Vol. 173, No. 8JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2562-25680021-9193/91/082562-07$02.00/0

Effects of Metal Ion Chelators on DNA Strand Breaks andInactivation Produced by Hydrogen Peroxide in Escherichia coli:

Detection of Iron-Independent LesionsNASSER R. ASAD AND ALVARO C. LEITAO*

Departamento de Radiobiologia, Instituto de Biofisica Carlos, Chagas Filho,Universidade Federal de Rio de Janeiro, 21949 Rio de Janeiro, RJ, Brazil

Received 11 September 1990/Accepted 2 February 1991

In order to study the role of metallic ions in the H202 inactivation of Escherichia coli cells, H202-sensitivemutants were treated with metal ion chelators and then submitted to H202 treatment. o-Phenanthroline,dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H202 treatmentwas not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coil. On theother hand, prior treatment with iron chelators protected the cells against the H202 lethal effect, indicating thatiron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNAdegradation studies indicated that these chelators did not completely block the formation of DNA single-strandbreaks by H202 treatment. Thiourea, a scavenger ofOH, caused a reduction in both H202 sensitivity and DNAsingle-strand break production. The breaks observed after treatment with metal chelators and H202 wererepaired 60 min after H202 elimination in xthA but not poLA mutant cells. Therefore, we propose that there areat least two pathways for H202-induced DNA lesions: one produced by H202 through iron oxidation and OHproduction, in which lesions are repaired by the products of the xthA and pol4 genes, and the other producedby an iron-independent pathway in which DNA repair requires poUM gene products but not those of the xthAgene.

The consecutive univalent reduction of molecular oxygento water produces three active intermediates: superoxideanion (02 ), hydrogen peroxide (H202), and hydroxyl rad-ical (OH'). These so-called oxidative oxygen species arepotent oxidants of lipids, proteins, and nucleic acids (14, 32,33).

Participation ofOH as the main damaging agent has beensuggested by studies using scavengers of OH'. Brakely et al.(3) have demonstrated that in vitro, dimethyl sulfoxidepartially inhibits DNA base damaging by H202. Oya et al.(35) have shown that the generation of chromosome aberra-tions by H202 treatment is efficiently suppressed by severalOH scavengers, such as ethanol, dimethyl sulfoxide, andmannitol.

Brandi et al. (4) have noted that Escherichia coli bacteriaare partially protected against the lethal effects of H202 bypretreatment with ethanol, dimethyl sulfoxide, or thiourea.The participation of transition metals, such as Cu(I) and

Fe(II), acting as reducing agents in the formation of OHthrough the Fenton reaction and the Harber-Weiss cycle hasbeen suggested previously (27, 32, 36, 40, 41):

O2- + Me"'-+02+ Me(n 1) (1)H202 + Me(n - 1) OH' + OH- + Men+ (2)

02- + H202-OH +OH-+ 02In fact, different metal chelators that can occupy metal

coordination sites exhibit protecting effects against H202lesions. For example, o-phenanthroline prevents the appear-ance of sister chromatid exchanges, DNA single-strandbreaks, and cell killing in a variety of mammalian cellsexposed to H202 (22, 32), and desferrioxamine inhibits lipid

* Corresponding author.

peroxidation (11). Imlay et al. (17) have shown that E. colibacteria are protected against the lethal effects ofH202 whenthe cells are pretreated with either o-phenanthroline ordesferrioxamine, confirming the role of iron in the generationof active oxygen species.

In E. coli, several studies indicate that the killing of cellsexposed to H202 is mainly due to damage on DNA (13, 18,23). Active species damage either sugar residues or bases,giving rise to different lesions, such as base loss and single-strand breaks (39).The H202-induced DNA lesions are efficiently repaired in

wild-type strains of E. coli. However, mutant strains lackingexonuclease III (product of the xthA gene) are hypersensi-tive to H202 (8, 9). This enzyme composes about 90% of thebacterial apurinic/apyrimidinic (AP) endonuclease activitywhich is necessary for the repair of H202 DNA lesions (9,21). Additionally, polA mutants (lacking DNA polymerase I)are also hypersensitive to H202, suggesting that the productof this gene is involved in the repair of H202-induced DNAlesions (12, 19).

In this work we analyzed the effect of metal ion chelatorson the production of lesions in H202-treated bacteria. Ourresults indicate that besides OH lesion production, H202generates at least one other kind of lesion which is efficientlyrepaired in polA+ bacteria.

