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The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide
Mutation Research, 190 (1987) 237-240 Elsevier
237
MTRL 0968
The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide G. Brandi 1, P. Sestili, M.A. Pedrini 2, L. Salvaggio 1, F. Cattabeni and O. Cantoni lstituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, lIstituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Universitit degli Studi di Urbino, Urbino, and 2Istituto di Genetica Biochimica ed Evoluzionistica, C.N.R., Pavia (Italy) (Accepted 4 November 1986)
Keywords: Temperature; Anoxia; (Escherichia coli); Hydrogen peroxide; Thiourea.
Summary The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure. Two modes of killing were detected following a 15-min challenge with H202 under either aerated or anoxic conditions. Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM. Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic ceils. Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H202. Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations. We suggest that hydroxyl radicals mediate mode two but not mode one killing by H202.
The cytotoxicity of H202 in prokaryotic cells is well documented (Repine et al., 1981; Carlsson and Carpenter, 1980; Demple et al., 1983; Demple and Halbrook, 1983; Christman et al., 1985; Imlay and Linn, 1986). Although the critical lesion produced by the oxidant has not been as yet identified, it is known that iron facilitates the bactericidal action by reacting with H202 to form a highly toxic radical species, the hydroxyl radical (OH') (Repine Correspondence: Dr. Orazio Cantoni, lstituto di Farmacologia e Farmacognosia, Universit/t degli Studi di Urbino, Via S. Chiara, 27, 61029, Urbino (Italy).
et al., 1981). Production of OH" occurs via the Fenton reaction: F e 2+ +
H202
~ Fe 3+ + OH" + O H -
Over the last few years various studies on E. coli have indicated that mutations in recA (Carlsson and Carpenter, 1980; Imlay and Linn, 1986), polA (Ananthaswamy and Eisenstark, 1977; Imlay and Linn, 1986) or xth (Demple et al., 1983; Imlay and Linn, 1986) strains confer hypersensitivity to H202, suggesting an important role for the DNArepair system in the protection of the cell against
0165-7992/87/$ 03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
238 the oxidative insult. Therefore, DNA seems to be the site of the H202-induced lethal damage. Recently, two modes of killing of E. coli by hydrogen peroxide were described (Imlay and Linn, 1986), the first occurring at concentrations of H202 below 3-5 mM and the second at concentrations higher than 10-20 raM. These modes of lethality differed in that unlike mode one, mode two killing did not require active metabolism and was not dramatically enhanced in strains with DNA-repair defects. In the present investigation we have attempted to characterize the effect of anoxia (during drug exposure) on cell death produced by H202. It is known that prechallenge growth in anoxic medium induces sensitivity to mode one killing by H202. We sought to determine whether anoxia or low oxygen levels had any effect on hydrogen peroxide lethality in cells whose growth had been achieved in aerated conditions. Our data demonstrate that aerobically grown cells display a higher vulnerability to H202-induced mode two killing when drug exposure is performed under aerated rather than anoxic conditions. On the contrary, 02 tension was not relevant as far as mode one killing by H202 is concerned. Furthermore, thiourea, a hydroxyl radical scavenger, was able to prevent mode two but not mode one lethality. We have also investigated the effect of H202 challenge at ice temperature on the survival of E. coli cells and found that at 4°C cells displayed an increased resistance to mode two killing, whereas mode one killing was not even detectable.
