Correlation between the anti-oxidant enzyme activities of blood fractions and the yield of bleomycin-induced chromosome damage

Mutation Research, 214 (1989) 129-136 Elsevier

129

MUT 02566

Correlation between the anti-oxidant enzyme activities of blood fractions and the yield of bleomycin-induced chromosome damage Marcelo L. Larramendy, Martha S. Bianchi and Juan Padr6n IMBICE (Instituto Multidisciplinario de Biologia Celular), C.C. 403, 1900 La Plata (Argentina) (Received 30 May 1988) (Accepted 16 February 1989)

Keywords: Anti-oxidant enzymes; Catalase; Peroxidase; Superoxide dismutase; Active oxygen species; Chromosomal damage; Bleomycin

Summary The concentration of the anti-oxidant enzymes catalase (CAT), peroxidases (POD) and superoxide dismutases (SOD) in different blood fractions, and the chromosomal sensitivity of lymphocytes to bleomycin-induced free radicals (expressed as frequency of dicentrics per bleomycin dose) were analyzed in 10 normal human donors. Our results demonstrate that the physiological concentration of the enzymes as well as the chromosomal sensitivity exhibited a wide interindividual variability. An inverse correlation between chromosomal sensitivity to bleomycin and SOD concentration in whole blood, plasma and red cells was found. On the other hand, no correlation between the yield of bleomycin-induced dicentrics and the concentration of CAT or POD was detected in any of the blood fractions analyzed. These findings suggest that the concentration of SOD may play an important role in the cellular susceptibility to DNA damage by free radicals.

In aerobic cells, the complete reduction of molecular oxygen to water by the univalent pathway may result in the formation of free radicals, the so-called active oxygen species. These hyperreactive species are neutralized by a system of intracellular defenses formed by the anti-oxidant enzymes (AOE): superoxide dismutases (SOD), catalase (CAT) and peroxidases (POD), and by several low-molecular-weight anti-oxidants (Frank, 1985). When the intracellular steady-state concentration of free radicals exceeds the detoxification capacity of the anti-oxidant system, the toxic-

Correspondence: Dr. Marcelo L. Larramendy, IMBICE, C.C. 403, 1900 La Plata (Argentina).

ity of active oxygen species becomes evident. A situation of this sort can be found during the exposure to hyperoxic conditions, to ionizing radiation or to certain antineoplastic drugs such as bleomycin, streptonigrin, adriamycin, etc. (Fricke, 1934; Oberley, 1982; Polard and Weller, 1967; Sugiura, 1979). The damage to the DNA induced by free radicals gives rise to DNA-strand breakage (Birboin, 1982; Meneghini and Hoffmann, 1980), chromosomal aberrations (Emerit et al., 1982; Estervig and Wang, 1984), an increase in sister-chromatid exchanges (Emerit, 1984; Larramendy et al., 1987) and mutagenesis (Moody and Hassan, 1982). Cellular death (Fridovich, 1975; Mello-Filho and Meneghini, 1985), carcinogenesis (Cerutti, 1985;

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

0 10 100 200

0 10 26 24

0 10 100 200

0 2 11 19

1(30 200

0 10 100 200

0 5 9 19

16 23

9

0 10 100 200

0 11 19 25

0

0 10 1(30 200

0 33 37 35

10

0 10 100 200

0 20 30 Failed

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14

8

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10

7

6

5

4

3

2

1

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0 3 11 17

Donor

BLM (/~g/ml)

Die,en tries (frequency per 100 cells)

30.63+0.23

17.275:0.37

10.855:0.34

14.425:0.52

6.535:0.70

6.335:0.05

10.255:0.44

10.675:0.48

10.415:0.26

196.85:9.8

128.1+ 2.0

143.85:1.5

234.85:30.5

394.55:21.6

611.95:4.9

132.05:8.0

89.95:8.6

20.55:1.0

11.15:2.0

2.255:0.04

1.275:0.01

1.73+0.01

1.695:0.01

2.625:0.02

1.56±0.05

1.485:0.05

1.725:0.06

4.765:0.36

2.965:0.08

28.93±0.08

17,195:0,06

10.635:0.12

13.955:0.04

6.765:0.93

7.7950.35

18.545:0.85

16.225:0.85

11.505:0.33

9.725:0.07

CAT ( U / m g Hb)

