Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper

Chemico-Biological Interactions 137 (2001) 75–88

Roles of vitamin C in radiation-induced DNAdamage in presence and absence of copper

Lu Cai a, James Koropatnick a,b,c, M. George Cherian a,b,*a Department of Pathology, Uni�ersity of Western Ontario, London, Ont, Canada N6A 5C1

b Department of Pharmacology and Toxicology, Uni�ersity of Western Ontario, London,Ont, Canada N6A 5C1

c Department of Oncology, Uni�ersity of Western Ontario, London, Ont, Canada N6A 5C1

Received 10 July 2000; received in revised form 20 February 2001; accepted 23 February 2001

Abstract

Exposure to either ionizing radiation or certain transition metals results in generation ofreactive oxygen species that induce DNA damage, mutation, and cancer. Vitamin C (areactive oxygen scavenger) is considered to be a dietary radioprotective agent. However, ithas been reported to be genotoxic in the presence of certain transition metals, includingcopper. In order to explore the capacity of vitamin C to protect DNA from radiation-in-duced damage, and the influence of the presence of copper on this protection, we investigatedvitamin C-mediated protection against radiation-induced damage to calf thymus DNA invitro in the presence or absence of copper(II). Vitamin C (0.08–8.00 mM, pH 7.0) signifi-cantly reduced DNA damage induced by �-irradiation (30–150 Gy) by 30–50%, similar tothe protective effect of glutathione. However, vitamin C plus copper (50 �M) significantlyenhanced �-radiation-induced DNA damage. Low levels of added copper (5 �M), orchelation of copper with 1-N-benzyltriethylenetetraine tetrahydrochloride (BzTrien) andbathocuprinedisulfonic acid (BCSA), abolished the enhanced damage without diminishingthe protective effect of vitamin C. These results indicate that vitamin C can act as: (1) anantioxidant to protect DNA damage from ionizing radiation; and (2) a reducing agent in thepresence of copper to induce DNA damage. These effects are important in assessing the roleof vitamin C, in the presence of mineral supplements or radioprotective therapeutic agents,particularly in patients with abnormally high tissue copper levels. © 2001 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: Vitamin C; Copper; Ionizing radiation; DNA damage; Radioprotection; Fenton reaction

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* Corresponding author. Tel: +1-519-6612030; fax: +1-519-6613370.E-mail address: [email protected] (M.G. Cherian).

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L. Cai et al. / Chemico-Biological Interactions 137 (2001) 75–8876

1. Introduction

Exposure to both ionizing radiation and certain transition metals can increasecellular formation of free radicals. Under aerobic conditions, this can lead toformation of reactive oxygen species. Reactivity of these oxyradicals leads todeleterious changes in living cells, including DNA strand breaks, protein oxida-tion and membrane damage [1]. Oxidative DNA damage has been suggested tobe critical in mutagenesis and carcinogenesis [2,3]. Ionizing radiation, includingboth high-LET (� particle) and low-LET (�- and X-irradiation), can cause cellu-lar damage. The toxic effects of high-LET radiation are generally considered tobe due to a direct action (i.e. direct energy deposition in critical macromolecules).For low-LET radiation the effects are predominantly caused by free radicalsproduced during the radiolysis of water. They interact with critical targetmolecules as they diffuse from their sites of production [3,4]. Radiation-inducedDNA damage by indirect mechanisms is therefore influenced by cellular antioxi-dant status [4,5].

Eukaryotic cells have different antioxidant defence systems to prevent the dam-age caused by active oxygen species. They include: (a) low molecularweight antioxidants such as vitamin C and glutathione (GSH) to scavenge reac-tive radicals; (b) enzymes to prevent or limit reactive free radical production(superoxide dismutase and catalase, for example); and (c) DNA repair processesto overcome radical-induced base damage [6–8]. As an antioxidant, vitamin Cplays broad biological roles, one of which is to restrict the propagation ofmultiple reactive electrophilic end-products from free radicals produced withinthe cells by normal cellular processes, or from toxic exogenous precursors [9,10].Protective effects against ionizing radiation damage have been extensively docu-mented in animals and in cultured cells [11–15]. Vitamin C was also shown toprevent cancer and clastogenic effects [9,14]. However, it is also reported thatvitamin C can enhance radiation-induced DNA damage at high concentration[15].

