INT . J. RADIAT . BIOL .,
1977,
VOL .
31,
NO.
5, 4 07-413
Action of hydrogen peroxide on degradation of DNA after irradiation in Escherichia coli
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KATHLEEN MAY KELLER and ERNEST C . POLLARD f Biophysics Laboratory, Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802, U .S .A. (Received 26 October 1976 ; accepted 31 January 1977) Hydrogen peroxide (H2O,), which produces breaks in cellular DNA, has not hitherto been shown to cause degradation of DNA . In this investigation it is shown that if transcription is blocked with rifampin, treatment with H2O, causes degradation of DNA to nearly the same extent as does gamma-radiation . Further, if cells are given a treatment with H 2 0 2 and incubated for 50 min, the amount of degradation in a second treatment is markedly less . This is attributed to the induction of the inhibitor of post-irradiation degradation of DNA (prd) by the first treatment . There is thus a double action of H 202 : first, to induce inhibition, and second, to cause degradation of DNA to begin in non-induced cells . The genetic dependence of induction by H 202 mimics that of ionizing radiation . Accordingly, the induction process does not occur in recA- and lex cells, because they are not inducible and is absent in recBcells because they lack exonuclease V, the major component of prd . Potassium iodide (KI), an OH radical scavenger, negates the action of peroxide on DNA . The results obtained in this study suggest a possible theory for the evolution of radiation response systems . 1 . Introduction Hydrogen peroxide (H 20 2) is a component of the products of the action of ionizing radiation on aqueous media, particularly if oxygen is present (Frey and Pollard 1966) . It can, moreover, be produced in the respiratory chain (Stanier, Douderoff and Adelberg 1970) . H2 02 has been shown to be concentrated from the medium by Escherichia coli cells (Frey and Pollard 1968) and to block the respiratory chain (Frey and Pollard 1966, 1968) . It causes a stoppage of transcription (Pollard and Barone 1966) and breaks in DNA (Pollard and Weller 1967), but several attempts failed to show that degradation of DNA, a very common event after ionizing radiation, also follows peroxide treatment (Pollard and Weller 1967) . Recent studies on the inhibition of post-irradiation degradation of DNA (prd) have shown that many agents (U .V ., ionizing radiation, nalidixic acid) cause the induction of an inhibition mechanism, which is dependent on the recA+lex+ genotype, and de novo protein synthesis, and which diminishes or prevents prd (Pollard and Randall 1973, Marsden, Pollard, Ginoza and Randall 1974) . It seemed possible that the failure to detect degradation of DNA after peroxide treatment might be due to the induction of inhibition superposed on the lesions which cause degradation of DNA . The use of rifampin to block transcription increases prd (Pollard and Weller 1969) by preventing the inhibition process (which requires protein synthesis) and therefore might be expected to t Supported by a grant from the U .S . Energy Research and Development Administration, E(11-1)-2362 . R.B .
2 E
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408
K . M. Keller and E . C. Pollard
reveal the presence of degradation if it is actually a factor in peroxide action . The results of these investigations are reported here . Since induction occurs only in the strains that are recA+ and lex+, strains which lack this genotype should not show the effects of rifampin to any marked extent . This has been verified . Further, the use of an inducing agent such as U .V . applied 40 min before peroxide treatment should inhibit degradation of DNA except in strains which are recA - or lex . This was also observed . If H2O, itself is an inducing agent, then a treatment with peroxide, followed by sufficient time for transcription, should modify the DNA degradation by a second treatment . This is found . One way in which peroxide may act is by the generation, within the cell, of hydroxyl radicals . These are effectively scavenged by KI which should, accordingly, remove the degradation . These studies show the above to be observed . 2.
