Mutation Research, 251 (1991) 109-113


© 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100220T MUT 05013

Iron is the intracellular metal involved in the production of D N A damage by oxygen radicals Alberto C. Mello-Filho and Rog6rio Meneghini Department of Biochemistry, Institute of Chemistry, University of Sdo Paulo, Sdo Paulo (Brazil)

(Received 24 August 1990) (Revision received 6 March 1991) (Accepted 25 April 1991)

Keywords: Oxy radical; Hydroxylradical; Iron; Copper; Electron spin resonance; Hydrogen peroxide

Summary The metal chelators 1,10-phenanthroline and 2,9-dimethyl-l,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical formation from hydrogen peroxide and Cu + or F2 z + (Fenton reaction) as determined by electron spin resonance, o-Phenanthroline prevented both Fe- and Cumediated Fenton reactions whereas neocuproine only prevented the Cu-mediated Fenton reaction. Because only 1,10-phenanthroline but not neocuproine prevented D N A strand-break formation in hydrogen peroxide-treated mammalian fibroblasts it appears that the Fe-mediated, as compared to the Cu-mediated, intranuclear Fenton reaction is responsible for D N A damage.

The production of D N A lesions by reactive oxygen species (ROS) has been intensely investigated in recent years. Several mechanisms have been suggested to explain the clastogenic effect of these species. One of them refers to a situation in which cells are exposed to an external source of ROS, as for instance at the inflammatory site when target cells are exposed to activated leuko-

Correspondence: Dr. R. Meneghini, Department of Biochemistry, Institute of Chemistry, University of S]o Paulo, C.P. 20780, S~o Paulo (Brazil). Abbreviations: ESR, electron spin resonance; DMPO, 5,5-di-

methyl-l-pirroline-l-oxide; PBS, phosphate-buffered saline (0.137 M NaC1, 2.7 mM KCI, 8 mM Na2HPO4, 1.5 mM KH2PO4); DETAPAC, diethylenetriamine pentaacetic acid; Desferal, desferrioxamine mesylate; ROS, reactive oxygen species.

cytes. In this case evidence has accumulated (Meneghini, 1988; Mello-Filho and Meneghini, 1984; Mello-Filho and Meneghini, 1985; Cochrane et al., 1988) to allow the proposal of a model according to which: (i) H 2 0 2 is the relevant ROS which carries into the cell the potential to produce D N A lesions (i.e., external 0 2 has no role other than to dismutate into H202); (ii) in the nucleus H 2 0 2 reacts with a transition metal to produce O H radical by a Fenton-type reaction and this radical is the species that ultimately damages DNA; (iii) an intracellular reducing agent is clearly necessary to provide ~educed metal ion for the Fenton reaction from a source of the more stable oxidized form. Among the several aspects of this model, one which remains unclear is the nature of the metal involved in this process. Both iron and copper form cellular complexes that have been shown to generate O H


We employed mammalian cell lines with 2 different origins: Val3 (an SV40-transformed WI38 human fibroblast) and 3T3 mouse fibroblasts. Virtually all experiments with cells shown here were repeated with these 2 cell lines and in some cases with other cell lines, and the results were essentially the same in all the cases. Therefore the results shown here are representative of a set of equivalent results. The cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal serum, 100 units/ml penicillin and 100 ~ g / m l streptomycin under a humidified atmosphere with 5% CO 2 at 37°C.

