Heredilus 82 (1976)
Brief reports I. NORDENSON, G. BECKMAN and L. BECKMAN: The effect of superoxide dismutase and catalase on radiation-induced chromosome breaks (Received October 30, 1975)
Although ionizing radiation is known to cause chromosome aberrations as well as cell death the sequence of events and the factors behind these damaging and detrimental effects are by no means fully elucidated. The damaging effect of ionizing radiation is mainly ascribed to the production of reactive radicals, especially the OH' radical. This radical is produced when ionizing radiation passes through water: H,O+H' +OH' The very reactive OH' radical is known to interfere with DNA (cf. MICHAELS and HUNT 1973). This effect of the OH' radical can be eliminated by OH' scavengers. Less attention has been paid to the other product, H'. In the presence of oxygen, H' rapidly transfers its electron to 0,, yielding 0;. The deleterious effect of the 0; radical has been discussed (1972, 1974). The 0; radical has been by FRIDOVICH shown to have sufficient survival time to diffuse and to cause cell damage through lipid peroxidation (FEEand TEITELBAUM 1972) and at least in bacterial cells also to cause damage to the DNA (WHITEet al. 1971). A scavenger for 0; radicals is superoxide dismutase (MCCORDand FRIDOVICH 1968, 1969), an enzyme which catalyzes the dismutation of the 0; radical to yield hydrogen peroxide and oxygen according to the following reaction: 0; +O; + 2 H + +02+ H 2 0 2
WHITEet al. (1971) have shown that 0; radicals cause single-strand breaks in DNA from bacteriophage and that superoxide dismutase can prevent these breaks. It is therefore of interest to study whether superoxide dismutase can protect against chromosome breaks in human cells. It is pertinent in this investigation also to consider the enzyme catalase. This enzyme is known to have a protective effect against cell damage following ionizing radiation. The protective effect has been ascribed to the ability ofcatalase to remove H,O,, which is cytotoxic. Furthermore, hydrogen peroxide is known to react with 0; radicals to give the reactive OH' radical according to the following reaction:
H + + 0, - + H,O,
+
+
0, H,O
+ OH'
A synergistic effect of superoxide dismutase and catalase in the prevention of lipid-peroxidation has (1972). been observed by FEEand TEITELBAUM The aim of this investigation was to find out if the enzymes superoxide dismutase and catalase have a protective effect on radiation-induced chromosome breakage in Man. Human lymphocytes were grown in vitro in medium 199, containing 20% AB serum, phytohemagglutinin and antibiotics. Lymphocyte cultures from the same individual were divided into five different groups, which were treated in the following way: ( I ) Control (2) Irradiation (3) Irradiation + superoxide dismutase (4) Irradiation+ catalase ( 5 ) Irradiation + catalase and superoxide dismutase At least two separate cultures were set up for each group. After 68-70 hours of incubation at 37°C the cells in group 2-5 were irradiated (Co6', total dose 135 R, 27 Rjmin). Immediately after irradiation, superoxide dismutase (SIGMA, 0.05 mg/ml medium) and/or catalase (SIGMA, 0.05 mg/ml medium) were directly added to the cultures. The cells were cultured for 2 hours after irradiation. Velbe (vinblastine sulfate, Lilly) was added 90 min before harvest. After hypotonic treatment for 15 min (0.075 M KCl) and centrifugation the cells were fixed (methano1:acetic acid, 3: 1) and thereafter spread on wet, cold slides. Preparations were made by the air-drying technique and stained in Giemsa solution. The cells were first studied at low magnification; then well-spread mitoses, considered to be complete, were karyotyped under high magnification ( x 1250). Slides from all five groups were coded and analyzed blindly. The chromosome aberrations observed and recorded were: gaps,chromatid breaks, isochromatid breaks, acentric fragments and chromatid exchanges. The chromosome aberrations observed in two separate
126
Hc~rc~d~los 82 ( I Y 7 6 )
BRIEF REPORTS
Tubk, 1. Effect of superoxide dismutase (SOD) and catalase o n radiation-induced chromosome breaks in two separate experiments Experimental conditions
Control Irradiation Irr. + S O D Irr. +catalase Irr. +SOD+catalasc
Number of cells analysed