MATERIALS AND METHODS

Bacterial strains. The E. coli K-12 strains used in this workare described in Table 1.Growth and radioactive labeling. Cells were grown over-

night in a shaking incubator at 37°C in M9 minimal medium(2) containing glucose (4 g/liter) supplemented with 2.5 mg ofCasamino Acids per ml and 1 ,xg of thiamine per ml for theAB1157 and BW9091 strains or 5 ,ug of niacin per ml and 20

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TABLE 1. E. coli K-12 strains

Designationa Repair genotype Other genotypes

AB1157 Wild type thr-J leuB6 thi-l argE3 his4proA2 lacYl galK2 mtl-l xyl-5ara-14 rspL31 tsx-33, supE44

BW9091 xthA Same as AB1157P3478 polA nia thy

a The AB strain was obtained from P. Howard-Flanders (University ofYale, New Haven, Conn.). The P3478 strain was obtained from R. Ley(Lovelace Medical Foundation, Albuquerque, N.Mex.), and the BW9091 wasobtained from A. G. Miguel (Biophysics Institute, UFRJ, Rio de Janeiro,Brazil).

,ug of thymine per ml for the P3478 strain; the supplementedmedia are designated M9S herein. A starting inoculum wastaken from these cultures, and the cells were grown in thesame medium until mid-exponential phase (1 x 108 2 x 108cells per ml). Radioactive cultures were prepared similarly,except that the growth medium was supplemented with 10,iCi of [methyl-3H]thymidine (New England Nuclear, Bos-ton, Mass.; 6.7 Ci/mmol) per ml and 200 ,ug of 2-deoxiade-nosine per ml for the AB1157 and BW9091 strains. TheP3478 strain was labeled by substituting thymine for 10 ,uCiof [methyl-3H]thymidine per ml in the growth medium.

Survival experiments. Cells in the mid-exponential phaseof growth were treated with 5 mM H202 (Perhidrol, 30%;Merck, Rio de Janeiro, Brazil) in M9S medium at 37°C undershaking. Samples (0.1 ml) were collected at different times ofincubation, appropriately diluted in M9 salts solution, andspread (in duplicate) on BT (rich) medium (29) solidified with1.5% agar. The colonies formed were counted after over-night incubation at 37°C.Treatment with metal ion chelators. Cells in the mid-

exponential phase of growth were treated for 20 min withmetal ion chelators, and then the survival experiments wereconducted as described above.Treatment with thiourea. Cells in the mid-exponential

phase of growth were treated concomitantly with H202 andthiourea. Survival experiments were conducted as describedabove.DNA sedimentation studies. The induction of DNA single-

strand breaks was followed by sedimentation through alka-line sucrose gradients by the procedure of MacGrath andWilliams (28) with slight modifications. Radioactive culturesin the mid-exponential phase of growth were submitted tosurvival experiments; 50-,ul samples were layered on top of0.2 ml of lysing solution (0.5 M NaOH, 0.01 M EDTA, and0.05% sodium dodecyl sulfate) on the top of a 4.4-ml gradientof 5 to 20% (wt/vol) sucrose in 0.4 M NaCl, 0.2 M NaOH,and 0.01 M EDTA. The tubes were maintained for 30 min atroom temperature and then centrifuged in an SW 50.1 rotorfor 120 min at 25,000 rpm and 20°C. After centrifugation, 30fractions were collected on paper strips (Whatman 17) pre-soaked with 5% trichloroacetic acid by using a peristalticpump. The paper strips were washed once in ice-coldtrichloroacetic acid, twice in 95% ethanol, and once inacetone. After drying, the radioactive content of each frac-tion was determined in a Beckman liquid scintillationcounter. The average molecular weights were calculatedaccording to the method described by Ley (25), and thenumber ofDNA single-strand breaks and alkali-labile bonds(DNA-SSB) per E. coli genome (2.5 x 109 Da) was calcu-lated as described by Ananthaswamy and Eisenstark (1).

In some experiments, after the H202 treatment, an excess

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H2 02 treatment (min)

FIG. 1. Survival of E. coli BW9091 (xthA) mutant cells treatedwith metal ion chelators and H202. Cultures in the mid-exponentialphase of growth were treated in M9S medium at 37°C with metal ionchelators for 20 min and then submitted to H202 (5 mM) treatmentfor different periods of time. 0, No addition; A, 1 mM dipyridyl; A,100 ,uM o-phenanthroline; O, 100 ,uM desferrioxamine; E, 100 ,uMneocuproine.

