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Superoxide dismutase and catalase activities in normal and cancerous tissues
Comp. Biochem. Physiol. Vol. 70B, pp. 819 to 820, 1981
0305-0491/81/120819-02502.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SUPEROXIDE DISMUTASE A N D CATALASE ACTIVITIES IN NORMAL A N D CANCEROUS TISSUES A. BARTKOWIAK1 and J. BARTKOWIAK2. ~Department of Biophysics, University of L6d~ and 2Department of Oncology, Medical Academy, 93-509 L6d~, Gagarina 4, Poland
(Received 11 May 1981) Abstract--Superoxide dismutase and catalase activities in hamster transplantable tumors were distinctly lower than in normal hamster liver, both quiescent and regenerating. activity assays tumor material was collected on the 8th day after transplantation, when tumor weighted approx. 3-5 g. For transplantation of tumor induced by SV 40 virus the neoplastic material was not dispersed and the enzymatic measurement was performed 4 weeks after operation. Partial hepatectomy was performed on normal hamsters under light ether anesthesia and 30--40~o of the liver was removed. The hamsters were killed at 12 hr after surgery and regenerated parts of liver were only collected. Livers of sham-operated animals were used as reference tissue. For SOD activity determination 10~ tissue homogenates in 0.05 M K2HPO4 buffer, pH 7.8, were prepared, centrifuged at 8000 @for 15 min, and appropriate aliquots of the supernatant were used for enzyme assay, by the method of Misra & Fridovich (1972). One unit of SOD activity was defined as the amount of the enzyme required for 50~ inhibition of the oxidation of adrenaline to adrenochrome, during 1.0 min. For catalase activity determination, cytosolic fraction was prepared in 0.01 M phosphate buffer pH 7.0, by the same technique as above. The changes of H202 level were measured according to Beers and Sizer (1952). The catalase activity was expressed in Bergmeyer units (BU) where one BU was defined as the amount of C which can decompose 1.0 g hydrogen peroxide in 1.0 rain. The activities of both enzymes were calculated per 1.0mg of total protein in the homogenates. Protein concentration was estimated by the method of Lowry (1951). All reported here data are the mean values of 6 experiments (3 parallel enzyme determinations in each case).
INTRODUCTION
During recent years the role of peroxide catabolic enzymes of various animal tissue have been studied. Substantial evidence has been provided that these enzymes are necessary for the survival of all oxygenmetabolizing cells, because the oxygen-derived radicals have the potential to key subcellular structures. Superoxide dismutase (SOD) (EC.I.15.1.1.) is a scavenger of superoxide anions which are generated during the univalent reduction of oxygen. Hydrogen peroxide---the product of this dismutation reaction--is mainly detoxified by catalase (C) (EC.I.ll.I.6.) and peroxidase (EC.I.11.1.7.) (Fridovich 1975; Matkovics et al., 1977). Cell damage caused by the activated metabolites of oxygen, may be also responsible for unascertained properties of cancer cells and certain biochemical differences between malignant and normal tissues (Oberley et al., 1980). Apparantly, there are differences in SOD activity in normal and cancer cells (Dionisi et al., 1975; Sahu et al., 1977). These differences are shown usually, but not always, as lower cytosolic Cu-Zn S O D activity in neoplastic material (Oberley et al., 1978). Similar changes of mitochondrial Mn S O D level are also observed (Van Balgooy & Roberts, 1979). However up to date the comparisons are based on a limited number of studies which disregard the peroxide catabolic enzymes other than SOD. We present results of preliminary studies on the association between S O D and catalase activities in hamster transplantable tumors and in normal and regenerating liver tissues. MATERIALS AND METHODS
Tumors investigated were: (i) The Kirkman-Robbins hamster hepatoma (obtained from the Department of Pathological Anatomy of Medical Academy in Wroclaw, where it was originally brought from the Chester Beatty Research Institute in London), (ii) The tumor induced by SV 40 virus (purchased from Institute of Oncology in Gliwice where it was originated), (iii) The amelanotic melanoma type Ab (obtained from Dr J. Bomirski from Medical Academy in Gdafisk). For cell line continuation a suspension of mechanically dispersed neoplastic cells was injected subcutaneously in the axilla of 2-month old syrian hamsters. For enzymes
RESULTS
The Kirkman-Robbins hamster hepatoma cells were highly dedifferentiated, readily transplantable and fast-growing. Activities of S O D and C were measured in tumor tissue and compared with activities in homologous normal tissue-liver, both quiescent and regenerating. Table 1 shows the values of S O D Table 1. SOD and catalase activities in hamster normal liver, regenerating liver and Kirkman-Robbins hepatoma Tissue Normal liver Regenerating liver Kirkman-Robbins Hepatoma
* Author to whom correspondence should be addressed.