Materials and methods Materials. H202 was purchased as a 30070 solution from J.T. Baker Chemicals B.V. (Deventer, The Netherlands); other chemicals and most reagent-grade biochemicals were from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and from Flow Labs (McLean, VA, U.S.A.). Bacterial strain and growth. The E. coli strain used, AB1157, was routinely grown in our laboratory at 37°C. Cells were initially grown
overnight (16-18 h) at 37°C in K medium (l°70 glucose, l°70 casamino acids, 1 /~g/ml thiamine hydrochloride, 25 #g/ml MgSO4"7H20 and 2 #g/ml CaCI2 in M9 salts). Samples were diluted 50-fold in fresh K medium and grown to about l0 s cells/ml under aerobic conditions. Aerated growth was achieved by incubation of 50 ml of K medium containing 1-5 × 107 cells/ml in a 500-ml Erlenmeyer flask with 200 rpm of shaking. Survival curves. Cells grown to about 108 cells/ml were harvested by centrifugation at room temperature, washed once with M9 salts and resuspended at 2-5 × 107 cells/ml (aerated conditions) or 7-9 × 107 cells/ml (anoxic conditions) in prewarmed M9 salts. Treatments with H202 (for 15 min at 37°C) were performed in 3 ml of cell suspension in either a 10-ml test tube with 150 rpm of shaking (anoxic conditions) or a 20-ml scintillation-counting vial with 200 rpm of shaking (aerated conditions). In other experiments cells were diluted in prechilled M9 salts (5-8 × 107 cells/ml) and treatments were for 15 min in scintillation-counting vials containing 3 ml of cell suspenion, at ice temperature (with 200 rpm of shaking). The challenge was terminated by dilution in M9 salts. Cells were plated in quadruplicate in LB agar plates and incubated at 37°C for 24 h to allow colony formation.
Results and discussion
Aerated E. coli cells were exposed for 15 min to various concentrations of H202 and the surviving fraction was measured as described in Materials and Methods. Results plotted in Fig. 1 indicate that toxicity was not a linear function of the drug concentration; rather, the curve was characterized by two regions of killing with an intervening zone of partial resistance. These results are in close agreement with those obtained by Imlay and Linn (1986) who have designated as mode one killing, the lethality occurring at low concentrations of H202 (<2.5 mM) and as mode two killing, the lethality which occurred at high, postshoulder concentrations (> l0 mM). We have investigated the
239 10-
g
_z _>
H202 (mM) Fig. I. Survival of E. coil cells treated for 15 min with H2Oz under aerated (ta) or anoxic (O) conditions. Each value is the average of 3-6 tests.
effect of anoxia during drug exposure on H202-induced cell death. It is known that under conditions of minimal agitation oxygen is rapidly depleted in culture medium at cellular density of approx. 5 x 107 cells/ml. Our experimental protocol, described in detail in the methods section, involved exposure of a 3-ml cell suspension ( - 10s cells/ml) in a 10-ml test tube to various concentrations of H202. Results, depicted in Fig. 1, indicate that mode one killing remained unaffected by reduction of 02 tension suggesting that this type of lethality is independent on exposure conditions (anoxic vs. aerated). In contrast, cells challenged with H202 in an anoxic medium were more resistant to mode two killing than cells treated in
aerobiosis. Thus, an oxygenated environment confers vulnerability to mode two killing. In other experiments, cells were exposed to two concentrations of H202, one representative of mode one killing (2.5 mM) and the other of mode two killing (25 mM), in the presence or absence of 35 mM thiourea (a scavenger of hydroxyl radicals) under either anoxic or aerated conditions. Results shown in Table 1 indicate that whereas mode two killing was markedly reduced by the hydroxyl radical scavenger under either anoxic or aerated conditions, mode one killing remained unaffected. This would suggest that OH radicals are involved in the production of mode two lethality but have hardly any effect on mode one lethality. We have also investigated the effect of H202 exposure at 4°C on E. coli viability. Results shown in Fig. 2 indicate that, at this temperature, mode one killing did not occur and that cells were more resistant to mode two killing. Hydroxyl radicals have often been indicated as the final DNA-damaging and cytotoxic species in H202 killing (Repine et al., 1981; Mello-Filho and Meneghini, 1984; Starke and Farber, 1985; Ward et al., 1985). In this paper we suggest that mode one and mode two killing are mediated by different cytotoxic species. We propose that mode two killing is induced by OH. and that mode one lethality is generated by a so far unknown species. Our suggestion is motivated by the following observations:
tOTABLE 1 EFFECT OF T H I O U R E A ON H202 KILLING IN E. coli CELLS a
z O
u.
