroxide and ascorbate (Fig. 3A). As little as 40 pg MT/ml showed a significant protective

z r’ / 02 mM H,O, 1 1

“0 50 100 150 200

Fig. 2. Copper induced DNA damage at different concentrations of Cu and H,O,. DNA damage induced by Cu(I1) in the presence of 0.2 or 2.0 mM H,02 for 30 min. The inset graph illustrates DNA damage at the low range of Cu(II) concentrations.

148 L. Cai et al. / Chemico-Biological Interartions 96 (1995) 143-155

.5 20

haO, (mM)

Fig 3. Effect of addition of Zn-MT on Cu(ll)-induced DNA damage. Zn-MT protected DNA from

damage induced by Cu(II) as CuClz in the presence of hydrogen peroxide and sodium ascorbate.

(A) DNA damage induced by 50 PM Cu(II) in the presence of 2 mM H,O, and varying amounts of

Zn-MT (o-1600 &ml). (B) DNA damage induced by 50 pM Cu(I1) in the presence of increasing amounts

of HzO, in the absence of MT (0) plus 4.1 eg Zn-MT/ml (A), or plus 41.2 pg Zn-MT/ml (0). *P < 0.05,

**P < 0.01.

effect up to 0.75 mM H202 (Fig. 3B). At a concentration of 500 &ml, Zn-MT effectively protected DNA damage, at a wide range of copper concentrations (Fig. 4).

Fig. 5 shows the protective effects of 500 &ml Zn-MT on Fe(III)- or Cu(II)- induced DNA damage in the presence of 2 mM H202 and 2 mM sodium ascorbate. Since Cu(I1) is more effective in inducing DNA damage than Fe(III), and 50 PM of Cu induced nearly 100% damage (Fig. l), it is difficult to compare directly the protective effects of Zn-MT at similar Fe or Cu concentrations. Therefore, sufficient Fe(II1) (100 PM) was added to induce the same level of DNA damage as 5 PM Cu(I1). Zn-MT (500 &ml) protected DNA from Cu(II)-induced damage, but did not protect DNA from Fe-induced damage at any concentration (Fig. 5).

The addition of Zn-MT (500 &ml) to a copper chloride/ hydrogen peroxide/ ascorbate system inhibited production of copper ion Cu(I) from Cu(I1) (Fig. 6).


.O 20 40 60 80 100 120 :40 160 180 200

Fig. 4. Protection of Zn-MT on Cu(II)-induced DNA damage. Zn-MT (500 &ml) protected DNA from

damage induced by Cu(II) at various concentrations (lo-200 PM) in the presence of 0.2 mM H,O, and

2 mM sodium ascorbate. DNA damage was measured as described in Fig. 1. 0 without Zn-MT; A with Zn-MT.

x 75- .

ii 5 50-

u

5 25- 0

O-

0 - Zn-MT

q + Zn-MT (500ug/ml)

=I T m 5 uM cu 5 NM Fe 50 MM Fe 100 uM Fe

Fig. 5. Effect of Zn-MT on copper- and iron-induced DNA damage. Calf thymus DNA was incubated

in the presence of Cu (5 PM) or Fe (5 PM-100 pM) in 2 mM H,O, and 2 mM sodium ascorbate for

30 min with or without Zn-MT (500 &ml). The asterisk indicates a significant difference between the

groups with and without Zn-MT (**P < 0.01).

150 L. Cai et al. / Chemico-Biological Inlet-actions 96 (1995) 143-155

-0 10 20 30 40 50

CuCI, (uM) Fig. 6. Effect of Zn-MT on generation of Cu(l) from Cu(l1). Cu(l) production from Cu(ll) in the absence

of MT (0) or the presence of 500 pg Zn-MT/ml (A). 2 mM H,O, and 2 mM sodium ascorbate were

included in reactions. Cu(1) formation was assessed by UV absorption (480 nm) in presence of

bathocuproinedisulfonic acid as described in Materials and methods.

3.3. Comparison of the protective effect of MT with other proteins

Compared to the marked protective effect of 100 &ml Zn-MT, other proteins such as calf thymus histone (100 &ml) did not protect DNA from Cu-induced damage, and bovine albumin (100 &ml) showed little protection as demonstrated

by DNA gel mobility (Fig. 7). These results were confirmed by changes in fluorescence measurements.

4. Discussion

Our results confirm that Fe(U), Fe(H) and Cu(l1) could induce DNA damage in vitro in the presence of H202 and ascorbic acid. However, there are two major differences in Cu and Fe induced damage: (1) Cu(I1) induces much more extensive DNA damage than Fe(I1) and Fe(III), confirming data from previous studies

[lo-14,35,43] and (2) DNA damage induced by Cu(I1) is markedly dependent on metal and H202 concentration. DNA damage in the presence of Fe(I1) and Fe(III), on the other hand, was minor at equimolar metal and hydrogen peroxide levels and became significant only at high concentrations of H20, (6 mM) and iron (125 FM).

Differences in characteristics of damage induction by these two metals have been discussed elsewhere by others [g-14,35,43).