SOD activity U/rag
Catalase activity BU- 10- 3/mg
58.5 + 3.5 49.7 _ 3.1 22.5 + 2.1
60.2 _ 4.7 52.1 _ 3.9 39.3 + 2.6
Units/mg protein, n = 6. 819
820
A BARTKOWIAKand J. BARTKOWIAK
Table 2. SOD and catalase activities in the hamster tumor induced by SV 40 virus and in the hamster amelanotic melanoma Tissue Tumor induced by SV 40 virus Amelanotic melanoma
SOD activity Catalase activity U/mg BU. 10- a/mg 19.7 +_ 2.2 24.5 _+ 2.6
16.5 _+ 1.9 38.2 _+ 3.0
n=6. and C activities. The results indicate that the SOD level in Kirkman-Robbins hepatoma is significantly lower than in normal hamster liver. The SOD activity in regenerating liver, where cells also proliferate rapidly, is comparable to that in quiescent liver, The comparison of catalase activities in all tissue show the same tendency as demonstrated for superoxide dismutase. In the case of amelanotic melanoma the transplantability and the rate of growth were comparable with features of Kirkman-Robbins hepatoma. Tumor induced by SV 40 virus was an example of a slowly growing tissue. Table 2 presents the results of measurement of SOD and catalase levels in the hamster tumors. Unfortunately, we have not been able to determine the activities of enzymes in homologous normal hamster tissues, because of their inaccessibility. The activities of S O D and C in both cancer tissues were similar to those observed in hepatoma cells but lower than in normal liver cells. DISCUSSION Our results suggest that there is a characteristic difference between hamster cancer cells and normal tissue, shown as a significant decrease in the activities of both S O D and C in neoplastic material. In the case of SOD our results confirmed previous findings from other laboratories, mentioned in Introduction. But our experiment also indicated that catalase activity-second enzyme involved in peroxide catabolism pathway--was closely correlated with alternations of S O D activity in studied tissues. Thus activities of described peroxide catabolic enzymes (SOD and C) are probably somehow linked functionally, but the level of that relation is unknown. There are not data now whether our observation express a general feature or not. Similar investigations on human cancer tissues
are required because they can be very useful, especially for a therapy. Radiotherapy and certain chemotherapeutic treatment of cancer depend on the action of free oxygen radicals, generated in cells by radiation or special chemical reagents (e.g. bleomycine; Sausvill et al., 1978). Efficacy of such treatment should be greater where tumors contain lower levels of S O D and catalase than in normal cells, especially when contiguous with the neoplasm. Moreover, such normal cells in the case of radiotherapy would be more resistant to the damaging action of superoxide radical (O2), since they possess greater activity of catabolic, defensive enzymes. REFERENCES
BEERS R. F. & SIZER I. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133-140. DIONISI D., GALEOTTIT., TERRANOVAT. & Azzt A. (1975) Superoxide radicals and hydrogen peroxide formation in mitochondria from normal and neoplastic tissue. Biochim. biophys. Acta 403, 292-300. FRIDOVlCH I. (1975) Superoxide dismutase. Ann. Rev. Biochem. 44, 147 159. MATKOVICS B., NOVAK R., HOANG DUE HANH SZABO L., VARGAS. I. & ZALE~NAG. (1977) A comparative study of some more important experimental animal peroxide metabolism enzymes. Comp. Biochem. Physiol. 56B, 31-34. MISRA H. P. & FRIDOVICH 1. (1972) The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175. LOWRY O. H., ROSENBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. OBERLEYL. W., BIZE I. B., SAHU S. K., LEUTHAUSERS. W. & GRUBERH. E. (1978) Superoxide dismutase activity of normal murine liver, regenerating liver and H6 hepatoma. d. natn. Cancer Inst. 61,375-379. OBERLEYL. W., OBERLEYT. D. (~ BUETTNERG. R. (1980) Cell differentiation, aging and cancer: the possible roles of superoxide and superoxide dismutases. Med. Hypoth. 6, 249-268. SAHU S. K., OBERLEYL. W., STEVENSR. H. ~z. RILEY E. F. (1977) Superoxide dismutase activity of Ehrlich ascites tumor cells. J. Natn. Cancer Inst. 58, 1125-1128. SAUSVILLE. A., PEISACHJ. & HORWITZS. B. (1978) Effect ot chelating agents and metal ions on the degradation oI DNA by bleomycin. Biochemistry 17, 2740-2745. VAN BALGt~V J. N. A. & ROBERTS E. (1979) Superoxide dismutase in normal and malignant tissues in different species. Comp. Biochem. Physiol. 62B, 263-268.