In addition to being essential for life, transition metals such as copper (Cu)and iron in lower oxidation state can participate in Fenton reactions to producefree radicals which may cause oxidative DNA damage [9,16–19]: Cu ions havebeen shown to be effective mediators of oxygen radical-induced cytotoxicity andgenotoxicity [16–19]. In addition, Cu may also enhance the effects of otheroxidizing agents, including ultraviolet [20] and ionizing [21] radiation. Therefore,it is important for any organism to maintain effective metal homeostasis, espe-cially Cu. This study was undertaken to investigate the role of vitamin C onradiation-induced DNA damage in the presence or absence of Cu in a cell-freesystem (calf thymus DNA in vitro). We found that vitamin C protected calfthymus DNA from damage induced by low LET radiation (�-rays) in vitro;however, vitamin C in combination with Cu enhanced radiation-induced DNAdamage.

L. Cai et al. / Chemico-Biological Interactions 137 (2001) 75–88 77

2. Materials and methods

2.1. Chemicals

Vitamin C (L-ascorbic acid, sodium salt), bathocuproinedisulfonic acid (BCSA,disodium salt), cupric chloride (dihydrate), calf thymus DNA (sodium salt), EDTA(disodium salt), ethidium bromide (EtBr), 1-N-benzyltriethylenetetraine tetrahy-drochloride (BzTrien) and luminol (5 amino-2,3 -dihydro-1,4 phthalazinedione)were all obtained from Sigma Co. (St. Louis, MO). K2HPO4 and KH2PO4 werepurchased from BDH Inc. (Toronto). Agarose was purchased from Life Technolo-gies, Inc. All reagents were analytical grade. Solutions were prepared in steriledeionized water. A concentrated stock solution of 80 mM phosphate buffer (pH7.0) was prepared and treated with a metal chelating resin (Bio-Rad) by the bathmethod to remove contaminating metal ions [22,23].

2.2. �-Irradiation

�-rays were generated from a 60Co source (Theratron Eldorado 6) at a dose rateof 3.0 Gy/min as determined by calibration using an air ionization chamber(Capintec PR-06C) connected to an electrometer (Capintex 192X). Calf thymusDNA in solution (1 ml, 300 �M DNA in 20 mM phosphate buffer) in capped 1.5ml polypropylene tubes was exposed to �-radiation under aerobic conditions for0–50 min to produce exposures of 0, 15, 30, 60, 120 and 150 Gy.

2.3. Reaction conditions

A 1 ml reaction mixture contained (in order of addition): 20 mM Chelex-treatedphosphate buffer (pH 7.0), 300 �M DNA, and either 0, 5, or 50 �M CuCl2 with orwithout 0.008–8 mM vitamin C. After radiation, EDTA (100 mM stock) wasimmediately added to produce a final concentration of 10 mM in a volume of 1 ml.Ten microliter of a 1 mM EtBr solution was then added and fluorescence measuredusing a Turner fluorescence spectrometer (excitation at 510 nm and emission at 590nm). In controls, 100% fluorescence was assessed in a solution containing allreagents (including DNA) not exposed to radiation. Zero fluorescence was assessedin a solution identical to the 100% reference solution except for the DNA. DNAdamage was measured as a loss in fluorescence [16,17].

2.4. Measurement of DNA damage

The EtBr binding assay for DNA damage (based on the formation of afluorescent complex between double-strand DNA and EtBr) was used [23]. DNAdamage after exposure to free radicals generated by radiation resulted in decreasedfluorescence because of decreased binding of EtBr. Thus, loss of fluorescence wasused as an indicator of DNA damage. Several forms of DNA damage, includingstrand scission, base oxidation, and base liberation, can contribute to the loss of


fluorescence [23]. Damage to DNA as determined by this method was confirmed byDNA gel electrophoresis which showed accelerated mobility with decreased averagemolecular weight due to double strand DNA breaks.

