dna base modifications and membrane damage in cultured mammalian cells treated with iron ions

Pergamon Free Radical Biology & Medicine, Vol. 18. No. 6, pp. 1013-1022. 1995

1995 Elsevier Science Ltd Printed in the USA. All rights reserved

0891-5849/95 $9.50 + .00

0891-5849(94)00241-x

Original Contribution

D N A B A S E M O D I F I C A T I O N S A N D M E M B R A N E D A M A G E I N

C U L T U R E D M A M M A L I A N C E L L S T R E A T E D W I T H I R O N I O N S

TOMASZ H. ZASTAWNY, *t STEVEN A. ALTMAN, ~: LISA RANDERS-E1CHHORN, + RAPTI MADURAWE, ~

JANICE A. LUMPKIN, ~ MIRAL DIZDAROGLU,* and GOVIND RAO *~

*Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA: ~Department of Clinical Biochemistry, Medical School, Poland; *Department of Chemical and Biochemical Engineering,

University of Maryland Baltimore County, Baltimore, MD, USA: and ~Medical Biotechnology Center of the Maryland Biotechnology Institute,

University of Maryland, Baltimore, MD, USA

(Received 12 August 1994; Revised 21 November 1994: Accepted 2 December 1994)

Abstract~We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 #M, 100 #M, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(lI) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(ll) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.

Keywords--Hydroxyl radical, Ferrous iron, In situ DNA damage, GC/MS-SIM, Lipid peroxidation, Superoxide radical, Frec radicals

INTRODUCTION

Oxygen-derived species formed in living cells by normal cellular metabolism or by exogenous sources are thought to play a causative role in biological processes such as mutagenesis, carcinogenesis, reproductive cell death, and aging.J-3 Of these species, superoxide radical (02"-) and H202 do not cause any DNA damage under physiological conditions. 4-7 The toxicity of 02"- and H202 is thought to result from their transition metal ion-catalyzed conversion (Haber-Weiss reaction) into hydroxyl radical ('OH), which reacts with components of DNA at or near diffusion-controlled rates, s Of the

Address correspondence to: Govind Rao, University of Maryland Baltimore County, Department of Chemical and Biochemical Engi- neering, Room 252 TRC Bldg., 5200 Westland Blvd., Baltimore, MD 21227, USA.

transition metal ions, iron and copper ions are the most likely candidates in vivo for catalyzing the conversion of 02"- and H202 into "OH, and of other poorly reactive species into reactive ones)

Iron is an essential component of many biological molecules including many enzymes. Under normal physiological conditions, the availability of iron ions for catalyzing free radical reactions in vivo is ex- tremely limited. Iron metabolism is well controlled in organisms by being bound to proteins such as transferrin, lactoferrin, and ferritin so as to minimize the amount of free intra- and extracellular iron. 2 Oxidative stress can cause release of iron from its storage sites. Thus 02"- can release iron from ferritin to serve as a catalyst for free radical reactions. 9-~ Heme proteins can be damaged by H202, releasing iron ions. 1213 On the other hand, while transferrin and lactoferrin do release iron at lower pH and in the presence of chela-

1013

1014 T.H. ZASTAWNY et al.

tors, they do not seem to be markedly affected by H202 and 02"-. 14

The ability of iron ions to cause mutagenesis, most likely by producing "OH, has been demonstrated. ~5 17 In the presence of H202, iron ions produce typical "OH- induced DNA base modifications and DNA-protein crosslinks in isolated DNA and chromatin. 4'~8'~9 Treat- ment of mammalian cells with H202 causes DNA strand breaks and modifies all four DNA bases, most likely due to site-specific generation of "OH in reactions of H202 with DNA-bound metal ions. 2° 22 Epide- miological studies have suggested a causal relationship between iron stores and cancer risk in humans. 23 Thus, free radical reactions stimulated by iron ions appear to be involved in the pathology of hemochromatosis (iron overload). 2

In the present work, we have examined the ability of exogenous iron ions in culture medium to produce DNA base modifications and membrane damage in cultured mammalian cells.

MATERIALS AND METHODS

Chemicals

Modified DNA bases, their stable isotope-labeled analogues, thymine-a,a,a,6-EI-L and materials for gas chromatography/mass spectrometry (GC/MS) were obtained as described. 24'25

Cell culture

Suspension cultured SP2/0 derived murine hybrido- mas, HyHEL-10, producing a monoclonal isotype 1 heavy chain anti-avian lysozyme IgG 26 were grown in Dulbecco's Modified Eagles Medium/Ham's F-12 (DMEM) supplemented with L-glutamine, fl-mercap- toethanol, NaHCO3, and 4% (v/v) fetal bovine serum (FBS). Cells were maintained at 37°C in 5% CO2 and were subcultured at midexponential growth phase. Cells were amplified in a 4 liter capacity CellStation ® bioreactor (Artisan Industries, Inc., Waltham, MA) as previously described. 27 Environmental and cell growth parameters were microprocessor controlled. Cultures were maintained throughout the growth cycle at 37°C, pH 6.90 with a dissolved oxygen (DO) concentration of 10% air saturation. Cells reached a density of approximately 8.0 × 105 viable cells/ml before samples were withdrawn for experimental use. Viability of cells treated with FeSO4 as determined by simultaneous fluorescent staining with fluorescein diacetate and propidium iodide, 28 typically exceeded 93%. No significant decline in cell viability occurred during the time course of any experiment.

