Modulation of DNA breakage induced via the Fenton reaction

Mutation Research, 295 (1993) 47-54 © 1993 Elsevier Science Publishers B.V. All rights reserved 0921o8734/93/$05.00

47

MUTAGI 00264

Modulation of D N A breakage induced via the Fenton reaction M.L. Muiras, P.U. Giacomoni and P. Tachon Laboratoires de Recherche Fondamentale de L 'Or~al, Aulnay-sous-Bois, France (Received 23 March 1992) (Accepted 19 June 1992)

Keywords: Fenton reaction; DNA breakage

Summary The conversion of the covalently closed circular double-stranded supercoiled DNA (pBR322) to a relaxed circle was used to investigate DNA nicking induced by Fe 2÷ and H202. In our experimental conditions of ionic strength (150 mM NaCI), pH = 7 and temperature (37°C), the dose-response curve for the ferrous iron mediated H202 dependent DNA nicking is peculiar. For a fixed concentration of ferrous iron (2/zM), the concentration of H 2 0 2 producing a maximum extent of DNA nicking was about 10-30 /zM. The DNA single-strand breakage decreased with an increase of H202 concentration. We have investigated the effects of several factors such as the nature of the buffer, ionic strength, temperature and pH. Buffer components leading to the autoxidation of ferrous iron to ferric iron (phosphate) or to the scavenging of reactive oxygen species (Tris) greatly alter the dose-response curve. The H202 concentrations required for producing the maximum extent of DNA single-strand breaks at 4°C and 56°C were respectively 30/zM and 3/zM. At pH = 10, the pattern of the dose-response curve was totally different. The data showed that the peculiar dose-response curve for the ferrous iron mediated H202 dependent DNA nicking greatly depended on the experimental conditions.

Ninety percent of molecular oxygen (30 2) consumed in mammalian cells is metabolized in the mitochondria. In the mitochondrial electron transport chain, oxygen is essentially reduced to water by cytochrome oxidase with a concomitant release of energy. However, a small fraction of oxygen can be partially reduced to produce su-

peroxide anion radical (O2), hydrogen peroxide ( H 2 0 2) and hydroxyl radical ( H O ) which are respectively the one, two and three univalent reduction intermediates of oxygen. 02

e-

+HOCorrespondence: Pierre Tachon, Laboratoires de Recherche Fondamentale de L'Or6al, 1, Avenue Eugene Schueller, 93600 Aulnay-sous-Bois, France. N.B. Throughout the article, O~- should be read as 0 3.

0 2 + 2H +

e - ~ H202

e - ~ HO"

(1)

It has been postulated that biological damage induced by oxygen species derived from molecular oxygen is involved in the aging process (Harman, 1981). Recently, this hypothesis has been strengthened by Ames and coworkers (Ames et

48

al., 1985; Adelman et al., 1988) who showed on the one hand a relationship between the urinary output of DNA oxidized bases and the metabolic rate of different species (mouse, rat, monkey and human) and on the other hand a correlation between metabolic rate and life span. Thus, they concluded that oxidative DNA damage is likely to be critical for aging. Among oxygen species, 0 2 and H 2 0 2 per se are not reactive but are implicated, via the Haber-Weiss reaction (reaction 2), in the production of a more strongly oxidizing species: hydroxyl radical ( H O ) 0 2 + H 2 0 2 --+ H O ' + H O - +

02

(2)

But reaction 2 is too slow (K a = 0.13 M -1 S-1) (Weinstein and Bielski, 1979) to be biologically important. As a matter of fact, the rate of the dismutation of the superoxide anion radical (reaction 3) has a rate constant K a = 4.5 • l0 s M - I S -1 0 2 + 0 2

2H +

, H202 + 02

(3)

It has been suggested that transition metals such as iron or copper act catalytically in free radical reactions. 0 2 + Fe(III) ~ 0 2 + Fe(II)

(4)

H202 + Fe(II) ~ H O ' + H O - + Fe(III)

(5)

