Protective role of zinc-metallothionein on DNA damage in vitro by ferric nitrilotriacetate (Fe–NTA) and ferric salts

Chemico-Biological Interactions 115 (1998) 141–151

Protective role of zinc-metallothionein on DNAdamage in vitro by ferric nitrilotriacetate

(Fe–NTA) and ferric salts

Lu Cai, Gina Tsiapalis, M. Geroge Cherian *

Department of Pathology, The Uni6ersity of Western Ontario, London, Ont. N6A 5C1, Canada

Received 9 February 1998; received in revised form 22 June 1998; accepted 24 June 1998

Abstract

Oxidative DNA damage can be caused by radicals generated by transitional metals likeiron in Fenton reaction. Metallothionein (MT) may play an important role in preventingoxidative DNA damage. Therefore, after comparing the effects of ferric salts (Fe), andcomplexes of ferric salts with nitrilotriacetic acid (Fe–NTA) on DNA damage, the protectiveeffects of zinc-MT (Zn-MT) on DNA damage of Fe salts or Fe–NTA were investigated invitro. DNA damage was measured by loss of fluorescence of DNA binding to ethidiumbromide, and also by increased DNA mobility in agarose gel electrophoresis. Both Fe saltsand Fe–NTA could induce calf thymus DNA damage in presence of hydrogen peroxide andascorbate. However, the degree of DNA damage was lower with Fe salts than that withFe–NTA complex. Addition of 50 mM Zn-MT could only protect DNA from Fe–NTA, butnot from Fe salt induced damage. The protective effect of MT was about five times betterthan that of glutathione (GSH). These results suggest a potential role for MT in protectionfrom Fe–NTA-induced DNA damage. © 1998 Elsevier Science Ireland Ltd. All rightsreserved.

Keywords: Metallothionein; Oxidative DNA damage; Iron complexes; Transitional metals

* Corresponding author. Tel.: +1 519 6612030; fax: +1 519 6613370; e-mail:[email protected]

0009-2797/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.

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L. Cai et al. / Chemico-Biological Interactions 115 (1998) 141–151142

1. Introduction

Oxidative DNA damage can act as an initiation of carcinogenesis or inpathogenesis of certain neurodegenerative disorders [1–3]. Thus, methods toreduce oxidative DNA damage can be used as a potential strategy forprevention of carcinogenesis and neurodegenerative disorders caused byoxidative stress conditions. Metallothioneins (MTs), intracellular proteins with ahigh content of thiol groups, are present mainly in cytoplasm of cells and alsoin nucleus under certain conditions [4]. They may be involved in bothdetoxification of potentially toxic heavy metal ions and homeostasis of essentialtrace metals. However, recent studies show a role for MT in protection againstoxidative damage from several types of free radicals including superoxide,hydroxyl and organic radicals [5]. We have reported previously the protectiverole of MT against radiation- and copper (Cu)-induced DNA damage [6,7].Iron (Fe) can cause oxidative damage to DNA by formation of hydroxylradicals through Fenton reaction [8]. In previous studies, we observed that zinc(Zn)-MT could protect calf thymus DNA from Cu-induced damage, but notfrom Fe-induced damage [6]. In contrast, Abel and Reuiter [9] reported thatMT could protect DNA damage caused by Fe–EDTA complex. These resultssuggest that MT can inhibit the Fe–chelate complex-induced DNA damage butnot that caused by Fe salts.

Nitrilotriacetate (NTA) is a synthetic aminotricarboxylic acid which can formwater-soluble chelate complexes with several metal cations at neutral pH. Itsprimary industrial use is in the treatment of water to prevent mineral build-upin boilers. An experimental model of Fe overload was developed using ferricnitrilotriacetate (Fe–NTA) to induce acute or subacute injury and formation ofrenal tumours [10–12]. The carcinogenicity of Fe–NTA seems to be associatedwith the interaction of NTA with Fe since no tumour was observed byadministration of NTA alone or NTA chelated to other metals [10]. Thecarcinogenicity of Fe–NTA was considered to be due to the oxidative DNAdamage [1,11]. This study was undertaken to investigate the role of Zn-MT inthe Fe–NTA-induced oxidative DNA damage in vitro, and this may beimportant to understand the preventive role of MT in Fe–NTA-induced renaltumours.