FeS04 treatment

Cells were aseptically transferred into sterile 75 cm 2 T-flasks, placed into a shaking water bath at 37°C, and equilibrated with air (100% DO) for 30 min prior to addition of FeSO4 stock solution to bring replicate flasks to a final concentration of 10 #M, 100 #M, or 1.0 mM FeSO4. FeSO4 was prepared as a l0 mM solution in distilled water just prior to the experiments. There was no visual precipitate formation, suggesting that the iron remained in the Fe(II) form at the time of addition to the cells in our experiment. Control cells were treated in the same manner except for Fe(II) treatment. Cells were gently agitated for 1 h, recovered by centrifugation, resuspended in phosphate buffered saline (PBS, 28 mM NaC1, 0.6 mM KCI, 1.6 mM NaEPO4, 0.3 mM KH2PO4) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM di- thiothreitol (DTT), rapidly frozen at -70°C, and stored at -20°C until chromatin extraction.

A second lot of cells was aliquoted into 75 cm 2 T- flasks containing sufficient ascorbic acid to bring the cells to a final concentration of 1.0 mM ascorbate. Flasks were incubated in a shaking water bath at 37°C at 100% DO for 30 min. Subsequently, cells were pelleted by centrifugation at 500 × g for 5 min, washed 2× in fresh DMEM/4% FBS medium, and resuspended in 4 0 - 5 0 ml of fresh medium. FeSO4 was then added to bring replicate flasks to a final concentration of l0 #M, 100 #M, or 1.0 mM. Control cells were treated in the same manner except for Fe(II) treatment. Cells were gently agitated for l h, recovered by centrifugation, resuspended in PBS, and stored until chromatin extraction. To determine the effect of dimethyl sulfoxide (DMSO) on DNA damage, the above procedure was repeated using aliquots of cells from the same reactor run except that cells were incubated in the presence of 100 mM or l M DMSO and were not washed prior to the addition of FeSO4. DMSO, therefore, was present in the medium during Fe(II) treatment.

To determine if residual iron ions may have an effect on DNA damage, experiments were conducted using deferoxamine. Cells were treated as described above with 1.0 mM FeSO4. This was followed by addition of deferoxamine to the medium to a concentration of 5 mM. Cells were incubated for 10 min, recovered by centrifugation, and sequentially washed once with each of the following: DMEM/4% FBS medium supplemented with 5 mM deferoxamine, DMEM/4% FBS medium and PBS. Cells were then frozen and stored until chromatin extraction. Controls consisted of untreated cells and of cells treated with Fe(II), but with no deferoxamine treatment. To remove possible iron ion residues from extracted chromatin, samples of

DNA base damage in FE(tl)-treated mammalian cells 1015

chromatin in 1 mM Tris-HCl were treated with 5 mM deferoxamine for 10 min at 4°C, centrifuged, washed once with 1 mM Tris-HCl, and stored for subsequent analysis. Controls consisted of chromatin from Fe(II)- treated cells without subsequent deferoxamine treatment and chromatin from untreated cells either with or without deferoxamine treatment.

Chromatin extraction

Chromatin was extracted from cells as described. 29 The DNA content of extracted chromatin was measured by UV absorbance at 260 nm (absorbance of 1 = 50 #g of DNA/ml) and was verified by fluorometric concentration analysis using Hoechst dye 33258 (Pierce).

Analysis by gas chromatography/mass spectrometry

Aliquots of thymine-a,t~,oz,6-ZH4 and stable isotope- labeled analogues of modified DNA bases were added as internal standards to chromatin samples containing 100 #g of DNA. 24'z5 Samples were hydrolyzed, deriva- tized, and then analyzed by GC/MS with selected ion- monitoring (SIM). 3° The quantification of modified DNA bases was done by isotope-dilution mass spectrometry. 24'25 Thymine-ot,a,t~,6-2H4 was used as an internal standard for thymine to verify the amount of DNA in chromatin samples. 3~