2.5. DNA gel electrophoresis

DNA samples were dissolved in Chelex-treated phosphate buffer. 1% non-dena-turing agarose (Ultrapure DNA grade from Bio-Rad) gels were prepared in TAEbuffer (10 mM Tris base, 4.4 mM acetic acid, 0.5 mM EDTA, pH 8.0). Samplealiquots (25 �l, including 3 �l loading buffer [0.25% bromophenol blue, 0.25%xylene cyanol FF and 30% glycerol in water]) were loaded into wells, andelectrophoresis carried out in TAE buffer plus 2% EtBr for 2–3 h. Photographswere taken under UV (312 nm) transillumination to visualize DNA mobility [16,17].

2.6. Measurement of reacti�e oxygen species

Luminol-enhanced chemiluminescence assay was used to measure the generationof reactive oxygen species (ROS) as described by Lundquist and Dahlgren [24]. Avolume of 200 �l of Luminol was dispensed into quadruplicate wells of a 96-wellpolyvinylchloride microtitre plate. An appropriate volume of copper chloride (1mM stock) or water vehicle was added to each well to obtain concentrations of0–50 �M copper. In duplicate wells, sodium ascorbate was added to obtain 2 mM(from 1 M stock) to copper containing wells. For appropriate controls the volumewas adjusted with water. The formation of reactive oxygen species was measuredcontinuously for 15 min using a Wallace 1420 Multilabel Counter (EG&G Wal-lace), and the data presented as counts per seconds (CPS) of luminescence (mean offour independent wells�standard deviation).

2.7. 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 t-test. The results of at least three measurements werepresented as mean�standard error (SE).

3. Results

3.1. Effect of �itamin C alone or Cu alone on EtBr/DNA fluorescence

In order to determine whether vitamin C alone, or Cu alone, induced DNAdamage, calf thymus DNA in solution was exposed to vitamin C (0.008–8 mM) orCuCl2 (5–100 �M) for 1.5 h and the DNA/EtBr fluorescence was measured. Nosignificant loss in EtBr/DNA fluorescence (indicative of DNA damage) was ob-served after exposure to vitamin C alone, or Cu alone (data not shown).


3.2. Effect of �itamin C alone or in the presence of Cu on radiation-induced DNAdamage

�-Radiation induced DNA damage (Fig. 1). A 50–70% loss in EtBr/DNAfluorescence occurred after treatment with ionizing radiation (60 and 150 Gyexposure). Addition of vitamin C (2 mM) markedly reduced DNA damage inducedby radiation at doses of 30–150 Gy (Fig. 1). In addition, a wide dose range ofvitamin C (0.08–8 mM) could effectively protect DNA damage induced by 150 Gyof �-radiation (Fig. 2).

The protective effect of vitamin C on radiation-induced DNA damage wasinvestigated in the presence of Cu. Vitamin C (2 mM) in the presence of 50 �M Cuadded to the DNA mixture prior to radiation enhanced DNA damage by 10–20%after radiation treatment (15–150 Gy) (Fig. 3). In order to explore the mechanismof increased DNA damage by Cu, the effects of low (5 �M) and high (50 �M) Culevels on DNA damage, with or without vitamin C, were measured. Cu aloneinduced no apparent DNA damage (Fig. 4). Two micromolar vitamin C in thepresence of Cu, but without radiation, resulted in significant DNA damage at both5 and 50 �M Cu (Fig. 4). However, vitamin C was able to decrease or increase�-radiation induced DNA damage, depending on the concentration of Cu. At lowCu level (5 �M), the protective effect of vitamin C was unaffected. At higher levels

Fig. 1. DNA damage induced by �-radiation and protection by vitamin C. DNA damage was measuredby a loss of DNA/EtBr fluorescence. When DNA in solution was exposed to �-radiation, DNA strandbreaks resulted in the loss of DNA/EtBr fluorescence (Section 2). Radiation at doses of 15–150 Gymarkedly induce DNA damage (squares). Two millimolar vitamin C added to DNA prior to irradiationprotected DNA from damage by 30–150 Gy �-rays (circles). Data points indicate mean�SE from threeseparate experiments. (a) significantly different from control, unirradiated DNA in the absence ofvitamin C (P�0.01, one-way ANOVA); (b) significantly different from DNA exposed to radiation only(P�0.01, one-way ANOVA).