0305-0491/81/120819-02502.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SUPEROXIDE DISMUTASE A N D CATALASE ACTIVITIES IN NORMAL A N D CANCEROUS TISSUES A. BARTKOWIAK1 and J. BARTKOWIAK2. ~Department of Biophysics, University of L6d~ and 2Department of Oncology, Medical Academy, 93-509 L6d~, Gagarina 4, Poland
(Received 11 May 1981) Abstract--Superoxide dismutase and catalase activities in hamster transplantable tumors were distinctly lower than in normal hamster liver, both quiescent and regenerating. activity assays tumor material was collected on the 8th day after transplantation, when tumor weighted approx. 3-5 g. For transplantation of tumor induced by SV 40 virus the neoplastic material was not dispersed and the enzymatic measurement was performed 4 weeks after operation. Partial hepatectomy was performed on normal hamsters under light ether anesthesia and 30--40~o of the liver was removed. The hamsters were killed at 12 hr after surgery and regenerated parts of liver were only collected. Livers of sham-operated animals were used as reference tissue. For SOD activity determination 10~ tissue homogenates in 0.05 M K2HPO4 buffer, pH 7.8, were prepared, centrifuged at 8000 @for 15 min, and appropriate aliquots of the supernatant were used for enzyme assay, by the method of Misra & Fridovich (1972). One unit of SOD activity was defined as the amount of the enzyme required for 50~ inhibition of the oxidation of adrenaline to adrenochrome, during 1.0 min. For catalase activity determination, cytosolic fraction was prepared in 0.01 M phosphate buffer pH 7.0, by the same technique as above. The changes of H202 level were measured according to Beers and Sizer (1952). The catalase activity was expressed in Bergmeyer units (BU) where one BU was defined as the amount of C which can decompose 1.0 g hydrogen peroxide in 1.0 rain. The activities of both enzymes were calculated per 1.0mg of total protein in the homogenates. Protein concentration was estimated by the method of Lowry (1951). All reported here data are the mean values of 6 experiments (3 parallel enzyme determinations in each case).
INTRODUCTION
During recent years the role of peroxide catabolic enzymes of various animal tissue have been studied. Substantial evidence has been provided that these enzymes are necessary for the survival of all oxygenmetabolizing cells, because the oxygen-derived radicals have the potential to key subcellular structures. Superoxide dismutase (SOD) (EC.I.15.1.1.) is a scavenger of superoxide anions which are generated during the univalent reduction of oxygen. Hydrogen peroxide---the product of this dismutation reaction--is mainly detoxified by catalase (C) (EC.I.ll.I.6.) and peroxidase (EC.I.11.1.7.) (Fridovich 1975; Matkovics et al., 1977). Cell damage caused by the activated metabolites of oxygen, may be also responsible for unascertained properties of cancer cells and certain biochemical differences between malignant and normal tissues (Oberley et al., 1980). Apparantly, there are differences in SOD activity in normal and cancer cells (Dionisi et al., 1975; Sahu et al., 1977). These differences are shown usually, but not always, as lower cytosolic Cu-Zn S O D activity in neoplastic material (Oberley et al., 1978). Similar changes of mitochondrial Mn S O D level are also observed (Van Balgooy & Roberts, 1979). However up to date the comparisons are based on a limited number of studies which disregard the peroxide catabolic enzymes other than SOD. We present results of preliminary studies on the association between S O D and catalase activities in hamster transplantable tumors and in normal and regenerating liver tissues. MATERIALS AND METHODS
Tumors investigated were: (i) The Kirkman-Robbins hamster hepatoma (obtained from the Department of Pathological Anatomy of Medical Academy in Wroclaw, where it was originally brought from the Chester Beatty Research Institute in London), (ii) The tumor induced by SV 40 virus (purchased from Institute of Oncology in Gliwice where it was originated), (iii) The amelanotic melanoma type Ab (obtained from Dr J. Bomirski from Medical Academy in Gdafisk). For cell line continuation a suspension of mechanically dispersed neoplastic cells was injected subcutaneously in the axilla of 2-month old syrian hamsters. For enzymes
RESULTS
The Kirkman-Robbins hamster hepatoma cells were highly dedifferentiated, readily transplantable and fast-growing. Activities of S O D and C were measured in tumor tissue and compared with activities in homologous normal tissue-liver, both quiescent and regenerating. Table 1 shows the values of S O D Table 1. SOD and catalase activities in hamster normal liver, regenerating liver and Kirkman-Robbins hepatoma Tissue Normal liver Regenerating liver Kirkman-Robbins Hepatoma
* Author to whom correspondence should be addressed.
SOD activity U/rag
Catalase activity BU- 10- 3/mg
58.5 + 3.5 49.7 _ 3.1 22.5 + 2.1
60.2 _ 4.7 52.1 _ 3.9 39.3 + 2.6
Units/mg protein, n = 6. 819
820
A BARTKOWIAKand J. BARTKOWIAK
Table 2. SOD and catalase activities in the hamster tumor induced by SV 40 virus and in the hamster amelanotic melanoma Tissue Tumor induced by SV 40 virus Amelanotic melanoma
SOD activity Catalase activity U/mg BU. 10- a/mg 19.7 +_ 2.2 24.5 _+ 2.6
16.5 _+ 1.9 38.2 _+ 3.0
n=6. and C activities. The results indicate that the SOD level in Kirkman-Robbins hepatoma is significantly lower than in normal hamster liver. The SOD activity in regenerating liver, where cells also proliferate rapidly, is comparable to that in quiescent liver, The comparison of catalase activities in all tissue show the same tendency as demonstrated for superoxide dismutase. In the case of amelanotic melanoma the transplantability and the rate of growth were comparable with features of Kirkman-Robbins hepatoma. Tumor induced by SV 40 virus was an example of a slowly growing tissue. Table 2 presents the results of measurement of SOD and catalase levels in the hamster tumors. Unfortunately, we have not been able to determine the activities of enzymes in homologous normal hamster tissues, because of their inaccessibility. The activities of S O D and C in both cancer tissues were similar to those observed in hepatoma cells but lower than in normal liver cells. DISCUSSION Our results suggest that there is a characteristic difference between hamster cancer cells and normal tissue, shown as a significant decrease in the activities of both S O D and C in neoplastic material. In the case of SOD our results confirmed previous findings from other laboratories, mentioned in Introduction. But our experiment also indicated that catalase activity-second enzyme involved in peroxide catabolism pathway--was closely correlated with alternations of S O D activity in studied tissues. Thus activities of described peroxide catabolic enzymes (SOD and C) are probably somehow linked functionally, but the level of that relation is unknown. There are not data now whether our observation express a general feature or not. Similar investigations on human cancer tissues
are required because they can be very useful, especially for a therapy. Radiotherapy and certain chemotherapeutic treatment of cancer depend on the action of free oxygen radicals, generated in cells by radiation or special chemical reagents (e.g. bleomycine; Sausvill et al., 1978). Efficacy of such treatment should be greater where tumors contain lower levels of S O D and catalase than in normal cells, especially when contiguous with the neoplasm. Moreover, such normal cells in the case of radiotherapy would be more resistant to the damaging action of superoxide radical (O2), since they possess greater activity of catabolic, defensive enzymes. REFERENCES
BEERS R. F. & SIZER I. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133-140. DIONISI D., GALEOTTIT., TERRANOVAT. & Azzt A. (1975) Superoxide radicals and hydrogen peroxide formation in mitochondria from normal and neoplastic tissue. Biochim. biophys. Acta 403, 292-300. FRIDOVlCH I. (1975) Superoxide dismutase. Ann. Rev. Biochem. 44, 147 159. MATKOVICS B., NOVAK R., HOANG DUE HANH SZABO L., VARGAS. I. & ZALE~NAG. (1977) A comparative study of some more important experimental animal peroxide metabolism enzymes. Comp. Biochem. Physiol. 56B, 31-34. MISRA H. P. & FRIDOVICH 1. (1972) The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175. LOWRY O. H., ROSENBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. OBERLEYL. W., BIZE I. B., SAHU S. K., LEUTHAUSERS. W. & GRUBERH. E. (1978) Superoxide dismutase activity of normal murine liver, regenerating liver and H6 hepatoma. d. natn. Cancer Inst. 61,375-379. OBERLEYL. W., OBERLEYT. D. (~ BUETTNERG. R. (1980) Cell differentiation, aging and cancer: the possible roles of superoxide and superoxide dismutases. Med. Hypoth. 6, 249-268. SAHU S. K., OBERLEYL. W., STEVENSR. H. ~z. RILEY E. F. (1977) Superoxide dismutase activity of Ehrlich ascites tumor cells. J. Natn. Cancer Inst. 58, 1125-1128. SAUSVILLE. A., PEISACHJ. & HORWITZS. B. (1978) Effect ot chelating agents and metal ions on the degradation oI DNA by bleomycin. Biochemistry 17, 2740-2745. VAN BALGt~V J. N. A. & ROBERTS E. (1979) Superoxide dismutase in normal and malignant tissues in different species. Comp. Biochem. Physiol. 62B, 263-268.