Thus, the Fenton reaction (reaction 5) is the main route for producing hydroxyl radical as well as related species such as the ferryl ion FeO 2÷. Imlay and Linn (1988) and Imlay et al. (1988) showed that the amount of DNA single-strand breaks induced by hydrogen peroxide in the presence of ferrous iron (Fenton reaction) has a characteristic aspect: 'The maximum extent of DNA nicking occurred at low H 2 0 2 concentrations and higher concentrations suppressed about half of the damage'. We have investigated this peculiar dose-response curve for the ferrous iron mediated H 2 0 2 dependent DNA nicking and particulary the effects of several factors: nature of the buffer, pH, ionic strength and temperature. These parameters could change the aqueous iron (II/III) redox

potential thus modifying the profile of the doseresponse curve. Materials and methods

Materials Tris (hydroxymethyl) amino methane (Tris), monosodium and disodium phosphates, sodium chloride were purchased from Merck (Darmstadt, Germany). Hydrogen peroxide was from Prolabo (Paris, France). Iron chloride (FeC13) and iron sulfate (FeSO 4) and other salts were analytical reagent grade. Chelex resin (50-100 mesh, sodium form) was from Sigma (Saint Louis, MO, USA). Bidistilled water was obtained from Biosedra (Malakoff, France) and was used for all purposes. The DNA used was the plasmid pBR322 purchased from Boehringer (Mannheim, Germany). It was ethanol precipitated once, centrifuged, decanted and dried overnight in order to lower as much as possible the concentration of ethanol in the final DNA solution. In our conditions, ethanol concentration was below 0.001% as measured by gas-phase chromatography in the laboratory of Dr. Goetz (L'Or6al, Aulnay-sous-Bois). DNA was resuspended in 40 mM NaCI and stored at 4°C. The concentration of DNA was estimated spectrophotometrically. DNA preparations typically contained about 80-85% double-stranded covalently closed circular (ccc) supercoiled molecules, 15-20% open relaxed circular molecules and no linear molecules.

Detection of DNA nicking DNA single-strand breaks were assayed by measuring the conversion of double-stranded ccc supercoiled DNA (form I) to double-stranded relaxed circular DNA (form II). The number of nicks introduced per molecule (r) can be calculated from the fraction of DNA molecules remaining supercoiled (Po) assuming a Poisson distribution of the nicks among the molecules: Po(r) = e -r.

The experiments were performed in 1.5 ml Eppendorf polypropylene tubes. In 20/~1, 0.2 /zg plasmid pBR322 (3.5 nM or 30.3 /zM in nucleotide concentration) was incubated in 150 mM NaC1 (pH = 7) except when the influence of ionic strength on cleavage activity was investigated. The

49

pH of the solutions was adjusted to 7 using NaOH 0.1 N. In order to investigate the effect of pH, HCI 0.1 N and NaOH 0.1 N were respectively used to lower the pH value to 4 or to increase it to 10. In order to learn about contaminating iron ions which could change the profile of the doseresponse curve, a set of experiments was done using stock DNA and buffer solutions treated with chelex resin and centrifuged at 10,000 rpm for 15 min to remove the resin before use. The pH of such a solution was adjusted to 7 using chelex treated 1 N HCI. To assess the effect of phosphate and Tris buffers, phosphate buffer at pH --- 7.4 or Tris-HC1 buffer at pH = 7.4 was added (final concentration: 10 mM). Hydrogen peroxide was added (final concentration: 0.3 /zM to 30 mM) and the reaction was started by the addition of the ferrous or ferric salt (final concentration: 2 /~M). The final volume was 20 ~1 and the incubation mixture was kept for 15 min at 37°C except when otherwise stated. The reaction was stopped by the addition of 10 /zl of electrophoresis sample buffer (4 M urea, 50% sucrose, 50 mM EDTA and 0.1% bromophenol blue). The electrophoresis and the quantification of the bands were per-

formed as described previously (Tachon, 1989; Boullard and Giacomoni, 1988). Experiments were done at least twice on different days. The figures in this paper show typical examples of the results obtained in the course of this investigation. Results The incubation of supercoiled ccc DNA (form I) in 150 mM NaC1, pH = 7, with 2 /~M ferrous iron and increasing H 2 0 2 concentrations for 15 min at 37°C results in the formation of singlestrand breaks as shown by neutral agarose gel electrophoresis (Fig. 1A). In the control lane (lane 1) 85% of DNA molecules are supercoiled. The addition of 2 ~M ferrous iron alone promotes a conversion of form I DNA to relaxed nicked DNA (form II) (lane 2). The quantitative analysis of the different bands shows that the addition of ferrous iron (2 ~M) produces about 0.2 nick per plasmid molecule, while the addition of 30 /xM H20 2 and 2 ~M ferrous iron introduces about 2 nicks per plasmid. Fig. 1A,B shows that the production of nicked DNA as a function of increasing H 2 0 2 concen-