Materials and methods Relevant characteristics of the cell strains are shown in the table . Cell strain
Relevant genetics
X-ray-mediated degradation of DNA
Inducible inhibitor (requires recA + lex + genotype)
13/r
Essentially wild type
+
+
B S_ 1
exr (lex )t
+
-
AB1157
Essentially wild type
+
+
AB2463
recA -
+
-
AB2470T
recB21 -
-
Not known
in strain B is the equivalent of lex in the K12 strains . I Lacks the recBC nuclease, exonuclease V, responsible for X-ray-mediated DNA degradation . f exr
Characteristics of cell strains used . Cells were grown in C-minimal medium (3 g NH 4 Cl, 6 g Na2 HPO 4 , 3 g KH 2 PO 4i 3 g NaCl, 100 mg MgCl,, 80 mg Na 2 SO 4 per litre) with 0 . 5 per cent glucose and supplemented with 0 . 2 per cent casamino acids and 20 µg/ml thiamine where required . For labelling the DNA, the cells were grown in the presence of 0 . 50 µCi/ml of 3H-thymidine and 200µg/ml of deoxyadenosine (Boyce and Setlow 1962) to early log phase . For observing degradation of DNA, 100µg/ml final concentration of rifampin was added and the cells were incubated at 37 ° C for 10 min, after which H2O, was administered and exposure continued for 10 min more . The cells were next washed twice and resuspended in medium containing 10 mg/ml final concentration of catalase . The cells were then incubated with aeration for 120 min, during which time samples
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
Peroxide and DNA degradation
409
were taken and assayed for trichloroacetic- acid (TCA)-soluble material . This was performed by taking 0 . 75 ml samples, adding to 4 . 5 ml 7 per cent TCA with 0 . 5 ml of a 6 mg/ml solution of bovine-serum albumin . The mixture was kept on ice for 30 min and then spun at 7000g for 5 min . 0 . 4-ml samples of the supernatant fluid were counted in a liquid scintillation counter using toluene, triton X-100, PPO and POPOP as the scintillation fluid . For U . V . induction, a fluence of 15 J /m 2 of U . V . from a germicidal lamp (G15T8 Sylvania) was given 50 min before the administration of rifampin . For peroxide induction, 6 mM peroxide was given for 10 min, the cells spun down and resuspended in medium for 50 min before treatment with rifampin . When KI was used it was added at a final concentration of 1 mM . Ionizing radiation was administered with a 50 kVp Picker X-ray machine with a beryllium window . 3.
Results Figure 1 (A) shows the effect of 6 mM H 20 2 on cells of E. coli B/r with and without rifampin . Without rifampin there was a slight DNA degradation, but in the presence of 100µg/ml rifampin there was nearly three times as much . Figure 1 (B) shows the effect of prior treatment with U .V . or a preliminary exposure to peroxide on the amount of DNA degradation . With no pretreatment, there is a clear increase in the amount of solubilized DNA . With
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(minutes) H2O,-mediated (6 mM) DNA degradation studies on B/r (A) with (A, •) and H202), and (B) with a dose of U .V . (15 J/m 2 ) and administration of H 2 0 2 (6 mM) 40 min before treatment with rifampin . 0, control, no H202 ; •, no U .V . +H 2 0 2 ; ∎, H2 0 2 +H202 ; A, U .V . + H 2 02 .
Figure 1 .
without (0, 0) the drug rifampin (circles =controls with no
41 0
K. M . Keller and E. C . Pollard
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
an inducing dose of 15 J/m 2 of U .V ., the amount of degradation is sharply reduced and a pre-dose of H 2 02 also shows curtailment, though not as effectively as was observed for U .V . U .V . alone, at this fluence, produces barely observable degradation (data not shown) . Figure 2 shows the effect of peroxide with and without rifampin on three mutant strains . Figure 2 (A) applies to E. coli B G _ 1 which is lex and uvr . There is considerable degradation but very little effect of rifampin . Figure 2 (B) is the case of AB2463 recA- . Here the degradation pattern is such that
(A) 4
B s-I
3 2 10 z 0 F
a ( ) AB2463
Q 4 0 M 0
0
3
z 20 0
z w w a
10 0 (C) 40
AB2470
30 20 10 0 20 40 60 80 100 120 POST-H 2 O 2 INCUBATION TIME (minutes)
Figure 2 . H 2 0 2 (3 mM) DNA degradation studies on (A) B 5_1 [exr(lex )], (B) AB2463 (recA- ), and (C) AB2470 (recB21 -) (6 mM H 2 0 2 ) with (A, •) and without (0, 0) rifampin .
Circles =controls .
rifampin actually diminishes peroxide-induced degradation . Moreover, in this strain, degradation of DNA is dose-dependent and is actually greatest at 0. 5 mM H2O, (data not shown) . Figure 2 (C) shows the effect of peroxide on strain AB2470 (recB - ) ; here there is very little DNA degradation either with or without rifampin . Several concentrations of H2O, were studied ; we show, for comparison, a rather higher concentration than for the other cells . Figure 3 shows the effect of 1 mM KI on peroxide-mediated degradation of DNA in strain B S _1 . The action of the peroxide is effectively eliminated . For comparison, the effect of KI on degradation of DNA produced by 23 krad of
Peroxide and DNA degradation
41 1
60 - (A) 50 40 z
30
0 Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
a
1977,
VOL .
31,
NO.
5, 4 07-413
Action of hydrogen peroxide on degradation of DNA after irradiation in Escherichia coli
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
KATHLEEN MAY KELLER and ERNEST C . POLLARD f Biophysics Laboratory, Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802, U .S .A. (Received 26 October 1976 ; accepted 31 January 1977) Hydrogen peroxide (H2O,), which produces breaks in cellular DNA, has not hitherto been shown to cause degradation of DNA . In this investigation it is shown that if transcription is blocked with rifampin, treatment with H2O, causes degradation of DNA to nearly the same extent as does gamma-radiation . Further, if cells are given a treatment with H 2 0 2 and incubated for 50 min, the amount of degradation in a second treatment is markedly less . This is attributed to the induction of the inhibitor of post-irradiation degradation of DNA (prd) by the first treatment . There is thus a double action of H 202 : first, to induce inhibition, and second, to cause degradation of DNA to begin in non-induced cells . The genetic dependence of induction by H 202 mimics that of ionizing radiation . Accordingly, the induction process does not occur in recA- and lex cells, because they are not inducible and is absent in recBcells because they lack exonuclease V, the major component of prd . Potassium iodide (KI), an OH radical scavenger, negates the action of peroxide on DNA . The results obtained in this study suggest a possible theory for the evolution of radiation response systems . 1 . Introduction Hydrogen peroxide (H 20 2) is a component of the products of the action of ionizing radiation on aqueous media, particularly if oxygen is present (Frey and Pollard 1966) . It can, moreover, be produced in the respiratory chain (Stanier, Douderoff and Adelberg 1970) . H2 02 has been shown to be concentrated from the medium by Escherichia coli cells (Frey and Pollard 1968) and to block the respiratory chain (Frey and Pollard 1966, 1968) . It causes a stoppage of transcription (Pollard and Barone 1966) and breaks in DNA (Pollard and Weller 1967), but several attempts failed to show that degradation of DNA, a very common event after ionizing radiation, also follows peroxide treatment (Pollard and Weller 1967) . Recent studies on the inhibition of post-irradiation degradation of DNA (prd) have shown that many agents (U .V ., ionizing radiation, nalidixic acid) cause the induction of an inhibition mechanism, which is dependent on the recA+lex+ genotype, and de novo protein synthesis, and which diminishes or prevents prd (Pollard and Randall 1973, Marsden, Pollard, Ginoza and Randall 1974) . It seemed possible that the failure to detect degradation of DNA after peroxide treatment might be due to the induction of inhibition superposed on the lesions which cause degradation of DNA . The use of rifampin to block transcription increases prd (Pollard and Weller 1969) by preventing the inhibition process (which requires protein synthesis) and therefore might be expected to t Supported by a grant from the U .S . Energy Research and Development Administration, E(11-1)-2362 . R.B .
2 E
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
408
K . M. Keller and E . C. Pollard
reveal the presence of degradation if it is actually a factor in peroxide action . The results of these investigations are reported here . Since induction occurs only in the strains that are recA+ and lex+, strains which lack this genotype should not show the effects of rifampin to any marked extent . This has been verified . Further, the use of an inducing agent such as U .V . applied 40 min before peroxide treatment should inhibit degradation of DNA except in strains which are recA - or lex . This was also observed . If H2O, itself is an inducing agent, then a treatment with peroxide, followed by sufficient time for transcription, should modify the DNA degradation by a second treatment . This is found . One way in which peroxide may act is by the generation, within the cell, of hydroxyl radicals . These are effectively scavenged by KI which should, accordingly, remove the degradation . These studies show the above to be observed . 2.
Materials and methods Relevant characteristics of the cell strains are shown in the table . Cell strain
Relevant genetics
X-ray-mediated degradation of DNA
Inducible inhibitor (requires recA + lex + genotype)
13/r
Essentially wild type
+
+
B S_ 1
exr (lex )t
+
-
AB1157
Essentially wild type
+
+
AB2463
recA -
+
-
AB2470T
recB21 -
-
Not known
in strain B is the equivalent of lex in the K12 strains . I Lacks the recBC nuclease, exonuclease V, responsible for X-ray-mediated DNA degradation . f exr
Characteristics of cell strains used . Cells were grown in C-minimal medium (3 g NH 4 Cl, 6 g Na2 HPO 4 , 3 g KH 2 PO 4i 3 g NaCl, 100 mg MgCl,, 80 mg Na 2 SO 4 per litre) with 0 . 5 per cent glucose and supplemented with 0 . 2 per cent casamino acids and 20 µg/ml thiamine where required . For labelling the DNA, the cells were grown in the presence of 0 . 50 µCi/ml of 3H-thymidine and 200µg/ml of deoxyadenosine (Boyce and Setlow 1962) to early log phase . For observing degradation of DNA, 100µg/ml final concentration of rifampin was added and the cells were incubated at 37 ° C for 10 min, after which H2O, was administered and exposure continued for 10 min more . The cells were next washed twice and resuspended in medium containing 10 mg/ml final concentration of catalase . The cells were then incubated with aeration for 120 min, during which time samples
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
Peroxide and DNA degradation
409
were taken and assayed for trichloroacetic- acid (TCA)-soluble material . This was performed by taking 0 . 75 ml samples, adding to 4 . 5 ml 7 per cent TCA with 0 . 5 ml of a 6 mg/ml solution of bovine-serum albumin . The mixture was kept on ice for 30 min and then spun at 7000g for 5 min . 0 . 4-ml samples of the supernatant fluid were counted in a liquid scintillation counter using toluene, triton X-100, PPO and POPOP as the scintillation fluid . For U . V . induction, a fluence of 15 J /m 2 of U . V . from a germicidal lamp (G15T8 Sylvania) was given 50 min before the administration of rifampin . For peroxide induction, 6 mM peroxide was given for 10 min, the cells spun down and resuspended in medium for 50 min before treatment with rifampin . When KI was used it was added at a final concentration of 1 mM . Ionizing radiation was administered with a 50 kVp Picker X-ray machine with a beryllium window . 3.
Results Figure 1 (A) shows the effect of 6 mM H 20 2 on cells of E. coli B/r with and without rifampin . Without rifampin there was a slight DNA degradation, but in the presence of 100µg/ml rifampin there was nearly three times as much . Figure 1 (B) shows the effect of prior treatment with U .V . or a preliminary exposure to peroxide on the amount of DNA degradation . With no pretreatment, there is a clear increase in the amount of solubilized DNA . With
60
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I
50 40
0 0a w
30 20 10
0
a 0 60
I
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I
I
I
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0
z 50
w v M
40
CL
30 20 U
10 I
I
I
I
0 20 40 60 80 100 120 POST-H 2 0 2 INCUBATION TIME
(minutes) H2O,-mediated (6 mM) DNA degradation studies on B/r (A) with (A, •) and H202), and (B) with a dose of U .V . (15 J/m 2 ) and administration of H 2 0 2 (6 mM) 40 min before treatment with rifampin . 0, control, no H202 ; •, no U .V . +H 2 0 2 ; ∎, H2 0 2 +H202 ; A, U .V . + H 2 02 .
Figure 1 .
without (0, 0) the drug rifampin (circles =controls with no
41 0
K. M . Keller and E. C . Pollard
Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
an inducing dose of 15 J/m 2 of U .V ., the amount of degradation is sharply reduced and a pre-dose of H 2 02 also shows curtailment, though not as effectively as was observed for U .V . U .V . alone, at this fluence, produces barely observable degradation (data not shown) . Figure 2 shows the effect of peroxide with and without rifampin on three mutant strains . Figure 2 (A) applies to E. coli B G _ 1 which is lex and uvr . There is considerable degradation but very little effect of rifampin . Figure 2 (B) is the case of AB2463 recA- . Here the degradation pattern is such that
(A) 4
B s-I
3 2 10 z 0 F
a ( ) AB2463
Q 4 0 M 0
0
3
z 20 0
z w w a
10 0 (C) 40
AB2470
30 20 10 0 20 40 60 80 100 120 POST-H 2 O 2 INCUBATION TIME (minutes)
Figure 2 . H 2 0 2 (3 mM) DNA degradation studies on (A) B 5_1 [exr(lex )], (B) AB2463 (recA- ), and (C) AB2470 (recB21 -) (6 mM H 2 0 2 ) with (A, •) and without (0, 0) rifampin .
Circles =controls .
rifampin actually diminishes peroxide-induced degradation . Moreover, in this strain, degradation of DNA is dose-dependent and is actually greatest at 0. 5 mM H2O, (data not shown) . Figure 2 (C) shows the effect of peroxide on strain AB2470 (recB - ) ; here there is very little DNA degradation either with or without rifampin . Several concentrations of H2O, were studied ; we show, for comparison, a rather higher concentration than for the other cells . Figure 3 shows the effect of 1 mM KI on peroxide-mediated degradation of DNA in strain B S _1 . The action of the peroxide is effectively eliminated . For comparison, the effect of KI on degradation of DNA produced by 23 krad of
Peroxide and DNA degradation
41 1
60 - (A) 50 40 z
30
0 Int J Radiat Biol Downloaded from informahealthcare.com by University of British Columbia on 10/30/14 For personal use only.
a