(5 ,ug/ml) of catalase (EC 1.11.1.6; Sigma Chemical Co., St.Louis, Mo.) was added. Then the cells were centrifuged,resuspended in M9S medium, and submitted to 60 min ofincubation in order to analyze the DNA-SSB repair.DNA degradation. The disappearance of trichloroacetic

acid-insoluble material was monitored by collecting 20-,usamples from bacterial radioactive suspensions at differentintervals and placing them on paper strips (Whatman 17).The radioactive content of each sample was determined asdescribed for the sedimentation studies.

RESULTS

Survival of H202-treated xthA mutant bacteria. The ironchelators used herein (o-phenanthroline, dipyridyl, and des-ferrioxamine) inhibited the H202 lethal effect (Fig. 1). Thisprotection was also observed in the wild-type (AB1157)bacteria treated with o-phenanthroline (data not shown). Incontrast, neocuproine, a copper chelator, did not inhibit thelethal effect of H202 (Fig. 1), even if concentrations higherthan 100 ,uM were used (data not shown). The metal ionchelators, per se, did not cause any cellular inactivation.

In order to confirm the production of OH by H202treatment, the cells were treated with thiourea (scavenger ofOH') and H202. Thiourea partially protected the cells,indicating that OH participates in the inactivation producedby H202 (Fig. 2).DNA strand break production by H202 treatment. Several

authors have shown that H202 treatment produces DNA-SSB in vitro (20, 24, 37, 42, 44) and in vivo (1, 12, 15, 23).Supposing that such breaks are produced by OH and thatmetal ion chelators block the production of such radicals, wecan expect that prior treatment with metal ion chelatorswould protect the cells against the DNA breaks produced byH202 treatment.

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100 10 20

H2 02 treatment (min)FIG. 2. Survival of E. coli BW9091 (xthA) mutant cells treated

with thiourea and H202. Cultures in the mid-exponential phase ofgrowth were treated in M9S medium at 37°C with thiourea (100 mM)and H202 (5 mM) for different periods of time or previously treatedwith o-phenanthroline (100 ,uM) for 20 min and then submitted tothiourea and H202 treatment for different periods of time. O, H202;O, thiourea; *0, thiourea plus H202; A, o-phenanthroline; A,o-phenanthroline plus thiourea plus H202.

Analysis of the DNA sedimentation profiles in alkalinesucrose gradients (Fig. 3) showed that prior treatment withmetal ion chelators did not affect the production of DNA-SSB in H202-treated xthA mutant bacteria. Moreover, DNAdegradation in these cells was as extensive as in cells treatedonly with H202 (about 18%). Similar results were obtained inwild-type cells pretreated with o-phenanthroline (data notshown). Metal ion chelators, per se, did not produce anyDNA-SSB or DNA degradation (data not shown).On the other hand, treatment with thiourea partially

protected the cells against the production of DNA-SSB byH202 (Fig. 4). However, in cells previously treated witho-phenanthroline, the number of DNA-SSB produced byH202 in the presence of thiourea was higher (107 DNA-SSB)than that observed without prior treatment with the metalchelator (61 DNA-SSB) (Fig. 4).

Repair of DNA-SSB by the xthA mutant bacteria. Sinceo-phenanthroline blocks the production of OH' and protectscells against the lethal effects of H202 treatment, the aboveresults suggest that in cells previously treated witho-phenanthroline the DNA-SSB observed may not be pro-duced by OH'. Once the xthA mutant is defective in repair-ing lesions produced by OH', one might expect that theDNA-SSB produced in the presence of both H202 and ironchelators should be repaired in these cells.

In order to measure the repair of DNA-SSB, the sizedistribution of DNA was analyzed in alkaline sucrose gradi-ents after treatment with H202 for 20 min and furtherincubation in the absence of H202 (excess of catalase).Reincubation in the absence of H202 resulted in a shift of theDNA sedimentation profile towards that of untreated cells(Fig. 5). After 60 min this profile was undistinguishable fromthat of control cells, indicating that DNA repair was almost

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FIG. 3. Alkaline sucrose gradient profiles of DNA from E. coliBW9091 (xthA) mutant cells after treatment with metal ion chela-tors. Cells in the exponential phase of growth were treated in M9Smedium at 37°C for 20 min with metal ion chelators and thensubmitted to H202 (5 mM) treatment for 20 min. 0, Untreatedcontrol; 0, control (20 min, H202 [230 DNA-SSB]); A, 1 mMdipyridyl (195 DNA-SSB); A, 100 ,M o-phenanthroline (186 DNA-SSB); l, 100 ,uM desferrioxamine (165 DNA-SSB); *, 100 ,Mneocuproine (218 DNA-SSB). Arrows indicate the direction ofsedimentation.

complete (seven DNA-SSB remaining), according to oursurvival results.When the cells were not previously treated with o-phenan-

throline, the same shift in the DNA sedimentation profiletowards that of untreated controls was observed. However,in this case, the repair was not complete, since 16 DNA-SSBper genome remained unrepaired after 60 min (data notshown), explaining the results found in the survival experi-ments. These results suggest that in the presence of iron,H202 produces at least two types of lesions: one thatrequires the product of xthA gene to be repaired (OHlesions) and other(s) that is repaired by different pathways.