7.485:0.26

Erythrocytes SOD ( U / r a g Hb)

CAT ( U / r a g Hb)

POD ( / x U / m g Hb)

Whole blood

1,5

103.9+ 4.6

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742.5+37.1

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2 6 9 , 8 ± 7.4

82.25:2.7

94.9 + 13.1

24.35:3.1

24.1+

POD ( / z U / m g Hb)

1.66+0.02

1.135:0.06

1.375:0.02

1.775:0.03

1.195:0.01

0.795:0.03

0.735:0.03

1.215:0.01

2.045:0.01

1,77±0.04

SOD ( U / r a g Hb)

10.685:1.71

2.81+0.26

14.895:0.35

5.845:0.93

4.25+0.08

6.145:0.28

9.575:0.40

1.15±0.12

0.375:0.04

0.345:0.06

CAT ( U / r a g Pt)

7.445:0.33

7.27+0.14

4.36+0.40

4.175:0.18

10,965:0.74

24.875:0.61

5.51±0.123

1.275:0.06

3,955:0.38

5.29d:0.16

POD ( m U / m g Pt)

M o n o n u c l e a r leukocytes

22.53±0.20

45.095:0.55

Failed

23,875:0.11

Failed

Failed

28.125:1.11

6.275:0.44

Failed

11.0350.15

SOD ( U / r a g Pt)

4.555:0.01

9.625:0.61

17.195:0.02

13.425:0.45

10.335:0.87

17.755:1.13

3.77±0.12

4.635:0.68

7.54+0.11

4.525:0.34

CAT ( m U / m g Pt)

Plasma

7.9 5:0.3

8.3:1:0.3

8.3 5:0.1

28.5 5:0.1

9.405:0.1

5.705:0.1

2.82+0.2

2.26 5:0.1

1.745:0.2

2,855:0.1

POD ( l a U / m g Pt)

0.575:0.01

0.385:0.01

0.415:0.01

0,465:0.01

0.315:0.01

0.185:0.01

0.155:0.01

0.155:0.01

0.385:0.02

0.245:0.02

SOD ( U / r a g Pt)

M E A S U R E M E N T S O F CAT, POD A N D S O D A C T I V I T I E S IN D I F F E R E N T B L O O D F R A C T I O N S A N D C H R O M O S O M A L S E N S I T I V I T Y O F H U M A N L Y M P H O C Y T E S T O B L M E X P R E S S E D AS F R E Q U E N C Y O F D I C E N T R I C S PER 100 C E L L S

TABLE 1

131 Weitzman et al., 1985) and depletion of the cellular nucleotide pool (Schraufstatter et al., 1980) are additional consequences of the aggression of free radicals to the cellular macromolecules. It has been reported that the concentration of AOE varies between different tissues of the same individual (Marklund, 1984) and also between the same tissues of different individuals in a population (Marklund, 1984; Lipecka et al., 1984). Moreover, there is evidence suggesting that the interindividual variations in SOD activity may play a role in the chromosomal sensitivity of lymphocytes to ionizing radiation (Lipecka et al., 1984). In this report we present data on 10 individuals. In each case we analyzed the concentration of AOE in different blood fractions and the chromosomal sensitivity of lymphocytes to bleomycininduced free radicals. Our results indicate an inverse correlation between SOD concentration and chromosomal sensitivity. On the other hand, no correlation of this sort was observed for the CAT and POD enzymes. Material

and methods

Blood samples Human blood samples were obtained from 10 healthy voluntary male donors (20-40 years old) selected according to the recommendations previously reported (Bianchi et al., 1979).