Fig. 2. Vitamin C protects calf thymus DNA from damage by �-irradiation. Prior to irradiation (150Gy), 0.008–8 mM vitamin C was added to DNA in solution. DNA damage was measured by loss ofDNA/ EtBr fluorescence (Section 2). Data indicate the mean�SE from three separate experiments. *,significantly different from DNA treated with 150 Gy �-rays alone (P�0.01, one-way ANOVA).

of Cu (50 �M), an increase in � radiation-induced DNA damage was found (Fig.4).

3.3. Comparison of capacity of �itamin C and GSH to protect calf thymus DNAfrom radiation-induced DNA damage

The ability of vitamin C or GSH (in the presence or absence of Cu) to protectcalf thymus DNA from radiation-induced damage was compared. Both GSH andvitamin C protected DNA from radiation damage (Fig. 5). However, vitamin C didnot protect DNA damage in the presence of 50 �M Cu, while GSH continued toprotect DNA from radiation damage in the presence of Cu (Fig. 5).

3.4. Effect of �itamin C on radiation-induced DNA damage after Cu remo�al bychelation

To investigate the effect of vitamin C on radiation-induced DNA damage in thepresence of complexed Cu with Cu-specific chelating agents, BCSA and BzTrienwere added prior to addition of vitamin C. When a 1:1 ratio of Cu to chelatingagents was used, the protective effect of vitamin C on radiation-induced DNAdamage was found only in the case of BzTrien (Fig. 6). When a 1:4 ratio of Cu tochelating agents was used, the protective effect could be found in both cases ofBCSA and BzTrien (Fig. 6).


Fig. 3. Enhancement of radiation-induced DNA damage in the presence of vitamin C plus Cu(II). Theeffect of 2 mM vitamin C�50 �M Cu(II) on the level of DNA damage induced by �-radiation (15–150Gy) was compared. DNA damage was measured by loss of DNA/EtBr fluorescence (Section 2). Dataindicate the mean�SE from three separate experiments. Asterisks indicate significant differences (*,P�0.05; **, P�0.01, Student’s t-test) compared to irradiated DNA in the absence of vitamin C andCu(II).

Fig. 4. Effect of vitamin C� increasing concentrations of Cu(II) on DNA damage by �-radiation. DNAdamage was measured in the absence of copper, and in the presence of low (5 �M) and high (50 �M)concentration of Cu(II) with or without added vitamin C (2 mM). DNA damage was measured by lossof DNA/EtBr fluorescence (Section 2). Data points indicate the mean�SE from three separateexperiments. (a) significantly different from DNA treated with radiation�Cu (P�0.05, Student’st-test).


Fig. 5. Comparison of effect of vitamin C or GSH in altering � radiation-induced calf thymus DNAdamage. Prior to � irradiation (120 Gy), vitamin C (2 mM) or GSH (2 mM), � Cu (50 �M), were addedto DNA. DNA damage was measured by loss of DNA/EtBr fluorescence (Section 2). Data pointsindicate the mean�SE from three separate experiments. (a) significantly less damage than DNA treatedwith radiation alone (P�0.01, Student’s t-test); (b) significantly greater damage than DNA treated withradiation alone (P�0.01, Student’s t-test).

Gel electrophoresis analysis of changes in DNA mobility confirmed the presenceof DNA damage demonstrated by the loss of EtBr/DNA fluorescence. As shown inFig. 7, DNA exposed to 60 Gy ionizing radiation migrated more quickly thancontrol, untreated DNA (Fig. 7, lanes A and B). Vitamin C (2 mM) markedlyprotected DNA from radiation-induced damage, resulting in significantly lowerDNA mobility (Fig. 7, lane C). However, vitamin C (2 mM) in the presence of Cu(50 �M) significantly enhanced radiation-induced DNA damage, as revealed byincreased DNA mobility (Fig. 7, lane D). When the Cu-chelating agent BzTrien wasadded prior to the addition of vitamin C, vitamin C continued to protect againstradiation-induced DNA damage. However, addition of BCSA did not have thesame effect.