Studies with the polA mutant cells. Several works haveshown that polA mutant cells are hypersensitive to H202treatment. Indeed, these mutant cells are more sensitive toH202 than the xthA mutant (12, 19, 23).DNA-SSB produced by H202 in the presence of o-phenan-

throline were repaired in the xthA mutants; however, suchlesions might not be repaired in the hypersensitive polA

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,, 60 40

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Fraction n°FIG. 4. Alkaline sucrose gradient profiles of DNA from E. coli

BW9091 (xthA) mutant cells after treatment with thiourea. Cells inthe exponential phase of growth were treated in M9S medium at37°C for 20 min in the presence of thiourea (100 mM) and H202 (5mM) or previously treated with o-phenanthroline (100 ,uM) for 20min and then submitted to thiourea and H202 treatment for 20 min.0, Untreated control; 0, control (H202 [285 DNA-SSB]); *,thiourea plus H202 (61 DNA-SSB); A, o-phenanthroline plus H202(234 DNA-SSB); O, o-phenanthroline plus thiourea plus H202 (107DNA-SSB). Arrows indicate the direction of sedimentation.

mutant cells, which should explain the extreme sensitivity ofsuch mutants to H202 treatment.To test this hypothesis, experiments with the polA mutant

were performed in which the killing and the DNA strandbreaks produced by H202 in the presence of o-phenanthro-line were determined.The polA mutant bacteria were more sensitive to H202

treatment than xthA mutant bacteria, and prior treatmentwith o-phenanthroline did not completely protect these cellsagainst the lethal effect of H202 (Fig. 6).The DNA-SSB produced by H202 in the presence of

o-phenanthroline were less repaired in polA mutant bacteriathan in the xthA mutant bacteria, as 30 breaks per genomeremained after 60 min postincubation (Fig. 7), which corre-lates with the survival observed.

DISCUSSION

Prior treatment with iron chelators protects E. coli cellsagainst the lethal effects of H202. These data indicate the

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FIG. 5. Alkaline sucrose gradient profiles of DNA from E. coliBW9091 (xthA) mutant cells treated with o-phenanthroline plusH202: kinetics of the repair. Cells in the exponential phase of growthwere treated in M9S medium at 37°C for 20 min with 100 ,uMo-phenanthroline and then exposed to H202 (5 mM) for an additional20 min. An excess of catalase (5 ,ug/ml) was then added. The cellswere centrifuged, resuspended in the M9S medium, and incubatedfor 10 min (U) (42 DNA-SSB), 30 min (A) (16 DNA-SSB), or 60 min(0) (7 DNA-SSB). 0, Untreated control; A, 20 min, o-phenanthro-line plus H202 (142 DNA-SSB). Arrows indicate the direction ofsedimentation.

participation of iron ions in the formation of OH by H202through the Fenton reaction in bacterial cells. The protectionobserved by prior treatment with o-phenanthroline anddipyridyl may be due to the capture of Fe(II) ions, resultingin the blockage of Fe-catalyzed H202 reduction to OH(equation 2). The protection by desferrioxamine could bedue to the capture of Fe(III) ions, blocking their reduction toFe(II) (equation 1).

Nevertheless, the copper chelator, neocuproine, did notprotect the cells against H202 lethality. Therefore, ourresults suggest that the copper effect on the OH productionin vivo in E. coli cells is not significant, which is consistentwith Mello-Filho's findings in mammalian cells (31).

Prior treatment with o-phenanthroline protected mamma-lian cells against the lethal effects of H202, and this protec-tion was correlated with the absence of DNA-SSB produc-tion (32). In E. coli we observed that prior treatment withiron chelators protects the cells against the lethal effects of

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0I-410)

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H2 0 2 treatment ( minFIG. 6. Effect of o-phenanthroline on the survival of E. coli

P3478 (polA) mutant cells treated with H202. Cells in the exponen-tial phase of growth were treated for 20 min at 37°C with 100 ,uMo-phenanthroline and then exposed to H202 for different periods oftime. 0, H202; A, o-phenanthroline plus H202.