A 1

3

5

7

9

11

13

FORM

II III

B =I

a

r, IN

H202 CraM) Fe II [2 j~M)

i 0

0

I

10. 3

I

I0_ 2

I0_ I

I

I

10 0

101

z

0 I

13

10 ,4.

+

+

+

4"

4"

4"

+

4"

+

4-

|

10.2

I1

10

f

10 °

I 1

10

H202 Concentration (mM)

Fig. 1. pBR322 DNA (0.2/.~g) was incubated in 150 mM NaC1 (pH = 7.0) for 15 min at 37°C. (A) Neutral agarose gel showing the production of single-strand breaks in pBR322 DNA induced by increasing H 2 0 2 concentrations in the presence of 2 tzM ferrous iron. Lane 1, no addition; lanes 2-13, 2/~M Fe(II); lanes 3-13, increasing concentrations of H 2 0 2 (0.3/~M, 1 ~M, 3 ~M, 10/~M, 30 ~M, 100 /~M, 300 /~M, 1 mM, 3 mM, 10 mM, 30 mM). (B) Dose effect of H 2 0 2 on the formation of nicks generated per plasmid molecule in the absence ((3) or the presence of either 2 ~ M Fe(III) ( • ) or 2 ~ M Fe(II) (o).

50

tration in the presence of ferrous iron is peculiar. The amount of single-strand breaks per plasmid increases with H202 concentration up to 10-30 #M. Above 30/zM, an increase of H 2 0 2 concentration yields a decrease of the number of nicks per plasmid molecule. At 30 mM H 2 0 2, the amount of DNA remaining supercoiled is virtually identical to that obtained in the presence of ferrous iron alone. In the absence of metal added or in the presence of ferric iron (2/zM), H 2 0 2 in this range of concentrations and for the same reaction time does not produce detectable DNA single-strand breakage (Fig. 1B).

Effects of chelex treated solutions and buffers on cleavage activity of 1-120e plus ferrous iron In a set of experiments, salt solutions, DNA and H202 solutions were treated with chelex resin prior to incubating DNA in the same experimental conditions as above (ionic strength 150 mM, pH = 7, temperature 37°C, incubation time 15 min) in the presence of ferrous iron (2 /zM) and increasing H202 concentrations. Since in our hands the treatment of solutions with chelex gave unreproducible bimodal dose-response curves, we have decided not to use chelex treated solutions, and to perform the control experiment with H 2 0 z and no exogenous iron. The buffering components such as phosphate buffer or Tris buffer at a final concentration of 10 mM greatly reduce the formation of nicks induced by H202 plus ferrous iron (Fig. 2).

Effect of ionic strength The influence of the ionic strength on the ferrous iron mediated HzO e induced DNA cleavage has been studied using NaCI in a concentration range from 50 mM to 500 mM. Under the same experimental conditions as above (pH = 7, temperature--37°C, incubation time = 15 min), decreasing the ionic strength to 50 mM increases the rate of the cleavage activity of H 2 0 2 plus ferrous iron. At 50 mM NaC1 and at H 2 0 z concentrations from 3 /~M to 300 /.~M, the supercoiled DNA was entirely converted to the relaxed nicked form (form II) and double-stranded linear form (form III) (Fig. 3B). By contrast, increasing the ionic strength to 500 mM reduces the amount

i

Z

2

0 I

I'

I

l

I

10 .3

10.2

10 "1

10 °

101

1-1202 Concentration (mM) Fig. 2. Effects of buffer components on the nicking of supercoiled pBR322 DNA induced by increasing H20 2 concentrations and 2 /~M Fe(II). DNA (0.2 /~g) was incubated for 15 min at 37°C in: 150 mM NaCI (pH = 7) (e); 150 mM NaCI containing I0 mM phosphate buffer (pH = 7.4) (z~); 150 mM NaCI containing I0 mM Tris buffer (pH = 7.4) ([3).

of single-strand breaks produced in 15 min (Fig. 3).