2. Materials and methods

2.1. Chemicals

L-Ascorbic acid (sodium salt), deoxyribonucleic acid (sodium salt from calfthymus), ethidium bromide (EB) and glutathione (GSH, reduced), were allobtained from Sigma (St. Louis, MO). Hydrogen peroxide (H2O2), K2HPO4,KH2PO4, ferric ammonium sulphate (FeNH4(SO4)2) and ferric chloride (FeCl3)were from Fisher (Mississauga, Ont.). Ferric nitrate (Fe(NO3)3) was obtained

L. Cai et al. / Chemico-Biological Interactions 115 (1998) 141–151 143

from Baker. All reagents were analytical grade. Solutions were prepared in steriledeionized water. Stock phosphate buffer solutions were treated with chelex resin(Bio-Rad) by a published method [13,14] to remove metal ions. MT was isolatedfrom rabbit liver as described earlier [15].

2.2. Fe(III)–NTA preparation

To prepare Fe–NTA, FeCl3, FeNH4(SO4)2 or Fe(NO3)3 was dissolved in 20mM chelex-treated phosphate buffer to achieve a molar ratio of 1:1 of Fe(III) toNTA. The pH was adjusted to 7.0 with 1 N HCl and 1 N NaOH.

2.3. Reaction conditions and measurement of DNA oxidation

EB binding assay [16], based on the formation of a fluorescent complex be-tween double-strand DNA and EB binding, was used to measure DNA damage.Exposure to free radicals generated by metal ion/ascorbate/peroxide damagedDNA and inhibited the binding of EB to DNA with decreased fluorescence.Several forms of DNA damage (including strand scission, base oxidation andbase liberation) are believed to contribute to the loss of fluorescence [13,14].Hence, the assay detects a broad range of different DNA lesions. A 2 mlstandard reaction mixture contained 20 mM chelex-treated phosphate buffer (pH7.0), 66.4 mg/ml DNA, 1–300 mM Fe(III) or Fe(III)–NTA, 2 mM H2O2, and 2mM sodium ascorbate. To measure the effect of MT on DNA damage, 50 mMrabbit liver Zn-MT was added to the reaction mixture after addition of Fe(III)or Fe(III)–NTA and before addition of both H2O2 and ascorbic acid. Reactionswere carried out at 30°C for 30 min. A total of 10 m l of a 1 mM EB solutionwas immediately added and fluorescence was measured using a Turner Fluorome-ter (excitation at 510 nm and emission at 590 nm). Since the enhancement in thefluorescence of EB was dose-dependent following interaction with DNA, it was agood measure of the integrity of DNA. In controls, 100% fluorescence wasassessed in a solution containing all reagents (including DNA) except for Fe(III)or Fe(III)–NTA. Zero fluorescence was assessed in a solution identical to the100% reference solution except DNA. The loss in fluorescence was used as ameasure of DNA damage.

2.4. DNA gel electrophoresis

Calf thymus DNA samples were prepared similar to that for fluorescenceanalysis, except for the addition of ethidium bromide. A total of 30 m l (including10 mg of DNA plus 3 m l loading buffer (0.25% bromophenol blue, 0.25% xylenecyanol FF, 30% glycerol in water)) were loaded onto agarose gels (1.0%) in TAEbuffer (40 mM Tris–acetate, 1 mM EDTA) and subjected to electrophoresis inthe presence of EB (0.5 mg per ml in TAE) for 2–3 h. Photographs were takenwith a UV (312 nm) transilluminator.