H202 (mM)
2.5 25
Survival (%)
(3 z
Aerated conditions
Anoxic conditions
- thiourea + thiourea
- thiourea + thiourea
50.6 3.4
49.5 14.2
51.2 74.3
D:
51.1 82.9
aE. coil cells were exposed to 2.5 or 25 mM H202 for 15 min (37°C) in the presence or absence of 35 mM thiourea under either aerated or anoxic conditions. Survival was determined as detailed in the methods section. Results are the mean of 3-5 Expts.
1'o
io
3'o
4'o
s~
H202 (mM}
Fig. 2. Survival of E. coil cells exposed for 15 min to various concentrations of H202 at 37°C (o) or 4°C (e). Each value is the average of 3-6 separate Expts.
240 (1) Since m o d e one killing is not a function o f 02 tension during drug treatment, the cytotoxic species must be f o r m e d with the same efficiency under aerated or anoxic conditions. Furthermore, the resulting D N A lesions must be repaired in a similar fashion. This is not consistent with the involvement o f O H . in m o d e one killing. (2) Cells treated under anoxic conditions are more resistant t h a n cells treated under aerated conditions to H202-induced m o d e two lethality. This is consistent with the involvement o f O H . in m o d e two killing. (3) Independently o f 02 tension the hydroxyl radical scavenger, thiourea, reduced m o d e two but did not change m o d e one lethality by H202. This indicates that O H . are involved in m o d e two but not in m o d e one killing. (4) Imlay and Linn (1986) have previously shown that m o d e one killing was abolished under conditions where metabolism was not active and, consistently, we find suppresion o f m o d e one killing following H202 exposure at 4°C. H y d r o x y l radicals, however, are also generated at this temperature and, in fact, m o d e two killing was produced, although in a m o r e relaxed fashion. In conclusion, utilizing various experimental conditions o f drug exposure, we have collected evidence that O H . are involved in m o d e two but not in m o d e one killing by H202. Experiments are in progress to prove further our hypothesis.
References Ananthaswamy, H.N., and A. Eisenstark (1977) Repair of hydrogen peroxide-induced single strand breaks in Escherichia coli deoxyribonucleic acid, J. Bacteriol., 130, 187-191. Carlsson, T., and W.S. Carpenter (1980) The rec-A gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity, J. Bacteriol., 142, 319-321. Christman, M.F., R.W. Morgan, F.R. Jacobson and B. Ames (1985) Positive control of a regulon for defenses against oxidative stress and some heat shock proteins in Salmonella typhimurium, Cell, 41,753-762. Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature (London), 304, 466-468. Demple, B., J. Haibrook and S. Linn (1983) Escherichia coli xth mutants are hypersensitive to hydrogen peroxide, J. Bacteriol., 153, 1079-1082. Imlay, J.A., and S. Linn (1986) Bimodal pattern of killing of DNA repair defective or anoxically grown Escherichia coli by hydrogen peroxide, 166, 519-527. Mello-Filho, A.C., and R. Meneghini (1984) In vivo formation of single strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction, Biochim. Biophys. Acta, 183, 383-392. Repine, J.E., A.B. Fox and E.H. Berger (1981) Hydrogen peroxide kills Staphylococcus aureus by reacting with staphylococcal iron to form hydroxyl radical, J. Biol. Chem., 256, 7094-7096. Starke, P.E., and J.L. Farber (1985) Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide, J. Biol. Chem., 260, 10099-10104. Ward, J.F., W.F. Blakely and J.E. Joner (1985) Mammalian cells are not killed by DNA single strand breaks caused by hydroxyl radicals, Radiat. Res., 103, 383-392. ¢
Acknowledgement This w o r k was supported by a grant f r o m A I R C .