Effect of temperature The effect of the temperature on the ferrous iron mediated HzO 2 induced DNA nicking has been investigated in a temperature range from 4°C to 56°C. Fig. 4 shows that temperature has no effect on the pattern of the dose-response curve but changes strikingly the H 2 0 2 concentration required for producing the maximum amount of single-strand breaks.

Effect of pH In order to characterize the effect of pH on the biphasic profile of the dose-response curve, experiments were performed in 150 mM NaC1 at 37°C for 15 min in the presence of ferrous iron (2 /xM) and increasing concentrations of H 2 0 z. The pH values were adjusted as mentioned in Materials and methods. At pH = 4, the profile of the

51 3 u

-

A

B

10

0

/-./ \ . l?o

0 ¢

I

I

10 .3

10 "2

I

10 "1

I

I

10 °

101

1:1202 Concentration (mM)

I

I

o I

10 "3

10 "a

o I

10 "1

I

I

10 °

101

H202 Concentration (mM)

Fig. 3. Effect of the ionic strength on the nicking of supercoiled pBR322 DNA induced by increasing H202 concentrations and 2 ~ M Fe(II). DNA (0.2 tzg) was incubated for 15 min at 37°C in: 50 mM NaCI (pH = 7) ( - ) ; 150 mM NaCl (pH = 7) (e); 500 mM NaCl (pH = 7) (©). (A) The number of nicks generated per plasmid molecule was plotted against H 2 0 2 concentration. (B) The percent of linear duplex DNA (form III) was plotted against H 202 concentration.

dose-response curve is the same as obtained at p H = 7. However, the cleavage activity of ferrous iron and low H 2 0 2 concentration is more important at pH = 4 than at pH = 7 as shown in Fig. 5B. At pH 10, the pattern of the dose-response curve is totally different. No single-strand breaks are observed up to 1 mM H 2 0 2 . At 3 mM H 2 0 2 , almost all supercoiled DNA is converted to relaxed nicked DNA (form II). At 10 mM and 30 mM H 2 0 2 , low amounts of form II and form IIl are observed, and one can see a smear indicating extensive DNA degradation (Fig. 5A, lanes 12 and 13).

Discussion Recently, Imlay and Linn (1988) have reported that D N A damage (as measured by phage inactivation and D N A single-strand breaks) induced by hydrogen peroxide through the Fenton reaction has a characteristic pattern. They showed that the maximum extent of D N A nicking, in the presence of ferrous iron, occurs at low H 2 0 2 c o n c e n t r a t i o n s and higher H 2 0 2 concentrations suppress

about half of the nicking. Furthermore, such a dose-response curve was also observed for the generation of hydroxyl radical monitored using the bleaching of p-nitrodimethylaniline. Using the conversion of the ccc doublestranded supercoiled D N A (pBR322) to a relaxed circle, we reproduced the same profile of the dose-response curve. We also observed the formation of linear duplex DNA. Although its accumulation could be indicative of double-strand breaks in form I, it could also derive from breaks opposite to nicks on the other strand in form II molecules. However, it has been calculated that it would take more than 100 random single-strand breaks per plasmid molecule to produce one double-stranded break (Kohen et al., 1986), and we observed form III D N A when there were~ about 1-2 nicks per plasmid (for example, see Fig. 3B). According to the redox potentials of the couples F e ( I I I ) / F e ( I I ) and H 2 0 2 / H O ' + H O - , which are respectively 0.11 V and 0.33 V at p H = 7 (Miller et al., 1990), the Fenton reaction may be thermodynamically favored. However, under physiological conditions, the autoxidation of ferrous iron to ferric iron (reaction 6) can occur,

52

of its concentration (Cher and Davidson, 1955; Taube, 1965). _

rate = k[Fe(II)]pO 2 [phosphate] 2

~

2-

/

o

Z 0 I

I

I

I

I I

t o "3

1o -2

10

lo

H 2 0 2 Concentration (mM) Fig. 4. Effect of the temperature on the nicking of supercoiled pBR 322 DNA induced by increasing H202 concentrations and 2/~M Fe(lI). DNA (0.2 ~g) was incubated 15 rain in 150 mM NaC1 (pH = 7) at 4° (O) or 56°C (e).