2.5. Statistical analysis

Data were analyzed according to one-way ANOVA and Tukey’s HSD multiplecomparison. When only two groups were compared, the data were analyzed byunpaired two-tailed Student’s t-test. The results of at least three measurements werepresented as mean9standard error.

3. Results

3.1. Comparison of Fe(III)–NTA-induced DNA damage with Fe(III) salts

The effect of non-chelated Fe salts and Fe–NTA on calf thymus DNA damagewas compared by measuring the loss of fluorescence after binding to EB. Fe–NTA(from Fe(NO3)3) caused significantly higher DNA damage than Fe(NO3)3 alone(Fig. 1). Fe–NTA prepared from FeCl3 or FeNH4(SO4)2 also showed more damagethan FeCl3 or FeNH4(SO4)2 themselves (data not shown). Fig. 2 shows the changesof DNA mobility caused by Fe salts and Fe–NTA (50, 100 and 200 mM) in thepresence of 2.0 mM H2O2 and 2.0 mM ascorbic acid. The results confirmed thoseobtained using the measurement of fluorescence intensity. In the experiment withFeCl3, DNA in groups treated with Fe salt moved slower than that in the groupstreated with Fe–NTA complex (Fig. 2). These results further confirmed theprevious finding that marked difference has been observed between DNA damage

Fig. 1. Comparison of DNA damage induced by Fe(NO3)3 and Fe–NTA. DNA damage was measuredby the loss of fluorescence after DNA binding to EB. Calf thymus DNA was incubated with indicatedconcentrations of Fe(NO3)3 in 2.0 mM ascorbic acid and 2.0 mM H2O2. A total of 10 m l of 1 mM EBsolution was immediately added and fluorescence was measured with the excitation at 510 nm andemission at 590 nm. In controls (i.e. 0% DNA damage), 100% fluorescence was assessed in a solutioncontaining all reagents (including DNA) except for Fe salts or Fe–NTA. Zero fluorescence was assessedin a solution identical to the 100% reference solution except DNA, presenting 100% DNA damage. Eachcolumn represents the mean of at least three experiments. The bars represent standard error. *,** PB0.05 or 0.01 vs. corresponding Fe(III) (one-way ANOVA).


Fig. 2. Electrophoretic migration profile of DNA damage induced by Fe salts and Fe–NTA. Calfthymus DNA was incubated for 30 min at 30°C with 50 (a), 100 (b) and 200 mM (c) Fe salts orFe–NTA in the presence of 2.0 mM H2O2 and 2.0 mM ascorbic acid. Lanes 7 and 14: normal calfthymus DNA standard is labelled as N.

induced by Fe salts and Fe–NTA [17–19]. The DNA damage was not due to theligand NTA since NTA alone did not cause any DNA damage (data not shown).This has also been demonstrated previously in vivo and in vitro [19,20].

3.2. Protection of MT from DNA damage caused by Fe(III)–NTA, but not byFe(III) salts

By the measurement of fluorescence intensity of DNA/EB binding, it wasobserved that addition of Zn-MT (50 mM) before exposure of the DNA toFe(NO3)3 did not protect Fe-induced DNA damage (Fig. 3A), and this wasconsistent with our previous study [6]. However, the same treatment resulted inprotection from Fe–NTA-induced DNA damage (Fig. 3B). This protective effect ofMT against Fe–NTA-, not Fe-, induced DNA damage was also confirmed by themeasurement of DNA mobility in gel electrophoresis (Fig. 4). These resultsindicated that MT did not protect DNA damage caused by Fe salts ([6];presentdata, Fig. 3A), but could markedly protect the DNA damage caused by chelated Fesuch as Fe–EDTA [9] and Fe–NTA (Fig. 3B).