H202; however, the number ofDNA-SSB produced by H202treatment was similar even in the presence or iron chelators.

Since under iron chelation conditions, DNA-SSB arerepaired in the xthA mutant bacteria, we suggest that in thiscase the H202 lesions do not constitute AP sites or breakscontaining 3'-phosphate ends or 3'-terminal sugar fragments,a kind of lesion that needs the prior action of exonuclease IIIfor its repair (10, 16, 34, 43). Alternatively, if these lesionsare formed, exonucleases other than exonuclease III areresponsible for their elimination, generating the 3'-OH endsrequired for the DNA polymerase I action. Another possi-bility is that H202 produces DNA breaks bearing 3'-OHends. In both cases, exonuclease III would not be necessaryfor the repair of these lesions, and this could explain thenormal repair observed in the xthA mutant cells. On theother hand, the inability to repair the DNA-SSB formed inpolA mutants is expected since DNA polymerase I would benecessary to perform the repair even in the presence of3'-OH termini.

Previous studies indicate the existence of different path-ways in the repair of H202-induced DNA lesions in E. coli(1, 5, 6, 15, 19, 26). Hagensee and Moses (13) suggested theexistence of two pathways in the repair of H202-inducedDNA damage, one using exonuclease III, DNA polymeraseIII, and DNA polymerase I while the other would be DNApolymerase I dependent, suggesting that these enzymescould be acting on different kinds of H202-induced DNAdamage.Our results suggest that there are at least two pathways in

the repair of H202-induced DNA lesions in E. coli: oneutilizing DNA polymerase I, acting on iron-independentlesions, and another utilizing exonuclease III and DNApolymerase I, acting on iron-dependent lesions. In the lattercase, DNA polymerase I is necessary for DNA polymeriza-tion after the action of the exonuclease III on the AP sites.Some iron-independent lesions are repaired even in the

polA mutant cells, as found in the survival and sedimentation

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FIG. 7. Alkaline sucrose gradient profiles of DNA from E. coliP3478 (polA) mutant cells treated with o-phenanthroline and H202:kinetics of repair. Cells in the exponential phase of growth weretreated in M9S medium at 37°C for 20 min with 100 ,uM o-phenan-throline and then exposed to H202 (5 mM) for an additional 10 min.After treatment, an excess of catalase (5 p,g/ml) was added, and thecells were centrifuged, resuspended in M9S medium, and reincu-bated for an additional 10 min (U) (76 DNA-SSB), 30 min (A) (38DNA-SSB), or 60 min (0) (30 DNA-SSB). 0, Untreated control; A,20 min with o-phenantroline plus 10 min with H202 (155 DNA-SSB).Arrows indicate the direction of sedimentation.

studies. This can be due to a residual activity of DNApolymerase I which is present in the polA mutant (7) or to theaction of DNA polymerase II and DNA polymerase III thatin some cases can substitute for DNA polymerase I (30, 38).Our results reinforce the suggestion of Hagensee and

Moses (13) in the sense that different repair pathways for theH202-induced DNA lesions may reflect different kinds oflesions produced.The fact that almost the same number of DNA-SSB is

formed in xthA cells by H202 even in the presence of ironchelators is difficult to explain. One possibility to consider isthat when iron is chelated, iron-independent lesions will beformed to a larger extent, once H202 is not consumed byFe(II) near the DNA.Moreover, the number of DNA-SSB observed in cells

previously treated with o-phenanthroline and submitted toboth H202 and thiourea treatment is also hard to explain. Inthis case, because of the absence of iron ions and possible

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EFFECT OF METAL CHELATORS AND H202 IN E. COLI

lack of OH, we could expect that the addition of thioureawould not modify the number of DNA-SSB formed. Onehypothesis to consider is that thiourea is active as a scaven-ger of some iron-independent radicals, that there is still OH-formation even in the presence of iron-chelators, once someiron remains in the cell, or that there is another mechanismby which OH is formed.

In summary, our results suggest that in E. coli, H202produces iron-dependent and iron-independent DNA lesionswhich are repaired by different processes.

ACKNOWLEDGMENTS

We are grateful to A. B. Silva and J. S. Cardoso for their experttechnical assistance, R. Meneghini, P. L. Moreau and D. P. Car-valho for help and discussion during the preparation of the manu-script, and R. Alcantara Gomes for determination of the numbers ofDNA-SSB.

'This work was supported by FINEP, CNPq, FAPERJ, andCEPG-UFRJ.

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