Accepted by A. Abbondandolo
237
MTRL 0968
The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide G. Brandi 1, P. Sestili, M.A. Pedrini 2, L. Salvaggio 1, F. Cattabeni and O. Cantoni lstituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, lIstituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Universitit degli Studi di Urbino, Urbino, and 2Istituto di Genetica Biochimica ed Evoluzionistica, C.N.R., Pavia (Italy) (Accepted 4 November 1986)
Keywords: Temperature; Anoxia; (Escherichia coli); Hydrogen peroxide; Thiourea.
Summary The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure. Two modes of killing were detected following a 15-min challenge with H202 under either aerated or anoxic conditions. Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM. Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic ceils. Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H202. Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations. We suggest that hydroxyl radicals mediate mode two but not mode one killing by H202.
The cytotoxicity of H202 in prokaryotic cells is well documented (Repine et al., 1981; Carlsson and Carpenter, 1980; Demple et al., 1983; Demple and Halbrook, 1983; Christman et al., 1985; Imlay and Linn, 1986). Although the critical lesion produced by the oxidant has not been as yet identified, it is known that iron facilitates the bactericidal action by reacting with H202 to form a highly toxic radical species, the hydroxyl radical (OH') (Repine Correspondence: Dr. Orazio Cantoni, lstituto di Farmacologia e Farmacognosia, Universit/t degli Studi di Urbino, Via S. Chiara, 27, 61029, Urbino (Italy).
et al., 1981). Production of OH" occurs via the Fenton reaction: F e 2+ +
H202
~ Fe 3+ + OH" + O H -
Over the last few years various studies on E. coli have indicated that mutations in recA (Carlsson and Carpenter, 1980; Imlay and Linn, 1986), polA (Ananthaswamy and Eisenstark, 1977; Imlay and Linn, 1986) or xth (Demple et al., 1983; Imlay and Linn, 1986) strains confer hypersensitivity to H202, suggesting an important role for the DNArepair system in the protection of the cell against
0165-7992/87/$ 03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
238 the oxidative insult. Therefore, DNA seems to be the site of the H202-induced lethal damage. Recently, two modes of killing of E. coli by hydrogen peroxide were described (Imlay and Linn, 1986), the first occurring at concentrations of H202 below 3-5 mM and the second at concentrations higher than 10-20 raM. These modes of lethality differed in that unlike mode one, mode two killing did not require active metabolism and was not dramatically enhanced in strains with DNA-repair defects. In the present investigation we have attempted to characterize the effect of anoxia (during drug exposure) on cell death produced by H202. It is known that prechallenge growth in anoxic medium induces sensitivity to mode one killing by H202. We sought to determine whether anoxia or low oxygen levels had any effect on hydrogen peroxide lethality in cells whose growth had been achieved in aerated conditions. Our data demonstrate that aerobically grown cells display a higher vulnerability to H202-induced mode two killing when drug exposure is performed under aerated rather than anoxic conditions. On the contrary, 02 tension was not relevant as far as mode one killing by H202 is concerned. Furthermore, thiourea, a hydroxyl radical scavenger, was able to prevent mode two but not mode one lethality. We have also investigated the effect of H202 challenge at ice temperature on the survival of E. coli cells and found that at 4°C cells displayed an increased resistance to mode two killing, whereas mode one killing was not even detectable.