3.3. Comparing the protecti6e effect of MT on Fe(III)–NTA-induced DNA damagewith that of GSH

GSH is a tripeptide which can also provide protection for oxidative DNAdamage. In order to compare the protective effect of MT and GSH, 50 mM GSH(same concentration as MT in Fig. 3) was added to DNA solution before additionof Fe(NO3)3 or Fe–NTA. It did not provide any protection in Fe(NO3)3-inducedDNA damage (Fig. 5A), but it showed protection against Fe–NTA induced DNAdamage (Fig. 5B). When GSH concentration was increased to about five times (i.e.250 mM), it provided the same protection as of 50 mM Zn-MT (Fig. 6). This isconsistent with the results of Abel and Reuiter [9], which showed that addition ofZn/Cd-MT inhibited Fe–EDTA-induced DNA damage and was more effectivethan GSH.


4. Discussion

In this in vitro study, we have shown that addition of Zn-MT can protect DNAdamage caused by Fe–NTA complex in presence of H2O2 but not DNA damagecaused by Fe salts. It is also shown that Zn-MT is about five times better thanreduced GSH in molar ratio to protect the DNA damage. The direct extrapolationof this in vitro experimental condition to in vivo systems may be difficult. However,since MT has been detected in cell nucleus in certain cell growth stages [4], theseresults have some relevance to protection of DNA damage by MT.

The protective role of MT in oxidative damage has been extensively documented[5,21,22], including protection of nephrotoxic effects of Cd and anti-cancer drugs

Fig. 3. Effects of Zn–MT on DNA damage induced by Fe(NO3)3, (Fe(III)), and Fe–NTA. DNAdamage was measured as described in Fig. 1. Calf thymus DNA was incubated with indicatedconcentrations of Fe(NO3)3 (A) or Fe–NTA (B) in the presence of 2.0 mM H2O2 and 2.0 mM ascorbicacid with or without addition of Zn–MT (50 mM). Each column represents the mean of at least threeexperiments. The bars represent standard error. ** PB0.01 vs. corresponding Fe–NTA alone (one-wayANOVA).


Fig. 4. Electrophoretic migration profile of DNA damage induced by Fe(NO3)3 and Fe–NTA. Calfthymus DNA was incubated for 30 min at 30°C with 50, 100 mM Fe(NO3)3 and Fe–NTA in thepresence of 2.0 mM H2O2 and 2.0 mM ascorbic acid without or with 50 mM Zn-MT. N, normal calfthymus DNA.

such as cisplatin and gentamicin [23–25]. The protective role of MT in oxidativeDNA damage has been investigated in recent years since oxidative DNA damagehas been considered as a critical event in certain chemical carcinogenesis. Theresults showed a protective effect of MT in Cd-, Cu-, H2O2-, and ionizingradiation-induced DNA damage [6,7,22,26–28]. In addition, the spontaneous muta-tion which can result from endogenous oxidative DNA damage can be reduced orenhanced by altering MT gene expression [29–31]. Thus, induction of MT candecrease both spontaneous and anti-cancer drug or radiation-induced tumourformation [32,33]. However, there is little information on whether MT can preventrenal tumours induced by Fe–NTA. We provide a direct in vitro evidence thatZn-MT can protect DNA from Fe–NTA-induced oxidative damage in this report.

Fe and ferritin have been detected in nuclei of several cells. DNA repair enzymeand transcription factor containing Fe-sulfur cluster have been described. SinceH2O2 itself is not toxic to cells, H2O2-induced oxidative DNA damage in cells hasbeen thought to result from the formation of hydroxyl free radicals through Fentonreaction with Fe [8]. Fe or Fe–chelate in nucleus bind with DNA and then reactwith H2O2 to generate hydroxyl radicals at close proximity to DNA [17]. In thepresent study, we report the differences in the degree of DNA damage caused by Fesalts alone and Fe–NTA complex (Figs. 1 and 2). To explain the results, wepropose the following hypothesises. Enhanced cellular damage by NTA may be dueto increased uptake of Fe into cell [20]. However, it cannot explain the presentresults because the current experiments were carried out in vitro using isolated calfthymus DNA. For the production of hydroxyl free radicals in the presence of H2O2,Fe–NTA and Fe–chelates of other aminopolycarboxylic acids were more effectivethan Fe salts because of their solubility [17,20]. Fe-NTA may bind with DNAdouble helix readily, and form the short-lived hydroxyl free radical at the site ofdamage to DNA [17]. Therefore, the difference in DNA damage observed herebetween Fe salts and Fe–NTA is mainly due to the chemical properties of theFe–NTA complexes.