Although metallothionein (MT) was isolated more than three decades ago, the

L. Cai et al. / Chemico-Biological Inreractions 96 (1995) 143-155 151

HISTONE (pg/ml) ALBUMIN (~g/ml) Zn-MT @g/ml)

Cuill) (uglml)

II----- __.~

0 0 I I 0 I loo 1 0 0 100 0

0 100 0 0

I 0110 20 0 10 20 0 10 20 0 10 20

23.1 kbp +

9.5 kbp +

4.3 kbp --,

Fig. 7. DNA gel pattern of DNA after various treatments. Calf thymus DNA fragmentation by Cu(ll)

as CuClz was inhibited by Zn-MT, but not bovine albumin or histone. All reactions were carried out in

the presence of 0.2 mM H,O, plus 2 mM sodium ascorbate. Increased mobility of DNA (detected by

EB-induced fluorescence) in a 1.0% agarose gel indicates increasing DNA fragmentation. The treatments

with various concentrations of copper and proteins are described in the upper panel.

cellular function(s) of this protein have not yet been fully defined. It is generally accepted that the principal roles of MT lie in the detoxication of potentially toxic heavy metal ions and in regulation of the metabolism of essential trace metals. However, there is increasing evidence that MT can reduce the toxic effects of several types of free radicals. For example, rat liver MT enhanced dismutation of superoxide radicals (02 :) in vitro [36]. The toxic effects of both free hydroxyl (‘OH) and superoxide radicals produced by the xanthine-xanthine oxidase reaction in vitro can be decreas- ed by addition of Cd/Zn-MT [26]. In cell culture experiments [24,25,37] and in whole animals [ 19,20,38] a role for MT in scavenging free radicals generated from a variety of chemicals, metals [39,40] and radiation also has been implicated. These reports suggest that MT is of importance in cellular defense mechanisms against free radicals.

In addition to the ability of MT to protect cellular damage, there are reports of the ability of MT to potentiate cellular damage under certain conditions. Cu, Zn-MT actually stimulated microsomal lipid peroxidation initiated by xanthine-xanthine oxidase in vitro [41] and DNA damage may be caused by unidentified radical species formed from Zn/Cd-MT [28]. Copper-MT can also increase lipid peroxidation in a microsomal system by hydroperoxides [34]. In addition, reports exist that overex-

152 L. Cai et al. / Chemico-Biological Interactions 96 (1995) 143-155

pression of foreign MT genes in transfected CHO cells did not increase cellular resistance to subsequent oxidative challenges [42]. These data suggest that MT may promote cellular damage under certain circumstances.

In the present investigation, however, it is clear that Zn-MT did not increase iron- or copper-induced DNA damage, but rather markedly reduced the copper induced damage. This shows that Zn-MT could directly protect calf thymus DNA from Cu- induced damage in an in vitro system. At the level of whole cells, Chinese hamster V79 cells transfected with antisense MT-l cDNA were sensitive to the DNA damaging action of HzOz while those treated with zinc exhibited both increased MT content (without increased GSH levels) and reduced susceptibility to HzOz-induced DNA damage [37]. The authors suggested that induction of MT synthesis and its presence in the nucleus may be the most important cellular defence against oxidative DNA damage.

The mechanism by which MT protects DNA from free radical-induced damage is considered to be through free radical scavenging: ionizing radiation studies have clearly shown that Zn,Cd-MT can effectively protect against radiation-induced damage to the sugar/phosphate DNA backbone [43]. However, the mechanism by which MT scavenges free radicals is still unclear. Although MT does not bind with Fe in vivo, an Fe-MT complex can be formed in vitro under anaerobic conditions [44]. The lack of protection of Fe-induced damage after addition of Zn-MT suggests little binding of Fe to Zn-MT under our experimental conditions. However, generation of Cu(1) from Cu(I1) was inhibited by addition of Zn-MT (Fig. 6). Because it is well known that Cu has a higher affinity for MT than Zn, these results suggest sequestration of Cu(II) by Zn-MT and thereby inhibition of the availability of Cu(I1) for reduction to Cu(1).

In addition to a role for MT in preventing formation of damaging radical species by interaction with metals, it is also possible that MT could interact with DNA directly and reduce DNA/Cu(I) interaction (considered to be a key step in Cu- induced DNA damage [45]). The site-specific DNA damage caused by Cu/peroxide suggests that Cu binds to specific sites in nucleic acids and mediates production of locally high concentrations of singlet oxygen or copper-peroxide radicals [46,47]. In our studies, DNA in the presence of Zn-MT formed a precipitate in the presence of 50 PM Cu(II) and 2 mM Hz02: it contained both DNA (assessed by DNA gel electrophoresis) and MT (assessed by Western blot analysis) (data not shown). It is possible that DNA complexed with MT can sequester metals and thus reduce radical-induced damage to DNA. Recent studies show that copper-MT can increase lipid peroxidation in vitro and can act as a pro-oxidant [34]. Thus, specific metals bound to MT may influence the properties of MT in oxidant stress.

In summary, we report that mammalian Zn-MT can inhibit DNA damage induced by reactive free radicals generated from oxygen donors in the presence of copper. It may do so both by sequestering copper to prevent its participation in redox reactions and by free radical scavenging. The localization of MT in cell nuclei under certain circumstances (early mammalian development, for example) and in some human tumours, may suggest a protective role for MT against DNA damage induced by free radicals.

L. Cai er al. / Chemico-Biological Interactions 96 (1995) 143-155 153

Acknowledgments

Thanks are due to Jane Pearson-Sharpe and Susanne Vesely for excellent technical assistance. This research was supported by grants to MGC and JK by the Medical Research Council of Canada.

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Metallothionein protects DNA from copper-induced but not iron-induced cleavage in vitro