Materials and methods Materials. H202 was purchased as a 30070 solution from J.T. Baker Chemicals B.V. (Deventer, The Netherlands); other chemicals and most reagent-grade biochemicals were from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and from Flow Labs (McLean, VA, U.S.A.). Bacterial strain and growth. The E. coli strain used, AB1157, was routinely grown in our laboratory at 37°C. Cells were initially grown
overnight (16-18 h) at 37°C in K medium (l°70 glucose, l°70 casamino acids, 1 /~g/ml thiamine hydrochloride, 25 #g/ml MgSO4"7H20 and 2 #g/ml CaCI2 in M9 salts). Samples were diluted 50-fold in fresh K medium and grown to about l0 s cells/ml under aerobic conditions. Aerated growth was achieved by incubation of 50 ml of K medium containing 1-5 × 107 cells/ml in a 500-ml Erlenmeyer flask with 200 rpm of shaking. Survival curves. Cells grown to about 108 cells/ml were harvested by centrifugation at room temperature, washed once with M9 salts and resuspended at 2-5 × 107 cells/ml (aerated conditions) or 7-9 × 107 cells/ml (anoxic conditions) in prewarmed M9 salts. Treatments with H202 (for 15 min at 37°C) were performed in 3 ml of cell suspension in either a 10-ml test tube with 150 rpm of shaking (anoxic conditions) or a 20-ml scintillation-counting vial with 200 rpm of shaking (aerated conditions). In other experiments cells were diluted in prechilled M9 salts (5-8 × 107 cells/ml) and treatments were for 15 min in scintillation-counting vials containing 3 ml of cell suspenion, at ice temperature (with 200 rpm of shaking). The challenge was terminated by dilution in M9 salts. Cells were plated in quadruplicate in LB agar plates and incubated at 37°C for 24 h to allow colony formation.
Results and discussion
Aerated E. coli cells were exposed for 15 min to various concentrations of H202 and the surviving fraction was measured as described in Materials and Methods. Results plotted in Fig. 1 indicate that toxicity was not a linear function of the drug concentration; rather, the curve was characterized by two regions of killing with an intervening zone of partial resistance. These results are in close agreement with those obtained by Imlay and Linn (1986) who have designated as mode one killing, the lethality occurring at low concentrations of H202 (<2.5 mM) and as mode two killing, the lethality which occurred at high, postshoulder concentrations (> l0 mM). We have investigated the
239 10-
g
_z _>
H202 (mM) Fig. I. Survival of E. coil cells treated for 15 min with H2Oz under aerated (ta) or anoxic (O) conditions. Each value is the average of 3-6 tests.
effect of anoxia during drug exposure on H202-induced cell death. It is known that under conditions of minimal agitation oxygen is rapidly depleted in culture medium at cellular density of approx. 5 x 107 cells/ml. Our experimental protocol, described in detail in the methods section, involved exposure of a 3-ml cell suspension ( - 10s cells/ml) in a 10-ml test tube to various concentrations of H202. Results, depicted in Fig. 1, indicate that mode one killing remained unaffected by reduction of 02 tension suggesting that this type of lethality is independent on exposure conditions (anoxic vs. aerated). In contrast, cells challenged with H202 in an anoxic medium were more resistant to mode two killing than cells treated in
aerobiosis. Thus, an oxygenated environment confers vulnerability to mode two killing. In other experiments, cells were exposed to two concentrations of H202, one representative of mode one killing (2.5 mM) and the other of mode two killing (25 mM), in the presence or absence of 35 mM thiourea (a scavenger of hydroxyl radicals) under either anoxic or aerated conditions. Results shown in Table 1 indicate that whereas mode two killing was markedly reduced by the hydroxyl radical scavenger under either anoxic or aerated conditions, mode one killing remained unaffected. This would suggest that OH radicals are involved in the production of mode two lethality but have hardly any effect on mode one lethality. We have also investigated the effect of H202 exposure at 4°C on E. coli viability. Results shown in Fig. 2 indicate that, at this temperature, mode one killing did not occur and that cells were more resistant to mode two killing. Hydroxyl radicals have often been indicated as the final DNA-damaging and cytotoxic species in H202 killing (Repine et al., 1981; Mello-Filho and Meneghini, 1984; Starke and Farber, 1985; Ward et al., 1985). In this paper we suggest that mode one and mode two killing are mediated by different cytotoxic species. We propose that mode two killing is induced by OH. and that mode one lethality is generated by a so far unknown species. Our suggestion is motivated by the following observations:
tOTABLE 1 EFFECT OF T H I O U R E A ON H202 KILLING IN E. coli CELLS a
z O
u.