Although MT is detected generally as a cytoplasmic protein, nuclear localizationof MT has been recognized in recent years [34,35]. The presence of MT in nucleushas been noted in several tissues, such as in human fetal liver, in both fetal andneonatal rat liver, in newborn mice and in certain tumours [4,34–37]. DNA flowcytometry studies demonstrated the localization of MT in the nucleus of bothdiploid and aneuploid cells during the G1/S phase of the cell cycle of adeno-carcinma of breast and colon [38]. The nuclear localization of MT may be relatedto the interaction of metal ions like zinc with various nuclear constituents duringcell cycle regulation and cell differentiation. This could also modulate gene expres-sion by exchange of Zn binding between MT, histone, transcription factors, andnuclear acidic proteins [4,37]. In addition, the presence of MT in nucleus may beimportant to protect DNA from Fe–chelate induced oxidative damage becausenucleus does not contain antioxidant enzymes such as superoxide dismutase,

Fig. 5. Effects of GSH on DNA damage induced by Fe(NO3)3, (Fe(III)), and Fe-NTA. DNA damagewas measured as described in Fig. 1. Calf thymus DNA was incubated with indicated concentrations ofFe(NO3)3 (A) or corresponding Fe–NTA (B) in the presence of 2.0 mM H2O2 and 2.0 mM ascorbic acidwithout or with the addition of GSH. Each column represents the mean of at least three experiments.The bars represent standard error. * PB0.05 vs. Fe–NTA alone (Student’s t-test).


Fig. 6. Comparison for the protective effects of Zn-MT and GSH on Fe–NTA-induced DNA damage.DNA damage was measured as described in Fig. 1. Calf thymus DNA was incubated with 100 mMFe–NTA, derived from Fe(NO3)3, in the 2.0 mM H2O2 and 2.0 mM ascorbic acid with and without MTor GSH. Each column represents the mean of at least three experiments. The bars represent standarderror. ** PB0.01 vs. Fe–NTA alone (Student’s t-test).

catalase and peroxidase [8]. Thus, the nuclear localization of MT may be animportant factor to determine the role of MT in protection from genotoxic effect ofchemicals [21,29,31].

The mechanisms involved in the protection of DNA damage by nuclear MT arenot yet understood [22,26–28]. It could be speculated that under oxidative stress,Zn may be displaced from MT, and both free sulfydryl groups and the released Znions could act as effective scavengers of free radicals, generated in the nucleus.Thus, MT may provide protection from free radical induced DNA damage, butmore studies are required to understand the biological significance of this finding,especially in relation to Fe–NTA induced renal tumours [18].

Acknowledgements

This work was supported by research grant from Medical Research Council ofCanada.

References

[1] S. Toyokuni, Iron-induced carcinogenesis: The role of redox regulation, Free Radic. Biol. Med. 20(1996) 553–566.

[2] P. Jenner, C.W. Olanow, Oxidative stress and the pathogenesis of Parkinson’s disease, Neurology47 (1996) 161–170.

[3] M.A. Smith, P.L.R. Harris, L.M. Sayre, G. Perry, Iron accumulation in Alzheimer disease is asource of redox-generated from radicals, Proc. Natl. Acad. Sci. USA 94 (1997) 9866–9868.


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

[5] M. Sato, I. Bremner, Oxygen free radicals and metallothionein, Free Radic. Biol. Med. 14 (1993)325–337.