Measurement of membrane permeability and lipid peroxidation

Approximately 9.0 × 106 cells cultured in a T-flask at midexponential growth stage were pelleted by centrifugation and incubated for 1 min in 25 ml of warmed (37°C) DMEM/4% FBS medium containing 12.5 ml of fluorescein diacetate working solution 31 made up in DMEM/4% FBS media. Cells were washed twice with fresh DMEM/4% FBS medium, resuspended in 600 ml of fresh medium, aliquoted into 75 cm 2 T-flasks in a shaking water bath at 37°C, and equilibrated at 100% DO for 30 min prior to addition of FeSO4 stock solution to bring replicate flasks to a final concentration of 10/.tM, 100/,tM, or 1.0 mM FeSO4. At 15 min inter- vals, 300 #1 samples of cell suspension in 1.8 ml amber Eppendorf tubes were centrifuged at 500 × g for 3 min to pellet cells with minimal lysis. Control cells were treated in the same manner except for FeSO4 treatment. Replicate aliquots of 200 #1 of supernatant were dispensed into a 96-well polystyrene MicroFluor B microtitration plate (Dynatech Laboratories, Chan- tilly, VA) and the fluorescent intensity recorded using a Pandex fluorescence concentration analyzer (Baxter

Health Care Corp., Mundelein, IL) at 485 nm excitation/595 nm emission. Replicate T-flasks containing DMEM/4% FBS medium were sampled as controls.

Lipid hydroperoxides formed in the membrane component of Fe(II)-treated cells were quantified using a modified iodometric assay. 33 Conditions were modified such that the assay was specific for lipid hydroperoxides and endoperoxides were excluded. Approximately 10 6 cells at midexponential growth stage were pelleted by centrifugation. The cell pellet was washed with PBS and resuspended in chloroform:methanol [2:1 (v/v)]. Phases were separated by centrifugation and the aqueous phase was aspirated away. The chloroform layer was transferred to a test tube and brought to dryness in vacuo at 45°C. The dried membrane fraction was resuspended in 1 ml of acetic acid:chloroform [3:2 (v/ v)] and 50 ml of saturated potassium iodide (1.2 g KI/ ml). The reaction was stopped after 5 min with 1 mM cadmium acetate. Phases were separated by centrifugation, and the absorbance of the aqueous phase was read at 353 nm against a blank of all reagents without the dried membrane fraction. The lipid peroxide content of Fe(II)-treated cells was quantitatively derived from a four-point standard curve constructed using 12.5, 25, 50, and 100 #M tert-butyl hydroperoxide. The total lipid peroxide content was estimated in cells treated with 10 mM tert-butyl hydroperoxide for 5 h.

Mathematical simulation of DNA damage

A kinetic simulation model of DNA damage by "OH in solutions containing Fe(II) was constructed to mathematically predict a dose-response relationship for DNA damage. The goal of the modeling was to determine if key reactions well established in the litera- ture could account for DNA base damage under biolog- ically relevant Fe(II) concentrations and predict the type of dose response for DNA damage that was exper- imentally observed. The reactions, their associated rate constants, and the resulting system of coupled ordinary differential equations (ODEs) included in this in vitro simulation model were previously described. 27 This system of ODEs along with the initial reactant concentrations (DNA, H202, Fe(II), 02) constituted the kinetic simulation model. The model was solved numerically on a SGI Crimson computer with the numerical ODE solver LSODES. 34 A solution of the model consisted of a list of concentrations for any reactant, intermediate ('OH) or product (DNA hits) at a chosen time of inte- gration. A DNA hit was defined as any product of the reaction "OH + DNA. The DNA concentration was held constant to allow for multiple hits. Values for the initial DNA, H202 and 02 concentrations used in the simulation were 3.5 × 10 -9 M, 1.0 × I0 -s M, and 2.8

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Fig. 1. The amounts of modified DNA bases in murine hybridoma cells. Treatment: 1, control; 2, 10 #M Fe(II); 3, 100 /.LM Fe(II); 4, 1 mM Fe(II); 5, ascorbic acid + 1 mM Fe(lI); 6, I mM Fe(lI) + 100 mM DMSO; 7, I mM Fe(II) + 1 M DMSO. Each data point represents the mean ± standard error from measurement of chromatin samples isolated from four separate batches of cells (1 nmol of a modified base/mg of DNA ~32 molecules of a modified base/105 DNA bases). Stars (*) above the error bars indicate that the treatment is significantly different (p -< 0.05 by Student 's t-test) from control. " n s " indicates treatments that appear different by comparison of error bars, but are not significantly different (by the Student 's t-test) relative to control (treatments 2 - 4 ) or to 1 mM Fe(ll) treatment (treatments 5-7) .

>( 10 -4 M, respectively. The DNA concentration was that reported in previous in vitro studies; ~5 the H202 concentration was the midvalue of the reported H202 concentration range in liver cells; 2 and the 02 concentration corresponded to air saturation (100% DO). The reaction was permitted to proceed for 60 min, the experimental treatment time and sufficient time for it to reach completion and the concentration of DNA hits for a given Fe(II) initial concentration was calculated. The concentration of DNA hits was then divided by the initial DNA concentration to give the DNA damage.