other chemicals at the specified concentrations. The titration of H 2 0 z was carried out in each experiment by the method of Cotton and Dunford (1973). For D N A strand-break determination the procedure previously described (Meneghini, 1976) was followed. The cells were treated with a solution containing 10 mM EDTA, 100 mM NaC1 and 0.5% Triton X-100 for 1 min at 37 ° C. This dissolves out the cytoplasm, leaving the nucleus attached to the bottom of the plate; for lysis 1.0 ml of a solution containing 1.0 M NaC1, 0.3 M N a O H and 10 mM E D T A was added for 1 h at 37 ° C; 0.2-ml aliquots of the lysates were layered on the top of 4.5-ml alkaline sucrose gradients (5-20% sucrose w / w in 0.3 M NaOH, 1.0 M NaCI, 10 mM E D T A ) and centrifuged at 25,000 rpm for 2 h at 2 0 ° C in an SW-50.1 rotor of a Beckman L-5 centrifuge. The radioactivity of the fractions collected from the bottom of the tube was measured as described elsewhere (Carrier and Setlow, 1971). The molecular weight of D N A for each fraction was determined by Studier's expression (Studier, 1965) using T7 DNA as standard. The number average molecular weights ( M , ) were obtained according to the expression M, = ~,ri/~,(ri/M i) where r i and M i are radioactivity and molecular weight of D N A in fraction i, respectively. For this calculation the top 3 fractions were ignored. The number of breaks was calculated by the formula: number of breaks = (M, u n t r e a t e d / M n treated - 1). The values were converted per 10 8 Da.

ESR spectrum


For O H radical measurements the spin trap D M P O was employed. The ESR signals were detected in an E4 Varian spectrometer. Samples were kept at room temperature in an 80-tzl flat quartz cuvette. Operation conditions were: microwave power, 20 mW; modulation amplitude, 1 G; time constant, 0.1 s; scan rate, 25 G / m i n .

The following chemicals were from Sigma Chem. Co.: o-phenanthroline, neocuproine, bathophenanthroline and DETAPAC. DMPO was from Aldrich Chemicals and was purified in an activated charcoal column and dissolved in bidistilled-deionized water. Desferal was a gift from Ciba-Geigy. [3H]Thymidine (50 C i / m m o l e ) was from New England Nuclear.

radical from H202 (Halliwell and Gutteridge, 1985). Moreover, both Fe (Mello-Filho and Meneghini, 1985) and Cu (Samuni et al., 1983) have been shown to mediate site-specific Fenton reactions, a situation in which the metal ion is bound to a macromolecule which will be the target of the generated O H radical. In the present work we have addressed this point by using 2 metal chelators which have very similar structures, o-phenanthroline (1,10-phenanthroline) and neocuproine (2,3-dimethyl-l,10-phenanthroline), but nevertheless exhibit different properties in terms of D N A protection against the effects of ROS and of inhibition of the in vitro Fenton reaction. Materials and methods


DNA strand breaks Cells were labeled for 1-2 days with 0.3 /~Ci/mi of [3H]thymidine (50 Ci/mmole). About 10 6 cells were seeded in 5-cm petri dishes and 12 h later washed with PBS. In the usual treatment the cells received 2 ml of PBS plus H 2 0 2 and

Results and discussion

In an attempt to determine the metal ion mediating the production of D N A strand breaks we compared o-phenanthroline and neocuproine


,5(;, Fig. 1. ESR spectra of the DMPO-OH adduct. The spectra were scanned approximately 5 rain after the beginning of the reaction, at room temperature. All reaction mixtures were 5×10 3 M HCI and 0.1 M DMPO. In addition A contained 10 -3 M FeSO 4 and 10 3 M H202, B contained 10 3 M H202 and C contained 10 3 M FeSO 4. Operating conditions are described in Materials and methods.

in terms of their capacity to prevent O H radical production by the Fenton reaction. In Fig. 1 the O H radical was produced by mixing FeSO 4 and H 2 0 2 and was detected by the spin trap DMPO (Bannister et al., 1982). The typical ESR spectrum of the D M P O - O H adduct is shown in Fig. 1A, and is characterized by the quartet with intensity ratios 1 : 2 : 2 : 1 and hyperfine splitting constants a N = a H = 14.9 G (Samuni et al., 1986). These spectra were recorded at 5 min of reaction and remained unaltered up to 10 min (not shown). We detected a vestige of the spectrum in Fig. 1B, without addition of the metal, probably due to