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

[7] L. Cai, M.G. Cherian, Adaptive response to ionizing radiation-induced chromosome aberrations inrabbit lymphocytes: Effect of pre-exposure to zinc, and copper, Mutat. Res. 369 (1996) 233–241.

[8] R. Meneghini, Iron homeostasis, oxidative stress and DNA damage, Free Radic. Biol. Med. 23(1997) 783–792.

[9] J. Abel, N. de Reuiter, Inhibition of hydroxyl-radical-generated DNA degradation by metalloth-ionein, Toxicol. Lett. 47 (1989) 191–196.

[10] J.L. Li, S. Okada, S. Hamazaki, Y. Ebina, O. Midorikawa, Subacute nephrotoxicity and inductionof renal cell carcinoma in mice treated with ferric nitrilotriacetate, Cancer Res. 47 (1987)1867–1868.

[11] S. Toyokuni, J.L. Sagripanti, DNA single- and double-strand breaks produced by ferric nitrilotriac-etate in relation to renal tubular carcinogenesis, Carcinogenesis 14 (1993) 223–227.

[12] Y. Ebina, S. Okada, S. Hamazaki, F. Ogino, J.I. Li, O. Midorikawa, Nephrotoxicity and renal cellcarcinoma after use of iron- and aluminium-nitrilotriacetate complexes in rats, J. Natl. Cancer Inst.76 (1986) 107–113.

[13] G.R. Buettner, Ascorbate autoxidation in the presence of iron and copper chelates, Free Radic.Res. Commun. 1 (1986) 349–353.

[14] L. Miline, P. Nicotera, S. Orrenius, M.J. Burkitt, Effect of glutathione and chelating agents oncopper-mediated DNA oxidation: Pro-oxidant and antioxidant properties of glutathione, Arch.Biochem. Biophys. 304 (1993) 102–109.

[15] G.F. Stephenson, H.M. Chan, M.G. Cherian, Copper-metallothionein from the toxic milk mutantmouse enhances lipid peroxidation initiated by an organic hydroperoxide, Toxicol. Appl. Pharma-col. 125 (1994) 90–96.

[16] R. Stoewe, W.A. Prutz, Copper-catalyzed DNA damage by ascorbate and hydrogen peroxide:Kinetics and yield, Free Radic. Biol. Med. 3 (1987) 97–105.

[17] S. Inoue, S. Kawanishi, Hydroxyl radical production and human DNA damage induced by ferricnitrilotriacetate and hydrogen peroxide, Cancer Res. 47 (1987) 6522–6527.

[18] S. Toyokuni, T. Mori, M. Dizdaroglu, DNA base modifications in renal chromatin of Wistar ratstreated with a renal carcinogen, ferric nitrilotriacetate, Int. J. Cancer 57 (1994) 123–128.

[19] T. Umemura, K. Sai, A. Takagi, R. Hasegawa, Y. Kurokawa, Formation of 8-hydrox-ydeoxyguanosine in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate,Carcinogenesis 11 (1990) 345–347.

[20] A. Hartwig, H. Klyszcz-Nasko, R. Schlepegrell, D. Beyersmann, Cellular damage by ferricnitrilotriacetate and ferric citrate in V79 cells: Interrelationship between lipid peroxidation, DNAstrand breaks and sister chromatid exchanges, Carcinogenesis 14 (1993) 107–112.

[21] M.A. Schwarz, J.S. Lazo, J.C. Yalowich, I. Reynolds, V.E. Kagan, V. Tyurin, Y.-M. Kim, S.C.Watkins, B.R. Pitt, Cytoplasmic metallothionein overexpression protects NIH 3T3 cells fromtert-butyl hydroperoxide toxicity, J. Biol. Chem. 269 (1994) 15238–15243.

[22] M.G. Cherian, P. Ferguson, Metallothionein in cytotoxicity and genotoxiciy of metals, in: N.D.Hadjiliadis (Ed.), Cytotoxic, Mutatgenic and Carcinogenic Potential of Heavy Metals Related toHuman Environment, NATO ASI series, 2. Environment, vol. 26, Kluwer, Dordrecht, 1997, pp.217–230.

[23] X.H. Du, C.Y. Yang, Mechanism of gentamicin nephrotoxicity in rats and the protective effect ofzinc-induced metallothionein synthesis, Nephrol. Dial. Transplant. 9 (1994) 135–140.

[24] M. Satoh, Y. Aoki, C. Tohyama, Protective role of metallothionein in renal toxicity of cisplatinum,Cancer Chemther. Pharmacol. 40 (1997) 358–362.

[25] C.D. Klaassen, J. Liu, Role of metallothionein in cadmium-induced hepatotoxicity and nephrotox-icity, Drug Metab. Rev. 29 (1997) 79–102.


[26] T.P. Coogan, R.M. Bare, M.P. Waalkes, Cadmium-induced DNA strand damage in cultured livercells: Reduction in cadmium genotoxicity following zinc pretreatment, Toxicol. Appl. Pharmacol.113 (1992) 227–233.

[27] T.P. Coogan, R.M. Bare, E.J. Bjornson, W.P. Waalkes, Enhanced metallothionein gene expressionis associated with protection from cadmium-induced genotoxicity in cultured rat liver cells, J.Toxicol. Environ. Health 41 (1994) 233–245.

[28] L.S. Chubatsu, R. Menechini, Metallothionein protects DNA from oxidative damage, Bichem. J.291 (1993) 193–198.

[29] E.I. Goncharova, T.G. Rossman, A role for metallothionein and zinc in spontaneous mutagenesis,Cancer Res. 54 (1994) 5318–5323.

[30] 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.

[31] T.G. Rossman, E.I. Goncharova, A. Nadas, N. Dolzhanskaya, Chinese hamster cells expressingantisense to metallothionein become spontaneous mutators, Mutat. Res. 373 (1997) 75–85.

[32] O. Kagimoto, A. Naganuma, N. Imura, T. Toge, O. Niwa, K. Yokoro, Effect of the administrationof bismuth on radiogenic thymoma induction in mice, J. Radiat. Res. 32 (1991) 417–428.

[33] M. Satoh, Y. Kondo, M. Mita, I. Nakagawa, A. Naganuma, N. Imura, Prevention of carcinogenic-ity of anticancer drugs by metallothionein induction, Cancer Res. 53 (1993) 4768–4769.

[34] D. Banerjee, S. Onosaka, M.G. Cherian, Immunohistochemical localization of metallothionein incell nucleus and cytoplasm of rat liver and kidney, Toxicology 24 (1982) 95–105.

[35] M. Panemangalore, D. Banerjee, S. Onosaka, M.G. Cherian, Changes in intracellualar accumula-tion and distribution of metallothionein in rat liver and kidney during postnatal development, Dev.Biol. 97 (1983) 95–102.

[36] N.O. Nartey, D. Banergee, M.G. Cherian, Immunohistochemical localization of metallothionein incell nucleus and cytoplasm of fetal human liver and kidney and its changes during development,Pathology 19 (1987) 233–238.

[37] M.G. Cherian, J.C. Lau, M.D. Apostolova, L. Cai, The nuclear-cytoplasmic presence of metalloth-ionein in cells during differention and development, in: C.D. Klaassen (Ed.), MT-97, Birkhauser,Basel, 1998 (in press).

[38] H. Haile Meskel, M.G. Cherian, V.J. Martinez, L.A. Veinot, J.V. Frei, Metallothionein as anepithelial proliferative compartment marker for DNA flow cytometry, Mod. Pathol. 6 (1993)755–760.

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Protective role of zinc-metallothionein on DNA damage in vitro by ferric nitrilotriacetate (Fe–NTA) and ferric salts