H202 (mM)
2.5 25
Survival (%)
(3 z
Aerated conditions
Anoxic conditions
- thiourea + thiourea
- thiourea + thiourea
50.6 3.4
49.5 14.2
51.2 74.3
D:
51.1 82.9
aE. coil cells were exposed to 2.5 or 25 mM H202 for 15 min (37°C) in the presence or absence of 35 mM thiourea under either aerated or anoxic conditions. Survival was determined as detailed in the methods section. Results are the mean of 3-5 Expts.
1'o
io
3'o
4'o
s~
H202 (mM}
Fig. 2. Survival of E. coil cells exposed for 15 min to various concentrations of H202 at 37°C (o) or 4°C (e). Each value is the average of 3-6 separate Expts.
240 (1) Since m o d e one killing is not a function o f 02 tension during drug treatment, the cytotoxic species must be f o r m e d with the same efficiency under aerated or anoxic conditions. Furthermore, the resulting D N A lesions must be repaired in a similar fashion. This is not consistent with the involvement o f O H . in m o d e one killing. (2) Cells treated under anoxic conditions are more resistant t h a n cells treated under aerated conditions to H202-induced m o d e two lethality. This is consistent with the involvement o f O H . in m o d e two killing. (3) Independently o f 02 tension the hydroxyl radical scavenger, thiourea, reduced m o d e two but did not change m o d e one lethality by H202. This indicates that O H . are involved in m o d e two but not in m o d e one killing. (4) Imlay and Linn (1986) have previously shown that m o d e one killing was abolished under conditions where metabolism was not active and, consistently, we find suppresion o f m o d e one killing following H202 exposure at 4°C. H y d r o x y l radicals, however, are also generated at this temperature and, in fact, m o d e two killing was produced, although in a m o r e relaxed fashion. In conclusion, utilizing various experimental conditions o f drug exposure, we have collected evidence that O H . are involved in m o d e two but not in m o d e one killing by H202. Experiments are in progress to prove further our hypothesis.
References Ananthaswamy, H.N., and A. Eisenstark (1977) Repair of hydrogen peroxide-induced single strand breaks in Escherichia coli deoxyribonucleic acid, J. Bacteriol., 130, 187-191. Carlsson, T., and W.S. Carpenter (1980) The rec-A gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity, J. Bacteriol., 142, 319-321. Christman, M.F., R.W. Morgan, F.R. Jacobson and B. Ames (1985) Positive control of a regulon for defenses against oxidative stress and some heat shock proteins in Salmonella typhimurium, Cell, 41,753-762. Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature (London), 304, 466-468. Demple, B., J. Haibrook and S. Linn (1983) Escherichia coli xth mutants are hypersensitive to hydrogen peroxide, J. Bacteriol., 153, 1079-1082. Imlay, J.A., and S. Linn (1986) Bimodal pattern of killing of DNA repair defective or anoxically grown Escherichia coli by hydrogen peroxide, 166, 519-527. Mello-Filho, A.C., and R. Meneghini (1984) In vivo formation of single strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction, Biochim. Biophys. Acta, 183, 383-392. Repine, J.E., A.B. Fox and E.H. Berger (1981) Hydrogen peroxide kills Staphylococcus aureus by reacting with staphylococcal iron to form hydroxyl radical, J. Biol. Chem., 256, 7094-7096. Starke, P.E., and J.L. Farber (1985) Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide, J. Biol. Chem., 260, 10099-10104. Ward, J.F., W.F. Blakely and J.E. Joner (1985) Mammalian cells are not killed by DNA single strand breaks caused by hydroxyl radicals, Radiat. Res., 103, 383-392. ¢
Acknowledgement This w o r k was supported by a grant f r o m A I R C .
Accepted by A. Abbondandolo