3.5. The formulation of reacti�e oxygen species

In order to demonstrate the formation of reactive oxygen species (ROS) withcopper alone or in presence of ascorbate, we used a Luminol-enhanced chemilu-minescence assay. The results show that copper alone (5 and 50 �M) did not showany chemiluminescence (Fig. 8). When 2 mM ascorbate was added, a significantincrease in chemiluminescence was observed in presence of copper (P�0.05), andthis increase was dependent on copper concentration while ascorbate alone in theabsence of copper did not show any increase in chemiluminescence. The data alsoshow that ascorbate enhances oxygen radical formation at a concentration ofcopper which can cause DNA fragmentation.


4. Discussion

The results presented here demonstrate the formation of reactive oxygen speciesin presence of both Cu and vitamin C, and this can be measured by luminol-en-hanced chemiluminescence. These free radicals can damage DNA. However, bothvitamin C and GSH can protect DNA from this damage in the absence or at lowconcentration of Cu.

A broad range of protective effects by vitamin C on cellular damage caused byionizing radiation has been reported, including inhibition of radiation-induced celltransformation [12,13], decrease in the incidence of some cancers [25,26], radiation-induced DNA damage, micronucleus formation, and chromosome aberrations[11,27,28], and prevention of radiation-induced damage to other cellular compo-nents [29,30]. These effects have been suggested to be related to the free radicalscavenging property of vitamin C, but direct evidence for this mechanism is lacking.Efficient vitamin C-mediated scavenging of free radicals induced by �-radiation inan in vitro system has been reported [31,32]. These reports, and the results of thepresent study on the protective effect of vitamin C on damage to calf thymus DNA

Fig. 6. Effect of vitamin C on radiation-induced DNA damage with Cu chelation. DNA damage inducedby 120 Gy �-rays was measured under conditions where: (1) Cu (50 �M) was added prior to the additionof vitamin C (2 mM) and irradiation; (2) Cu specific chelators (BCSA, BzTrien) were added before theaddition of Cu (50 �M), vitamin C (2 mM) and irradiation. DNA damage was measured by loss ofDNA/EtBr fluorescence (Section 2). Data points indicate the mean�SE from three separate experi-ments. (a) significantly greater damage than that induced by radiation alone (P�0.05, Student t-test);(b, c) significantly less damage than that induced by radiation alone; (b, P�0.05; c, P�0.01, Studentt-test).


Fig. 7. Electrophoretic migration profile of DNA after exposure to radiation�vitamin C. (A) Controlcalf thymus DNA; (B) DNA exposed to 60 Gy �-rays; (C) DNA with vitamin C (2 mM) prior toexposure to � radiation (60 Gy); (D) DNA with added Cu (50 �M) and vitamin C (2 mM) prior toexposure to � radiation (60 Gy); (E) DNA with BzTrien added prior to addition of Cu (BzTrien:Cu=1:1) and vitamin C, followed by exposure to � radiation (60 Gy); (F) DNA with BCSA added prior toaddition of Cu (BCSA:Cu=1:1) and vitamin C, followed by exposure to � radiation (60 Gy).

following in vitro �-radiation exposure (Figs. 1, 2 and 5), provide further evidencefor the protective effect of vitamin C and GSH against radiation-induced DNAdamage by scavenging free radicals.

Vitamin C alone (0.008–8.0 mM) did not induce damage to calf thymus DNA.This is in accord with previous investigations in where there is no evidence foreither vitamin C-induced chromosome damage, in rodents, or induction of spermabnormality in humans [10,33]. Contrary to studies in animals, in vitro investiga-tions have indicated that vitamin C, under certain conditions, can be mutagenic andcytotoxic [34], especially in the presence of Cu [9,16,18,35]. The present study alsodemonstrated that vitamin C in the presence of Cu can generate reactive oxygenradicals and induce DNA damage, particularly at higher concentration of Cu (50�M). We have previously shown that certain transition metals, such as Cu and ironsalts, in the presence of vitamin C/hydrogen peroxide can cause calf thymus DNAdamage in an in vitro system [17,18]. In these experiments, vitamin C reduces Cu(II)to Cu(I), which then participates in a Fenton reaction to produce reactive hydroxyradicals that cause DNA damage.