leading to reaction 6 in competition with the Fenton reaction. Fe(II) + 0 2 -~ Fe(III) + 0 2

Tris buffer also influences the biological damage mediated by the Fenton reaction. Indeed, Tris is able to chelate iron (Miller et al., 1990) allowing the Fenton reaction to occur close to it. Moreover, it was shown that Tris reacts with H O with a rate constant of 1.1 × 109 M -1 s -1 (Hicks and Gebicki, 1986) generating a carbon-centered radical. The latter species can be converted to CO 2 via formaldehyde intermediate (Schiicker et al., 1991). All these Tris properties lower the flux of H O or related species able to reach the biological target such as DNA. The suppressive effect of phosphate and Tris buffer on DNA nicking at low H 2 0 2 concentrations (10-30 ~M) in the presence of ferrous salts is in agreement with the properties mentioned above. In the absence of buffer, the rate of Fe(II) autoxidation in NaC1 salt is low (Miller et al., 1990), but could occur in the presence of DNA at low ionic strength. As a matter of fact, at low ionic strength, ferrous iron may be chelated to the phosphodiester backbone leading to an autoxidation of ferrous to ferric iron and to the production of reactive oxygen species in the vicinity of DNA following the sequence of reactions: DNA-Fe(II) + 0 2 ~ DNA-Fe(III) + 0~-

(6) 0 2 + 0 2

The redox potentials of the different couples (Fe(III)/Fe(II), H 2 0 2 / H O ' + H O - , 0 2 / 0 2) vary greatly with pH, ionic strength, temperature and some buffer components (Reed, 1982). So we decided to investigate these factors in DNA breakage mediated by the Fenton reaction. Phosphate and Tris buffers are mostly used in biochemical experiments, particularly with isolated DNA. The ability of phosphate buffer to catalyze rapid Fe(II) oxidation is well known (Cher and Davidson, 1955). The rate for the autoxidation of ferrous iron in the presence of inorganic phosphate is dependent on the square

2H +

' H202 + O 2

(7) (3)

DNA-Fe(II) + H 2 0 2 DNA-Fe(III)... H O + O H -

(8)

Thus, when H 2 0 2 w a s added to the reaction mixture, at low ionic strength (< 50 mM NaCl), the autoxidation of ferrous iron competes with the oxidation of ferrous iron by exogenous hydrogen peroxide (Fenton reaction). In order to learn about DNA single-strand breakage induced by the Fenton reaction and to reduce the effect of the ferrous autoxidation process, hydrogen perox-

53 A

1

3

5

7

9

11

13

FORM

!

II III

H202

/

0 0

I

I

10-3 10-2 10-1

I

B

I

100

101

(mM) Fe II (2qM)

4"

+

4"

4"

+

+

4"

4,

÷

+

÷

I

+

I

I

I

I

1 0

10-3 10.2 10"~ 10

1

10

H202 Concentration (mM)

Fig. 5. Effect of pH on the nicking of supercoiled pBR322 DNA induced by increasing H202 concentrations and 2 ~M Fe(ll). DNA was incubated for 15 min in 150 mM NaCI at 37°C. (A) Neutral agarose gel showing the production of breaks in pBR322 DNA induced by increasing H202 concentrations and 2/xM Fe(II) at pH = 10. Lane 1, no addition; lanes 2-13, 2 ~M Fe(ll); lanes 3-13, increasing concentration of H202 (0.3 ~M, 1 /~M, 3/zM, 10/zM, 30 ~M, 100 ~zM,300/xM, 1 mM, 3 mM, 10 mM, 30 raM). (B) DNA was incubated at pH = 4 ([]), pH = 7 (o) and pH = 10 ( zx).