the reaction of H 2 0 2 with traces of contaminant metal. In Fig. 1C it can be seen that without addition of H 2 0 2 no signal is observed, showing that there is no significant contribution of Fe 2+ autoxidation in Fig. 1A. The height of the signal in the D M P O - O H ESR spectrum was employed to quantify the production of O H radical via the Fenton reaction. Both copper (CuSO 4) and iron (FeSO4) ions were employed to generate O H radical from H202. Cuprous ion should have been used as the ultimate Fenton reactant but cuprous salts are readily converted to the cupric form. We employed cupric salt which in the presence of H 2 0 2 and D M P O produced a D M P O - O H adduct with an ESR spectrum identical to that shown in Fig. 1A. It is likely that the production of O H radical was mediated by a reduction of Cu 2+ by H 2 0 2 , followed by a Fenton reaction of Cu + with H 2 0 2 : (i) H 2 0 2 + C u 2 + - - ) Cu+--[ -- 0 2 + 2H+; (ii) C u + + H 2 0 2 ~ C u 2 + + O H - + O H - The experiment in Fig. 2A showed that in the case of a copper-mediated Fenton reaction the compounds o-phenanthroline and neocuproine were equally effective in inhibiting the production of O H radical. In fact, when the chelator:metal molar ratio was greater than 3 virtually no signal was detected. A different situation was found in the case of ironproduced O H radical (Fig. 2B). In this case increasing concentrations of o-phenanthroline progressively inhibited OH-radical formation, a corn-




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Fig. 2. Prevention by o-phenanthroline and neocuproine of the DMPO-OH adduct production by U202 and iron/copper. The height of the signal with intensity 2 in the DMPO-OH ESR spectrum was normalized to quantify the production of OH radical. The concentrations were 10 3 for H202, CuSO4 (A) and FeSO4 (B). The concentrations of o-phenanthroline (e) and neocuproine (0) varied according to the indicated chelator:metal ratio.



Added chelator

D N A strand breaks

% Inhibition

None 10 5 M o-phenanthroline 10 - 4 M o-phenanthroline 1 0 - 4 M neocuproine 10 -3 M desferal 10 4 M bathophenanthroline disulfonate 10 -3 M D E T A P A C

1.00 0.13 0 1.20 0.77

0 87 100 0 23

0.71 0.90

29 10

VA13 h u m a n fibroblasts, prelabeled with [3H]thymidine, were exposed to 3 × 1 0 -5 M H 2 0 2 in PBS for 30 min at 3 7 ° C in the presence of the indicated concentrations of transition metal chelators. The frequency of D N A strand breaks was determined as described in Materials and methods and normalized in relation to the control value (no chelator added) which produced approximately 3 single-strand breaks per 108 Da.

plete inhibition being observed at a chelator: metal molar ratio equal to or greater than 3. This is in agreement with the stoichiometry of the complex of 3 o-phenanthroline to 1 iron. However, neocuproine at a concentration 10 times higher than Fe 2+ showed no effect on the DMPO-OH signal, revealing a marked distinction between these 2 chelators in preventing OH-radical generation from the iron-mediated Fenton reaction. We then determined how protective o-phenanthroline and neocuproine were against the production of DNA strand breaks by H 2 0 z. For this purpose sedimentation analysis of the DNA was employed. Typical DNA sedimentation profiles have been shown in previous works from this laboratory (Mello-Filho and Meneghini, 1984, 1985). The experiment in Table 1 shows that among several metal chelators only o-phenanthroline prevented DNA strand-break production very efficiently. In fact, 10 -5 M o-phenanthroline afforded 87% protection whereas 10 -4 M neocuproine was completely ineffective, having induced even a slight stimulation (this was reproducible but we cannot explain the results). Control experiments were run in which the chelators