R E S UL T S

Ten pyrimidine- and purine-derived lesions were identified and quantified in chromatin samples isolated from Fe(II)-treated and control cells. These were 5- hydroxy-5-methylhydantoin (5-OH-5-MeHyd), 5-hy-

droxyhydantoin (5-OH-Hyd), 5-hydroxyuracil (5-OH- Ura), 5-(hydroxymethyl)uracil (5-OHMeUra), 5-hydroxycytosine (5-OH-Cyt), 5,6-dihydroxyuracil (5,6- diOH-Ura), 4,6-diamino-5-formamidopyrimidine (Fa- pyAde), 8-hydroxyadenine (8-OH-Ade), 2,6-diamino- 4-hydroxy-5-formamidopyrimidine (FapyGua), and 8- hydroxyguanine (8-OH-Gua). Figure 1 illustrates the amounts of the modified bases in control cells and in cells treated with Fe(II) under various conditions. Pretreatment of cells with ascorbic acid did not affect the background levels of modified bases (data not shown). Treatment of cells with 10/zM Fe(II) caused no significant increases in the background amounts of modified bases except for 5-OH-5-MeHyd (Fig. 1). In the case of 5-OH-Ura, 5-OH-Cyt, FapyGua, and 8- OH-Gua, the error bars of 10 #M Fe(II) treatment samples did not overlap with the error bars of control samples. Nevertheless, the Student's t-test showed no significant difference. These cases are designated with

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1018 T.H. ZASTAWNV et al.

" n s " (not significant) in Fig. 1. Significant increases in the amounts of 5-OH-5-MeHyd, 5-OH-Ura, 5-OH- Cyt, 8-OH-Ade, and 8-OH-Gua over their background levels were observed in cells treated with 100 #M Fe(II). In this case, although the error bars for FapyAde and FapyGua did not overlap with the error bars of control samples, the Student's t-test yielded no significant difference. All 10 modified bases were significantly formed in cells upon treatment with 1 mM Fe(II) (Fig. 1). The increase of product yields over background levels varied from 1.4- to 3.9-fold. The highest ratio of increase was observed in the yields of 8-OH-Ade (3.9-fold), 5-OH-Cyt (3.4-fold), 5-OH-Ura (3.3-fold), and 8-OH-Gua (2.6-fold). In terms of the net yield produced at 1 mM Fe(II) treatment, 8-OH- Gua was the major product (0.44 nmol/mg of DNA) followed by 5-OH-Hyd (0.3 nmol/mg of DNA), Fapy- Ade (0.21 nmol/mg of DNA), 8-OH-Ade (0.20 nmol/ mg of DNA), 5-OH-Cyt (0.18 nmol/mg of DNA), and 5-OH-Ura (0.14 nmol/mg of DNA).

Product yields in cells, which were treated with ascorbic acid and then with 1 mM Fe(II), were similar to those in cells exposed to 1 mM Fe(II) only (Fig. 1). The same was true of cells exposed to 10 #M or 100 #M Fe(II) (data not shown). To test the effect of an "OH scavenger on product formation, cells were treated with l mM Fe(II) in the presence of DMSO at a concentration of 100 mM or 1 M in the culture medium. Product yields observed under these conditions are also shown in Fig. 1. No significant inhibition of product formation by DMSO was observed.

The effect on product yields of any possible Fe(II) residues in the culture medium during sample prepara- tion for GC/MS analysis was tested. This was done by measuring the yields of products in cells treated with 1 mM Fe(II) and subsequently incubated with deferoxamine to remove possible Fe(II) residues. For comparison, aliquots of the same lot of Fe(II)-treated cells without incubation with deferoxamine were also used to measure the yields of products. As examples, Fig. 2 illustrates the yields of several products in cells treated under these conditions. No significant change in product yields was observed in cells incubated with deferoxamine following Fe(II) treatment. Furthermore, chromatin samples isolated from cells were incubated with deferoxamine to remove any possible Fe(II) residues. Subsequently, the yields of products were measured. Again, no change in product yields was observed when these results were compared with those obtained with chromatin samples that were not incubated with deferoxamine (data not shown).

Cell membrane permeability, measured as increased fluorescence at 485 nm excitation/535 nm emission, increased in response to treatment with varying con-

centrations of Fe(II) over a 2-h time period (Fig. 3). No increase in membrane permeability relative to controls was apparent in cells treated with 10 #M Fe(II) for the first 90 min following the start of treatment and increased only slightly during the final 30 min of the experiment. In contrast, the membrane permeability of cells treated with either 100 #M or 1 mM Fe(II) increased significantly after 15 min of treatment and continued to increase throughout the period of observation (Fig. 3). A plot of fluorescence vs. concentration of Fe(II) at four representative treatment times revealed a rapid increase in membrane permeability up to 100 /.tM Fe(lI) and then a slow increase up to 1 mM Fe(II) (Fig. 4). Concomitant with the increase in membrane permeability, production of lipid peroxides was observed in cells upon treatment with various concentrations of Fe(II) (Fig. 5). The increase in lipid peroxides depending on Fe(II) concentration was similar to the increase in membrane permeability.