ide is added first and the ferrous salt is added last. However, at 150 mM NaCI, the reversion of the sequence of addition of reagents does not change the profile of the dose-response curve (data not shown), suggesting that in our experimental conditions, at physiological ionic strength (NaCI 150 mM), ferrous iron reacts principally with H 2 0 2 in the bulk solution. The effect of temperature is important. According to the redox potential determination by the Nernst equation, this effect of temperature is expected. On the other hand, one cannot eliminate the possibility that the temperature, by changing the twist of the double strand, modifies the secondary or tertiary structure of pBR322, facilitating the D N A single-strand breakage mediated by the Fenton reaction. As a matter of fact, increasing temperature by 30°C from 20°C to 50°C will modify the twist of pBR 322DNA by about 4, as can be calculated assuming that p B R 322 D N A contains 4000 base pairs and that the angle between base pairs changes by 0.01 degree per degree Celsius (Depew and Wang, 1975). High pH totally changes the profile of the dose-response curve. Indeed, at pH = 10, no

D N A nicking occurs at low H 202 concentrations. This effect could be due to the important shift of the aqueous iron ( I I I / I I ) redox potential, because of its precipitation as hydroxide. For example, the aqueous iron ( I I I / I I ) redox potentials are 0.77 V and 0.11 V respectively at pH = 1 and pH = 7. In conclusion, whatever the reactive species generated via the Fenton reaction, D N A breakage is maximum at low H 2 0 2 concentrations (1030/zM). The smaller effect of higher H 2 0 2 concentration could be due to its property to scavenge hydroxyl radical or related species as suggested by Imlay and Linn (1988). So it appeared that the pattern of the peculiar bimodal dose-response curve greatly depends on the experimental conditions. References

Adelman, R., R.L. Saul and B.N. Ames (1988) Oxidative damage to DNA: Relation to species metabolic rate and life span, Proc. Natl. Acad. Sci. USA, 85, 2706-2708. Ames, B.N., R.L Saul, E. Schwiers, R. Adelman and R. Cathcart (1985) Oxidative DNA damage as related to cancer and aging: the assay of thymine glycol, thymidine glycol, and hydroxymethyluracil in human and rat urine,

54 in: R.A. Sohal, L.S. Birnbaum and R.G. Cutler (Eds.), Molecular Biology of Aging: Gene Stability and Gene Expression, Raven Press, New York, NY, pp. 137-144. Boullard, A., and P.U. Giacomoni (1988) Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA, J. Photochem. Photobiol. Biol., 2, 491-501. Cher, M., and N. Davidson (1955) The kinetics of the oxygenation of ferrous iron in phosphoric acid solution, J. Am. Chem. Soc., 77, 793-798. Depew, R.E., and J.C. Wang (1975) Conformational fluctuations of DNA helix, Proc. Natl. Acad. Sci. USA, 72, 4275-4279. Harman, D. (1981) The aging process, Proc. Natl. Acad. Sci. USA, 78, 7124-7128. Hicks, M., and J.M. Gebicki (1986) Rate constants for reaction of hydroxyl radicals with Tris, Tricine and Hepes buffer, FEBS Lett., 199, 92-94. Imlay, J.A., and S. Linn (1988) DNA damage and oxygen radical toxicity, Science, 240, 1302-1309. Imlay, J.A., S.M. Chin and S. Linn (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro, Science,-240, 640-642.

Kohen, R., M. Szyf and M. Chevion (1986) Quantitation of single- and double-strand DNA breaks in vitro and in vivo, Anal. Biochem., 154, 485-491. Miller, D.M., G.R. Buettner and S.D. Aust (1990) Transition metals as catalysts of 'autoxidation' reactions, Free Radical Biol. Med., 8, 95-108. Reed, C.A. (1982) Oxidation states, redox potentials, and spin states, in: H.B. Dunford, D. Dolphin, K.M. Raymond and L. Sieker (Eds.), The Biological Chemistry of Iron, D. Riedel Publishing Co., pp. 25-42. Sch~icker, M., H. Foth, J. Schliiter and R. Kahl (1991) Oxidation of Tris to one-carbon compounds in a radical-producing model system, in microsomes, in hepatocytes and in rats, Free Radical Res. Commun., 6, 339-347. Tachon, P. (1989) Ferric and cupric ions requirement for DNA single-strand breakage by H202, Free Radical Res. Commun., 7, 1-10. Taube, H. (1965) Mechanism of oxidation with oxygen, J. Gen. Physiol., 49, 29-35. Weinstein, J., and B.H.J. Bielski (1979) Kinetics of the interaction of perhydroxyl and superoxide radicals with hydrogen peroxide. The Haber-Weiss reaction, J. Am. Chem. Soc., 101, 58-62.