without H 2 O 2 w e r e shown to produce no strand breaks (results not shown). Other chelators, desferal, DETAPAC and bathophenanthroline disulfonate, which we determined to effectively prevent OH production by Fe-directed Fenton reaction (ESR experiments not shown), were only slightly protective against DNA strand-break formation. However, as previously discussed (Mello-Filho and Meneghini, 1985), these compounds should exhibit a low capacity to cross the plasma membrane, bathophenanthroline sulfonate and DETAPAC because of their charged structures and desferal, a 600-MW polypeptide, because of its size as well. However, this should not be the case with neocuproine, which differs from ophenanthroline only by 2 additional methyl groups. These groupsshould give neocuproine an even stronger hydrophobic character and therefore a greater capacity to cross the cell membrane. In fact, we ran control experiments to show that neocuproine does enter the cell (not shown). In these experiments the cells were preloaded with neocuproine and then, after washing, exposed to the strong clastogenic complex o-phenanthroline-Cu (Que et al., 1980). A significant protection against the clastogenic action was observed when the cells were preloaded with neocuproine, due to the intracellular formation of the inert complex neocuproine-Cu (Que et al., 1980). The close parallelism exhibited by o-phenanthroline and neocuproine in their effects on HzO2-produced DNA strand breaks and F e / C u mediated production of OH radical is a strong argument in favor of iron being the intracellular metal involved in the production of DNA lesions b y H 2 O 2.

A possible explanation is that o-phenanthroline is preventing intracellular iron from participating in Fenton-type reactions responsible for DNA damage. Little is known about intranuclear concentrations of Fe and Cu. Sissoef et al. (1976) reported that mammalian DNA, extracted in a way to prevent binding of contaminant metal, contained 32 /zg of iron per g of DNA. The reason for the different behavior of these 2 chelators may lie in the fact that o-phenanthroline binds strongly to Fe z+ (3 o-phenanthroline: 1 Fe 2+) and Cu + (2 o-phenanthroline:l Cu +)

113 forming complexes with high stability constants, /33 = 21.1 a n d /32 = 15.8, respectively (Sillen, 1964). N e o c u p r o i n e also forms a stable 2 : 1 complex with C u + with /32 = 19.1 b u t its b i n d i n g c o n s t a n t to F e z+ is very low, only the first association step b e i n g observed (Sillen, 1964). W e had already a n t i c i p a t e d the i n v o l v e m e n t of iron instead of c o p p e r in the m e d i a t i o n of D N A s t r a n d breaks ( M e l l o - F i l h o a n d M e n e g h i n i , 1985). I n fact, had c o p p e r b e e n the m e t a l r e s p o n s i b l e for the catalysis, o - p h e n a n t h r o l i n e w o u l d have enh a n c e d D N A s t r a n d - b r e a k p r o d u c t i o n instead of affording p r o t e c t i o n ( Q u e et al., 1980). A l t h o u g h o - p h e n a n t h r o l i n e chelates C u + into a form that does n o t react with H 2 0 2 (Fig. 2), the t e r n a r y complex formed between D N A , o-phenanthroline a n d Cu is extremely clastogenic t h r o u g h a reaction that involves oxygen ( Q u e et al., 1980). It thus seems that c o p p e r as a F e n t o n r e a c t a n t a n d o - p h e n a n t h r o l i n e - c o m p l e x a b l e is virtually a b s e n t in the n u c l e u s of h u m a n fibroblasts u n d e r the e x p e r i m e n t a l c o n d i t i o n s described. T h e s e results with o - p h e n a n t h r o l i n e a n d n e o c u p r o i n e also reinforce the a r g u m e n t that an i r o n - m e d i a t e d F e n t o n r e a c t i o n may be the m a i n c o n t r i b u t o r to D N A s t r a n d breaks i n d u c e d by R O S .

Acknowledgements W e t h a n k Dr. O h a r a A u g u s t o for h e l p i n g with the e l e c t r o n spin r e s o n a n c e e x p e r i m e n t s . This work was s u p p o r t e d by grants from by F I N E P , F A P E S P a n d CNPq, Brazilian agencies of science support.

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Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals.

The metal chelators 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical forma...
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