Lymphocyte cultures, bleomycin treatments and cytogenetic analysis For each donor, aliquots of 1 ml of whole blood were mixed with 9 ml of Ham's F10 medium (Gibco, Grand Island, NY) and treated with bleomycin (BLM, Lab. Dr. Gador, Argentina). Stock solutions of BLM in Hanks' saline were freshly prepared before use; 0.1 ml of the corresponding solution was added to blood cell suspensions to obtain a final concentration of 10, 100 or 200 #g/ml. Control samples were mock-treated with 0.1 ml of Hanks' saline without BLM. After 3 h at room temperature, the cells were washed 3 times with Hanks' saline and resuspended in complete culture medium (80% Ham's F10, 17% fetal calf serum, 3% PHA-M (Gibco), 100 units penicillin/ml and 100 #g streptomycin/ml (Gibco)). Colchicine (0.1/xg/ml, Sigma, St. Louis, MO) was

added at 45 h and harvesting was performed 48 h after starting the culture. At this time, more than 95% of human lymphocytes are in the first mitotic division (Bianchi et al., 1979). Cultures were set up in duplicate for each sample. A total of 100 metaphases were analyzed per sample and the frequency of dicentric chromosomes was estimated. The analysis of BLM-induced chromosome aberrations was restricted to dicentric chromosomes as indicators of chromosome damage according to the recommendations of UNSCEAR (1969, cited by Kucerova and Polivkova, 1976).

Anti-oxidant enzyme activity assay CAT, POD and SOD activities were measured in whole blood, erythrocytes, plasma and mononuclear leukocytes. Whole blood was diluted 1 : 10 in M/15 phosphate buffer solution (pH 7.0). Cells were lysed with 0.1% Triton X-100 (Sigma) and sonication (W-225 R Sonicator, Heat Systems, Ultrasonics). Enzyme activity determinations were made on supernatants after centrifugation at 20,000 x g for 15 rain (4°C). Mononuclear leukocytes were isolated from whole blood by the FicoU-Hypaque (Sigma) gradient separation technique (Bryum, 1968). Erythrocytes were obtained from erythrocyte pellets of Ficoll-Hypaque gradients (no attempt to separate contaminant polymorphonuclear leukocytes was made). After separation the cells were resuspended, lysed and sonicated in the same buffer solution used for whole blood samples. The analysis of AOE activities was performed in the supernatants after centrifugation at 20,000 × g for 15 rain (40C). Plasma samples for AOE measurements were obtained from whole blood centrifuged at 20,000 x g for 15 min (4 ° C). Blood and blood fractions were processed immediately before the enzyme assay or stored at - 70 ° C until use. CAT and POD activities were measured as indicated by Liick (1965a,b, respectively). SOD activity was determined by following up the inhibition of epinephrine autoxidation to adrenochrome (Misra and Fridovich, 1972). One unit of SOD activity was defined as the amount of en-

132

zyme inducing a 50% inhibition in the rate of epinephrine autoxidation. Enzyme activities were expressed as units per milligram of hemoglobin (U/mg Hb) for whole blood and erythrocyte samples, and as units per milligram of protein (U/mg Pt) for plasma and mononuclear leukocyte samples.

The correlation between chromosomal sensitivity to BLM (expressed as frequency of dicentrics per 100 cells per dose of BLM) and AOE activities was evaluated with an IBM PC by using the Statgraphics software (Statistical Graphics Corporation). Unless otherwise stated, the level of significance chosen was 0.05.

Statistical analysis Analysis of variance (ANOVA) was done for chromosome aberrations and AOE data. Tukey's test for comparisons was used to determine the significance of dicentric value differences between control and BLM-treated samples.

Results and discussion

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blood and blood fractions from each donor are presented in Table 1. As shown by other authors (Dresp et al., 1978), a 3-h pulse treatment of Go lymphocytes with BLM produced a dose-responsive increase in the frequency of dicentrics over control values ( p <

0.05) in 9 of the 10 donors tested. In the remaining case the chromosomal sensitivity to BLM was evident but the dose-response curve was not clear (case 5, Table 1). Using ANOVA we could demonstrate that interindividual variations in chromosomal sensitivity

134

to BLM were significant ( p < 0.05) for each one of the drug doses employed. Likewise, the interdonor variabilities in CAT, POD and SOD activities were also found to be statistically significant ( p < 0.05).