Aberrant cells ",,
Chromosome aberrations per cell
Expt I
Expt 2
Expt I
Expt 2
Expt I
Expt 2
16 21 40 41
28 51 68 80 46
0 43 35 37
0 41 32 28 17
0
0 0.49 0.35 0.31
-
~~
0.71 0.50 0.44
n 17
-
TubIe 2. Decrease of radiation-induced chromosome breakage (per cent) caused by superoxide dismutase (SOD) and catalase Experimental conditions
SOD Catala\e SOD+catalase
Number of aberrant cells
Number of chromosome aberrations
Expt I
Expt 2
Expt 1
Expt 2
19 14
22 32 59
30 38
29 37 65
experiments are summarized in Table 1. The observed decrease in radiation-induced chromosome breakage, caused by superoxide dismutase and catalase, is shown in Table 2. The combined effect of both enzymes was studied only in the second experiment. There was no significant difference between the two experiments concerning the effect of superoxide dismutase and catalase, hence the results have been pooled in the statistical analysis. The frequency of aberrant cells was 42 per cent after irradiation and decreased to 35, 31 and 17 per cent in the presence of superoxide dismutase, catalase and both enzymes, respectively. Only the combined effect was statistically significant (PO.Ol) but not in the presence of superoxide dismutase. The combined effect was highly significant (P>O.OOl). The mechanism behind the protective effect of superoxide dismutase and catalase is not known. Our working hypothesis is that chromosomal damage continues after irradiation has been discontinued due to the production of OH' radicals in the cell environment (medium) through the interaction of hydrogen peroxide and the superoxide radical, and that superoxide dismutase and catalase protect against this kind of damage since both enzymes can interfere with the production of OH' radicals.
~
A ~ ~ k n o ~ i ~ l e d ~ , ? r rThis n t . study was supported by the Swedish Medical Research Council (Project N o . 03X-2725). -
Department of Medical Genetics, University of Umei, Sweden Literature cited FEE, J. A. and TEITELBAUM, H . D. 1972. Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis. - Biochrm. Biophp. Re.?. Comniun. 49: 150- 158 FRIDOVICH. I . 1972. Superoxide radical and superoxide A c c . Chrm. R
Brief reports I. NORDENSON, G. BECKMAN and L. BECKMAN: The effect of superoxide dismutase and catalase on radiation-induced chromosome breaks (Received October 30, 1975)
Although ionizing radiation is known to cause chromosome aberrations as well as cell death the sequence of events and the factors behind these damaging and detrimental effects are by no means fully elucidated. The damaging effect of ionizing radiation is mainly ascribed to the production of reactive radicals, especially the OH' radical. This radical is produced when ionizing radiation passes through water: H,O+H' +OH' The very reactive OH' radical is known to interfere with DNA (cf. MICHAELS and HUNT 1973). This effect of the OH' radical can be eliminated by OH' scavengers. Less attention has been paid to the other product, H'. In the presence of oxygen, H' rapidly transfers its electron to 0,, yielding 0;. The deleterious effect of the 0; radical has been discussed (1972, 1974). The 0; radical has been by FRIDOVICH shown to have sufficient survival time to diffuse and to cause cell damage through lipid peroxidation (FEEand TEITELBAUM 1972) and at least in bacterial cells also to cause damage to the DNA (WHITEet al. 1971). A scavenger for 0; radicals is superoxide dismutase (MCCORDand FRIDOVICH 1968, 1969), an enzyme which catalyzes the dismutation of the 0; radical to yield hydrogen peroxide and oxygen according to the following reaction: 0; +O; + 2 H + +02+ H 2 0 2
WHITEet al. (1971) have shown that 0; radicals cause single-strand breaks in DNA from bacteriophage and that superoxide dismutase can prevent these breaks. It is therefore of interest to study whether superoxide dismutase can protect against chromosome breaks in human cells. It is pertinent in this investigation also to consider the enzyme catalase. This enzyme is known to have a protective effect against cell damage following ionizing radiation. The protective effect has been ascribed to the ability ofcatalase to remove H,O,, which is cytotoxic. Furthermore, hydrogen peroxide is known to react with 0; radicals to give the reactive OH' radical according to the following reaction:
H + + 0, - + H,O,
+
+
0, H,O
+ OH'
A synergistic effect of superoxide dismutase and catalase in the prevention of lipid-peroxidation has (1972). been observed by FEEand TEITELBAUM The aim of this investigation was to find out if the enzymes superoxide dismutase and catalase have a protective effect on radiation-induced chromosome breakage in Man. Human lymphocytes were grown in vitro in medium 199, containing 20% AB serum, phytohemagglutinin and antibiotics. Lymphocyte cultures from the same individual were divided into five different groups, which were treated in the following way: ( I ) Control (2) Irradiation (3) Irradiation + superoxide dismutase (4) Irradiation+ catalase ( 5 ) Irradiation + catalase and superoxide dismutase At least two separate cultures were set up for each group. After 68-70 hours of incubation at 37°C the cells in group 2-5 were irradiated (Co6', total dose 135 R, 27 Rjmin). Immediately after irradiation, superoxide dismutase (SIGMA, 0.05 mg/ml medium) and/or catalase (SIGMA, 0.05 mg/ml medium) were directly added to the cultures. The cells were cultured for 2 hours after irradiation. Velbe (vinblastine sulfate, Lilly) was added 90 min before harvest. After hypotonic treatment for 15 min (0.075 M KCl) and centrifugation the cells were fixed (methano1:acetic acid, 3: 1) and thereafter spread on wet, cold slides. Preparations were made by the air-drying technique and stained in Giemsa solution. The cells were first studied at low magnification; then well-spread mitoses, considered to be complete, were karyotyped under high magnification ( x 1250). Slides from all five groups were coded and analyzed blindly. The chromosome aberrations observed and recorded were: gaps,chromatid breaks, isochromatid breaks, acentric fragments and chromatid exchanges. The chromosome aberrations observed in two separate
126
Hc~rc~d~los 82 ( I Y 7 6 )
BRIEF REPORTS
Tubk, 1. Effect of superoxide dismutase (SOD) and catalase o n radiation-induced chromosome breaks in two separate experiments Experimental conditions
Control Irradiation Irr. + S O D Irr. +catalase Irr. +SOD+catalasc
Number of cells analysed
Aberrant cells ",,
Chromosome aberrations per cell
Expt I
Expt 2
Expt I
Expt 2
Expt I
Expt 2
16 21 40 41
28 51 68 80 46
0 43 35 37
0 41 32 28 17
0
0 0.49 0.35 0.31
-
~~
0.71 0.50 0.44
n 17
-
TubIe 2. Decrease of radiation-induced chromosome breakage (per cent) caused by superoxide dismutase (SOD) and catalase Experimental conditions
SOD Catala\e SOD+catalase
Number of aberrant cells
Number of chromosome aberrations
Expt I
Expt 2
Expt 1
Expt 2
19 14
22 32 59
30 38
29 37 65
experiments are summarized in Table 1. The observed decrease in radiation-induced chromosome breakage, caused by superoxide dismutase and catalase, is shown in Table 2. The combined effect of both enzymes was studied only in the second experiment. There was no significant difference between the two experiments concerning the effect of superoxide dismutase and catalase, hence the results have been pooled in the statistical analysis. The frequency of aberrant cells was 42 per cent after irradiation and decreased to 35, 31 and 17 per cent in the presence of superoxide dismutase, catalase and both enzymes, respectively. Only the combined effect was statistically significant (PO.Ol) but not in the presence of superoxide dismutase. The combined effect was highly significant (P>O.OOl). The mechanism behind the protective effect of superoxide dismutase and catalase is not known. Our working hypothesis is that chromosomal damage continues after irradiation has been discontinued due to the production of OH' radicals in the cell environment (medium) through the interaction of hydrogen peroxide and the superoxide radical, and that superoxide dismutase and catalase protect against this kind of damage since both enzymes can interfere with the production of OH' radicals.
~
A ~ ~ k n o ~ i ~ l e d ~ , ? r rThis n t . study was supported by the Swedish Medical Research Council (Project N o . 03X-2725). -
Department of Medical Genetics, University of Umei, Sweden Literature cited FEE, J. A. and TEITELBAUM, H . D. 1972. Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis. - Biochrm. Biophp. Re.?. Comniun. 49: 150- 158 FRIDOVICH. I . 1972. Superoxide radical and superoxide A c c . Chrm. R