A mathematical model simulating the dependence of DNA damage on Fe(II) concentration was solved over a concentration range of 10 #M to 1 mM Fe(II). The obtained plot exhibited an initial rapid increase followed by a slower, nearly linear increase in DNA damage (Fig. 6). The simulation result was in good agreement with the experimental results. This is clearly demonstrated in Fig. 7, where, as examples, plots of the amounts of three modified DNA bases vs. the concentration of Fe(II) are illustrated. A pattern similar to that in Fig. 6 was also observed with the change in cell membrane permeability and with the formation of lipid peroxides (Figs. 4 and 5, respectively).

DISCUSSION

The results of this study show that treatment of mammalian cells in culture with Fe(II) causes modification of all four DNA bases in their chromatin. A similar pattern of modified DNA bases was observed previously in cultured mammalian cells, which were treated with H202, ionizing radiation, activated leukocytes or tert-butyl hydroperoxide. 27'3°'36"37 Identified base lesions are typically produced in DNA by well- known "OH-generating systemsfl ~ This fact implicates "OH as the reactive species responsible for DNA base modifications in cells treated with Fe(I1). Because of short diffusion distance of "OH in cells (~2 nm), 3~ DNA base modifications are expected to occur in a site-specific manner due to formation of "OH in close proximity to DNA. 2° The failure of DMSO to inhibit DNA base damage may be due to this type of generation of "OH. Calculations based on nonhomogeneous kinetics also support the hypothesis that added "OH scavengers may not protect DNA against the action of "OH in ce l l s . 39

DNA base damage in FE(II)-treated mammalian cells 1019

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Fig. 2. The amounts of modified DNA bases in murine hybridoma cells. Treatment: 1, control; 2, 1 mM Fe(II); 3, 1 mM Fe(II) + deferoxamine. Each data point represents the mean _+ standard error from measurement of chromatin samples isolated from four separate batches of cells (1 nmol of a modified base/rag of DNA ~32 molecules of a modified base/105 DNA bases). Stars above bars indicate that the treatment is significantly different (/9 <_ 0.05 by Student's t-test) from control.

Formation of typical "OH-induced products in nuclear D N A leads to the question as to what mechanisms underlie the generation of "OH in nuclei o f cells upon Fe(II) treatment in culture. A likely mechanism may involve electron transfer reactions o f Fe(II) with molecular oxygen, giving rise to formation of 0 2 ° , which can dismutate to HzO2 .2 In in vitro studies, H 2 0 2 w a s

found to be an essential intermediate in Fe(II)-mediated D N A damage and mutagenesis. ~Sm H 2 0 2 c a n dif- fuse through the cell, cross the nuclear membrane, and react with DNA-bound metal ions, causing site-specific

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formation of "OH with subsequent damage to nuclear DNA. 2°'22 On the other hand, oxidative stress may bring about the release o f metal ions from their storage sites in cells. 2'~ Increased membrane permeability by Fe(II) treatment of cells as found in the present work may also permit diffusion of Fe(II) into cells resulting in an increase in the level of intracellular iron ions. Free iron ions may bind to cellular components including DNA, increasing the amount o f metal ions bound to DNA. By autoxidation, DNA-bound iron ions may give rise to formation of H202 and finally "OH, causing site-specific damage to DNA. 2'~5'2° In vitro studies have demonstrated that treatment of DNA with iron ions causes formation of promutagenic D N A lesions by involvement o f H 2 O 2 and "OH. 15-j7 This is in agreement with the fact that some of the D N A base lesions identified in the present work have been found to have promutagenic properties. 4°'4z-44

Another mechanism underlying D N A damage may involve lipid peroxidation. As discussed above, the interaction o f Fe(II) with oxygen can result in formation of O2" , H 2 0 2 , and, finally, "OH or a similarly reactive species, which may bring about membrane lipid peroxidation. 2"45 In the present work, Fe(II)-mediated membrane lipid peroxidation of cells was concomitant with an increase in cell membrane permeability. Lipid peroxidation has been implicated in various stages o f carcinogenesis. 45 This implies that D N A damage may occur as a result of lipid peroxidation in vivo. In isolated systems such as liver nuclei, mitochondria, and liver slices, D N A damage was detected when lipid peroxidation was induced. 46 49 However, previous work has not provided any evidence as to the nature of DNA-damaging species. Peroxyl and alkoxyl radicals, which may be generated by reactions o f iron ion corn-

1020 T.H. ZASTAWNY e t al.

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p lexes with l ip id hydroperoxides , z are unl ike ly to reach nuclear D N A even i f they are formed f rom the nuclear membrane . 45 Some other products o f l ip id peroxida t ion such as react ive carbonyl products possess diffusibi l i ty and are poss ib le mutagens. 45"5° It is not known, however, whether such carbonyl products or a lkoxyl and peroxy l radicals can cause D N A base modif icat ions observed here, even i f they reach nuclear DNA.

Recen t ly , I toh et al. have r epo r t ed that m i t o c h o n d r i a l D N A was d a m a g e d upon e x p o s u r e o f rat h e p a t o m a ce l l s to e i the r 100 /~M Fe( I I ) or Fe ( I I I ) ions. 5~ The i r resu l t s suppor t the obse rva t i on s m a d e in our s tudies . As in our s tudies , the i r s tudies were based on i ron ions a d d e d to the m e d i u m wi thou t add i t i on o f any HzO2.

A remarkab le aspect o f the present work is that s imi lar kinet ics with respect to Fe(II) concentra t ion

were observed in terms o f D N A base damage, membrane permeabi l i ty , and product ion o f l ipid peroxides . Fur thermore , the mathemat ica l s imulat ion of D N A damage upon Fe(II) t reatment was in good agreement with exper imenta l results.

Fe(I I ) is ox id ized to Fe(III ) by react ing with molecular oxygen. Its subsequent fate remains unknown at this point. W e did not observe any precipi ta t ion at the highest level of Fe(II) s tudied (1 mM). Interest ingly, in every case where we a t tempted to use Fe(III) , the cel ls rapidly lost v iabi l i ty and so a meaningful experiment could not be performed. The damaging sequence o f react ions is l ike ly to involve electron transfer reac- t ions o f Fe(II) with molecu la r oxygen, giving rise to format ion o f Fe(III ) and O2"-, which can dismutate to HEOz. z The resul t ing HzOz would then react with Fe(II) to form damag ing "OH. At h igher concentrat ions of Fe(II) , the accumula t ion o f Fe(I I I ) p roduced by the first react ion can potent ia l ly act as a scavenger o f Oz ' - by compet ing with other Oz ' - molecules and thereby decrease the overal l rate o f HzOz formation. This would lead to the decrease in D N A damage as well as l ipid peroxida t ion observed be tween 1 0 0 / z M and 1 mM.

In conclusion, the results show that the t reatment o f cells in cul ture with Fe(I I ) causes damage to all four bases in nuclear DNA, resul t ing in format ion o f a variety o f products . Treatment o f cells with iron ions may increase the intracel lular iron ion concentrat ion and/or cause oxida t ive stress re leas ing metal ions f rom their s torage sites with subsequent b inding to DNA. Mechan i sms o f D N A base damage appear to involve "OH as the damaging species p roduced by D N A - b o u n d metal ions. Identif ied D N A lesions may be potent ial ly promutagenic . Exposure o f mammal i an cells to iron ions may thus contr ibute to pa thologic processes.

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Initial Concentration of Fe(II) (BM)

Fig. 6. Mathematically simulated dependence of DNA damage on concentration of Fe(II).

0 . 3 . ,I . . . ,I . . . ,h . . . ,I . . . ,I . . . ,I .

. ~ A 5-OH-Cyt ~ 8-OH-Gua ~- * FapyGua

0 I ' ' ' 1 ' ' ' 1 ' ' ' 1 ' ' ' 1 ' ' ' 1 ' 0 200 400 600 800 1000

Concentration of Fe(II) (IxM)

Fig. 7. The amounts of 5-OH-Cyt, FapyGua, and 8-OH-Gua in cells treated with Fe(II) at different concentrations. The data in Fig. 1 were used to obtain these plots.

0.25 <

0.2

0.15

" 0.1

0.05

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

DNA base damage in FE(II)-treated mammalian cells 1021

Acknowledgements - - Support from the National Institutes of Health Grant RR-06562 and a National Science Foundation Presidential Young Investigator Award to G.R. with matching funds from Artisan Industries, Inc. Waltham, MA, is acknowledged. Support from the National Institutes of Health Research Supplement for Underrepre- sented Minority Investigators to J.L. is acknowledged. M.D. ac- knowledges support from the Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy, Washington, DC.

Certain commercial equipment or materials are identified in this article to adequately specify experimental procedures. Such identifi- cation does not imply endorsement by the National Institute of Stan- dards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

REFERENCES

1. Frenkel, K. Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol. Ther. 53:127-166; 1992.

2. Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine, 2nd ed. Oxford: Clarendon; 1989.

3. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362:709-715; 1993.

4. Aruoma, O. I.; Halliwell, B.; Dizdaroglu, M. Iron ion-dependent modification of bases in DNA by the superoxide radical-generating system hypoxanthine/xanthine oxidase. J. Biol. Chem. 264:13024-13028; 1989.

5. Blakely, W. F.; Fuciarelli, A. F.; Wegher, B. J.; Dizdaroglu, M. Hydrogen peroxide-induced base damage in deoxyribonucleic acid. Radiat. Res. 121:338-343; 1990.

6. Lesko, S. A.; Lorentzen, R. J.; Ts'o, P. O. P. Role of superoxide in deoxyribonucleic acid strand scission. Biochemistry 19:3023- 3028; 1980.

7. Sagfipanti, J. L.; Kraemer, K. H. Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide. J. Biol. Chem. 264:1729-1734; 1989.

8. von Sonntag, C. The chemical basis of radiation biology. Phila- delphia: Taylor and Francis; 1987.

9. Biemond, P.; Van Eijk, H. A.; Swaak, A. J. G.; Koster, J. F. Iron mobilization from ferritin by superoxide derived from stimulated polymorphonuclear leukocytes: Possible mechanism in in- flammation diseases. J. Clin. Invest. 73:1576-1579; 1984.

10. Buettner, G. R.; Saran, M.; Bors, W. The kinetics of the reaction of ferritin with superoxide. Free Radic. Res. Commun. 2:369- 372; 1987.

1 I. Reif, D. W. Ferritin as a source of iron for oxidative damage. Free Radic. Biol. Med. 12:417-427; 1992.

12. Gutteridge, J. M. C. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides. FEBS Lett. 201:291-295; 1986.

13. Puppo, A.; Hailiwell, B. Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron: Is haemoglobin a biological Fenton reagent? Biochem. J. 249:185-190; 1988.

14. Aruoma, O. I.; Halliwell, B. Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron: Are iactoferrin and transferrin promoters of hydroxyl-radical generation? Biochem. J. 241: 273-278; 1987.

15. Loeb, L. A.; James, E. A.; Waltersdorph, A. M.; Klebanoff, S. J. Mutagenesis by the autoxidation of iron with isolated DNA. Genetics 85:3918-3922; 1988.

16. McBride, T. J.; Preston, B. D.; Loeb, L. A. Mutagenic spectrum resulting from DNA damage by oxygen radicals. Biochemistry 30:207-213; 1991.

17. Reid, T. M.; Loeb, L. A. Tandem double CC--*TT mutations are produced by reactive oxygen species. Proc. Natl. Acad. Sci. USA 90:3904-3907; 1993.

18. Dizdaroglu, M.; Rao, G.; Halliwell, B.; Gajewski, E. Damage to the DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions. Arch. Biochem. Biophys. 285:317-324; 1991.

19. Nackerdien, Z.; Rao, G.; Cacciuttoio, M. A.; Gajewski, E.; Diz- daroglu. M. Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions. Biochemistry 30:4873-4879; 1991.

20. Chevion, M. A site-specific mechanism for free radical induced biological damage: The essential role of redox-active transition metals. Free Radic. Biol. Med. 5:27-37; 1988.

21. Dizdaroglu, M. Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275:331-342; 1992.

22. Meneghini, R.; Martins, E. L. In: Halliwell, B.; Aruoma, O. I., eds. Hydrogen peroxide and DNA damage. DNA and free radicals. New York: Ellis Horwood; 1993:83-93.

23. Stevens, R. G.; Jones, D. Y.; Micozzi, M. S.; Taylor, P. R. Body iron stores and the risk of cancer. N. Engl. J. Med. 319:1047- 1052; 1988.

24. Dizdaroglu, M. Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry. FEBS Len. 315:1-6; 1993.

25. Dizdaroglu, M. Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Methods Enzy- mol. 234:3-16; 1994.

26. Smith-Gill, S. J.; Malnhart, C. R.; Lavoie, T. B.; Rudikoff, S.; Potter, M. VL-VH expression by monoclonal antibodies recog- nizing avian lysozyme. J. lmmunol. 132:963-967; 1984.

27. Altman, S. A.; Zastawny, T. H.; Randers, L.; Lin, Z.; Lumpkin, J. A.; Remacle, J.; Dizdaroglu, M.; Rao, G. tent-Butyl hydroperoxide-mediated DNA base damage in cultured mammalian cells. Mutat. Res. 306:35-44; 1994.

28. Altman, S. A.; Randers; L.; Rao, G. Comparison of trypan blue dye exclusion and fluoromeu'ic assays for mammalian cell viability determinations. Biotech. Prog. 9:671-674; 1993.

29. Gajewski, E.; Rao, G.; Nackerdien, Z.; Dizdaroglu, M. Modifi- cation of DNA bases in mammalian chromatin by radiation- generated free radicals. Biochemistry 29:7876-7882; 1990.

30. Nackerdien, Z.; Olinski, R.; Dizdaroglu. M. DNA base damage in chromatin of gamma-irradiated cultured human cells. Free Radic. Res. Commun. 16:259-273; 1992.

31. Djuric, Z.; Luongo, D. A.; Harper, D. A. Quantitation of 5- (hydroxymethyl)uracil in DNA by gas chromatography with mass spectral detection. Chem. Res. Toxicol. 4:687-691; 1991.

32. Jones, K. H.; Senft, J. A. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate- propidium iodide. J. Histochem. Cytochem. 33:77-79; 1985.

33. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Meth- ods Enzymol. 53:302-310; 1978.

34. Hindmarsh, A. C. ODEPACK, a systematized collection of ODE solvers. In: Stepleman, R. S., ed. Scientific computing: Applica- tions of mathematics and computing to the physical sciences. IMACS transactions on scientific computation, vol. 1. New York: North Holland Publishing Co.; 1983:55-65.

35. Muiras, M. L.; Giacomoni, P. U.; Tachon, P. Modulation of DNA breakage induced via the Fenton reaction. Mutat. Res. 295:47-54; 1993.

36. Dizdaroglu, M.; Nackerdien, Z.; Chad, B.-C.; Gajewski, E.; Rao, G. Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated mammalian cells. Arch. Biochem. Biophys. 285:388-390; 1991.

37. Dizdaroglu, M.; Olinski, R.; Doroshow, J. H.; Akman, S. A. Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes. Cancer Res. 53:1269-1272; 1993.

38. Ward, J. F.; Blakely, W. F.; Joner E. I. Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide. Radiat. Res. 1113:383-392; 1985.

39. von Sonntag, C.; Schuchmann H.-P. Radical-mediated DNA damage in presence of oxygen. Methods Enzymol. 233:47-56; 1994.

40. Loeb, L. A.; McBride, T. J.; Reid, T. M.; Cheng, K. C. Muta- genic spectra of oxygen free radicals. In: Probst, G. S.; Vodicnik,

1022 T.H. ZASTAWNV et al.

M. J.; Dorato, M. A., eds. New horizons in molecular toxicology. Bethesda, MD: FASEB; 1991:35-39.

41. Toyokuni, S.; Sagripanti, J.-L. Iron-mediated DNA damage: Sensitive detection of DNA strand breakage catalyzed by iron. J. lnorg. Biochem. 47:241-248; 1992.

42. Grollman, A. P.; Moriya, M. Mutagenesis by 8-oxoguanine: An enemy within. T1G 9:246-249; 1993.

43. Purmal, A. A.; Kow, Y. W.; Wallace, S. S. Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro. Nucleic Acids Res. 22:72-78; 1994.

44. Wallace, S. S. The biological consequences of oxidized DNA bases. Br. J. Cancer 55:118-125; 1987.

45. Cheeseman, K. H. Lipid peroxidation and cancer. In: Halliwell, B.; Aruoma, O. I., eds. DNA and free radicals. New York: Ellis Horwood; 1993:109-144.

46. Fraga, C. G.; Tappel, A. L. Damage to DNA concurrent with lipid peroxidation in rat liver slices. Biochem. J. 252:893-896; 1988.

47. Hruszkewycz, A. M. Evidence for mitochondrial DNA damage by lipid peroxidation. Biochem. Biophys. Res. Commun. 153:191-197; 1988.

48. Hruszkewycz, A. M.; Bergtold, D. S. The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation. Mutat. Res. 244:123-128; 1990.

49. Shires, T. K. Iron-induced DNA damage and synthesis in isolated rat liver nuclei. Biochem. J. 205:3321-3329; 1982.

50. Esterbauer, H.; Eckl, P.; Ortner, A. Possible mutagens derived from lipids and lipid precursors. Mutat. Res. 238:223-233; 1990.

51. Itoh, H.; Shioda, T.; Matsura, T.; Koyama, S.; Nakanishi, T.; Kajiyama, G.; Kawasaki, T. Iron ion induces mitochondrial

DNA damage in HTC rat hepatoma cell culture--Role of anti- oxidants in mitochondrial DNA protection from oxidative stresses. Arch. Biochem. Biophys. 313:120-125; 1994.

ABBREVIATIONS

D M E M - - D u l b e c c o ' s Modified Eagles Medium/ H a m ' s F-12

D M S O - - d i m e t h y l sulfoxide

5 ,6 -d iOH-Ura- -5 ,6 -d ihydroxyurac i l D O - - d i s s o l v e d oxygen

FapyAde- -4 ,6 -d i amino-5 - fo rmamidopyr imid ine FapyGua- -2 ,6 -d i amino-4 -hydroxy-5 -

formamidopyr imidine F B S - - f e t a l bovine serum

G C / M S - S I M - - g a s chromatography-mass spectrometry with selected-ion monitor ing

O2"- - - superox ide radical

" O H - - h y d r o x y l radical

5 -OH-5-MeHyd- -5 -hydroxy-5 -me thy lhydan to in 5-OH-Cyt - - 5-hydroxycytosine

5 - O H - H y d - - 5 - h y d r o x y h y d a n t o i n

5 - O H - U r a - - 5-hydroxyuracil 5 - O H M e U r a - - 5-(hydroxymethyl)uracil

8 - O H - A d e - - 8-hydroxyadenine

8 - O H - G u a - - 8-hydroxyguanine

dna base modifications and membrane damage in cultured mammalian cells treated with iron ions

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