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The correlations between the yield of BLM-induced dicentric chromosomes and the SOD, CAT and POD contents for the different blood fractions analyzed are depicted in Figs. 1-3. For the 3 BLM doses used, the regression test shows a lin-

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135 ear, negative correlation between the yield of BLM-induced dicentrics and the concentration of SOD in whole blood, erythrocytes and plasma. The SOD activity of mononuclear leukocytes could be only determined in 6 out of the 10 individuals analyzed. In the other 4 cases we observed a wide variability between successive assays made with the same cell fraction. Therefore, no reliable mean values could be obtained for these individuals. The small size of the sample (6 cases) prevented us from obtaining a trustworthy correlation between sensitivity to BLM and SOD activity for mononuclear blood cells. N o significant correlation between the yield of BLM-induced dicentrics and the concentration of CAT (Fig. 2) or POD (Fig. 3) was found in any of the blood fractions analyzed. The existence of a relationship between the A O E activity and the frequency of chromosomal damage induced in human lymphocytes by active oxygen species was first described by Nordenson et al. (1976). They reported a decrease in chromosome breakage by the addition of CAT or CAT plus SOD immediately after X-ray exposure. Nevertheless, the authors were not concerned with studying the possible dependence between the yield of radiation-induced aberrations and the A O E content of different blood fractions. Recently, Lipecka et al. (1984) reported the existence of a wide interindividual variability in SOD activities in lymphocytes and red cells from healthy human donors. Besides, a negative correlation between the SOD activity of these white cells and the frequency of y-ray-induced aberrations was also shown. Our results regarding the variation of A O E levels and the linear, negative relationship between BLM-induced dicentrics and the level of SOD in whole blood fractions are in good agreement with the data of Lipecka et al. (1984). The above findings suggest that SOD activity in peripheral blood may be responsible for the chromosomal sensitivity to active oxygen species. The confirmation of this assumption could be of practical importance. Thus, for instance, an assay of SOD activity in blood fractions could be used as a simple method for evaluating the potential clastogenic risk of exposure to BLM, ionizing radiations or any other chemical agent inducing the production of active oxygen species.

Acknowledgements We wish to thank Mr. Livio De Rossi (Laboratorios Dr. Gador, Argentina) for providing us bleomycin samples and Lic. M. Reigosa for technical assistance. This work was supported by grants from C O N I C E T and CIC.

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136 radiation-induced chromosome aberrations, Studia Biophys., 100, 211-217. Li~ck, H. (1965a) Catalase, in: H. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, pp. 885-894. LUck, H. (1965b) Peroxidase, in: H. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, pp. 895-897. Marklund, S.L. (1984) Mammalian superoxide dismutases, in: W. Bors, M. Saran and D. Tait (Eds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter and Co, Berlin, pp. 765-777. Mello-Filho, A., and R. Meneghini (1985) Protection of mammalian cells by o-phenanthroline from lethal and DNAdamaging effects produced by active oxygen species, Biochim. Biophys. Acta, 847, 82-89. Meneghini, R., and M.E. Hoffmann (1980) The damaging action of hydrogen peroxide on DNA of human fibroblasts is mediated by a non-dialyzable compound, Biochim. Biophys. Acta, 608, 167-173. Misra, H., and I. Fridovich (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase, J. Biol, Chem., 247, 3170-3175.

Moody, C.S., and H.M. Hassan (1982) Mutagenicity of oxygen free radicals, Proc. Natl. Acad. Sci. (U.S.A.), 79, 2855-2859. Nordenson, I., G. Beckman and L. Beckman (1976) The effect of superoxide dismutase and catalase on radiation-induced chromosome breaks, Hereditas, 82, 125-126. Polard, E.C., and P.K. Weller (1967) Chain scission of ribonucleic acid and deoxyribonucleic acid by ionizing radiation and hydrogen peroxide in vitro and in Escherichia coli cells, Radiation Res,, 32, 417-440. Oberley, L. (1982) Superoxide dismutase and cancer, in: L. Oberley (Ed.), Superoxide Dismutase, Vol. II, CRC Press, Boca Raton, FL, pp. 127-165. Schraufstatter, V., D.B. Hinshaw, P.A, Hyslop, R.G. Spragg and C.G. Cochrane (1986) Oxidant injury to cells, J. Clin. Invest., 77, 1312-1320. Sugiura, Y. (1979) Production of free radicals from phenol and tocopherol by bleomycin-iron(I1) complex, Biochem. Biophys. Res. Commun., 87, 649-653. Weitzman, S,A., A.B.. Weitberg, E.P. Clark and T.P. Stossel (1985) Phagocytes as carcinogens: malignant transformation produced by human neutrophils, Science, 227, 1231-1233.