In this report, we demonstrate a protective effect of vitamin C against DNAdamage induced by �-radiation (Figs. 1 and 2), but enhancement of radiation-in-duced DNA damage in the presence of a high concentration of Cu (50 �M) (Figs.


3 and 4). This suggests that vitamin C can play a dual role. One effect of vitaminC is to reduce Cu(II) to Cu(I) in a Fenton reaction, and a second effect is to act asa free radical scavenger to protect DNA from damage induced by radiation. Arecent report indicated that vitamin C at low concentration could protect DNAfrom radiation-induced damage in mouse bone marrow cells, but at high concentra-tion enhanced the radiation-induced effect [15]. The authors hypothesized that highconcentration of vitamin C could participate in the production of hydroxyl freeradicals through a Fenton reaction [15], and this is in agreement with our results(Fig. 8). At low Cu concentrations (5 �M) the protective effect of vitamin C wasstill predominant, but when Cu was added at high concentrations (50 �M), theprotective effect was negated by the damage resulting from the formation ofhydroxyl free radicals by Cu/vitamin C redox activity. Furthermore, the addition ofCu(I) specific chelating agent, (BSCA), prevented the formation of free radicals byFenton reaction. Under these conditions, vitamin C exhibited primarily a protectiveeffect against radiation-induced DNA damage.

All these results suggested that vitamin C may mediate multiple biological effectsunder different experimental conditions. These in vitro results, if they are indicativeof similar effects in humans in vivo, are important to consider in the use of vitaminC as a radioprotective or therapeutic agent.

Previous studies have demonstrated that oxidative damage in human tissues playsa major role in certain metabolic diseases such as Cu overload. For example, ascompared to control mice, 90% of 1 year old tx mice (which share many of thecharacteristics of human Wilson’s disease) had high hepatic Cu and developednodules with marked gross abnormal liver structures, with a high incidence ofapoptotic bodies [36,37]. These effects were considered to be due to the oxidativedamage from accumulated Cu which can generate active Cu(I) in the presence of a

Fig. 8. Formation of reactive oxygen species in presence of copper and vitamin C. Luminol-enhancedchemiluminescence was measured at different concentrations of copper (0, 5 and 50 mM) with orwithout 2 mM vitamin C. Reactive oxygen species (ROS) is shown as counts per second (cps). Theasterisks indicate values significantly different (P�0.05) from those without vitamin C.


reductant such as vitamin C [37,38]. Similarly, the sensitivity of LEC rats (anothermodel of Wilson’s disease) to radiation-induced DNA damage and chromosomeaberrations was increased [39]. These effects can be explained by Cu-mediatedoxidative stress.

In contrast to results with vitamin C, GSH could provide marked protectionfrom radiation-induced DNA damage even in the presence of Cu (Fig. 5). Consid-ering the reductive capacity of GSH, it has the theoretical capacity to generatedamaging Cu(I) radicals from Cu(II) in the same fashion as vitamin C. However,spin-trapping experiments have indicated that, while DNA strand scission by Cu(II)complexed with ethylemediamine or L-histidyl-glycine-glycine was enhanced byascorbate, GSH had no effect [40]. Furthermore, GSH, unlike vitamin C has thecapacity to form a 1:1 chelate with Cu(I) [41], and this may explain why vitamin Ccan exacerbate DNA damage under our experimental conditions where as GSH canprotect. Thus, the difference between GSH and vitamin C on protective effectCu-indued DNA damage depends on the effective chelation of Cu. Our results withCu-specific chelators also support that vitamin C can protect DNA damage, if theCu(I) is chelated. Thus, the Cu chelators may be useful both in reducing Cuaccumulation in tissues and toxic events in Wilson’s disease [42]. The effectivechelation of Cu could also protect cells from oxidative injury when the individualswith aberrant Cu accumulation are exposed to radiation or other stress conditionsresulting in increased oxidation.

Acknowledgements

This study was supported by operating grants from the Canadian Institutes ofHealth Research to M.G. Cherian and J. Koropatnick.

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Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper