Copper- and Zinc-containing Superoxide Dismutase and Manganese- containing Superoxide Dismutase in Human Tissues and Human Malignant Tumors1

Superoxide dismutases might conceivably protect against both ionizing radiation and free radical-producing antibiotic antitumor drugs. Copperand zinc-containing Superoxide dismutase (CuZn Superoxide dismutase) and manganese-contain ing Superoxide dismutase (Mn Superoxide dismutase) were specifically assayed in human malignant tumors and for com parison in human tissues. The tumors possessed less CuZn Superoxide dismutase than did the more metabolically active tissues, but there was a large overlap between the tissue and the tumor levels. Mn Superoxide dismutase was found in all tumors, and the ratio between the activities of CuZn Superoxide dismutase and Mn Superoxide dismutase was not different from that of the normal tissues. Human tumors are thus different from tumors from other species which have been reported to be deficient or very low in Mn Superoxide dismutase. There was no obvious relation between sensitivity to ionizing radiation and content of the enzymes among the tumors and the tissues, nor did tumor types known to be responsive to radical-produc ing drugs possess less CuZn Superoxide dismutase or Mn Superoxide dismutase than other tumors. INTRODUCTION The Superoxide aniÃ3n radical Cv~ and its corresponding acid HO2 [pKa, 4.88 (3)] are formed directly and through secondary reactions by ionizing radiation. The presence of oxygen in a solution greatly increases the formation of the radical by secondary reactions. Oxygen is also known to in crease the biological effects of ionizing radiation. The mecha nisms of the oxygen enhancement are not fully known. There are indications that toxic effects of Cv~ or products derived from the radical might contribute to it. The disproportionation of the Superoxide radical, 2O2-~ + 2H* -> O2 + H2O2 (20), is very efficiently catalyzed by Superoxide dismutases. Superox ide dismutase (EC 1.15.1.1) has been shown to protect DNA (39), proteins (11), and cell membranes (29) against ionizing radiation. Myoblasts (21), Mycoplasma (28), and bacteria (22, 26) are protected by Superoxide dismutase in the medium. Mice given Superoxide dismutase parenterally show increased radioresistance (30, 31). Variation of Superoxide dismutase content in bacteria, induced by growth in media lacking the prosthetic metals of Superoxide dismutase (26), has indicated the importance of endogenous enzyme in the protection against the oxygen effects. Mammalian cells in culture exposed to diethyldithiocarbamate, which inhibits CuZn Superoxide dis mutase,2 show increased radiosensitivity (12, 41). Both anthracycline antibiotics and bleomycin appear to exert their toxic action on DNA by way of reactive free radicals derived from oxygen. Superoxide dismutase has been shown in vitro to protect DNA against bleomycin Fe2+ complex (13, 35, 36). The anthracyclines, daunomycin and doxorubicin, apparently take part easily in oxidation-reduction cycles pro ducing Superoxide radicals (1, 9, 10, 23), and Superoxide dismutase has been shown to protect DNA (14). In eukaryotes, 2 forms of Superoxide dismutase are generally found, one cytoplasmic and mitochondrial enzyme containing copper and zinc and one mitochondrial enzyme containing manganese (40). At least in primates, the Mn Superoxide dismutase is also found in the cytoplasm (19). Against the above background, a study of the activities of CuZn Superoxide dismutase and Mn Superoxide dismutase in human tumors seems highly motivated. The enzymes might conceivably be of importance, for both the radiation response and the response to the radical-producing antitumor drugs. The present paper reports specific analyses of CuZn superoxide dismutase and Mn Superoxide dismutase in 32 human tumors and for comparison human tissues. MATERIALS AND METHODS Tissues and Tumors. Human tissues from accident or sui cide victims without known physical diseases were obtained within 24 hr after death from the Department of Forensic Medicine. Macroscopically homogeneous pieces of tumor (0.5 g) were cut out of operation preparations within an hr after surgery. The tissues and tumors were kept at -90° before assay. They were homogenized in an Ultra-Turrax with 20 volumes of 10 mw potassium phosphate, pH 8.0, plus 30 rriM KCI. The homogenates were then sonicated with a Branson B30 under cooling with ice. After extraction for 30 min at 4°, the homogenates were centrifuged (20,000 x g for 15 min), and enzyme and protein analysis was performed on the supernatants. The procedure has been found to extract efficiently both CuZn Superoxide dismutase and Mn Superoxide dismu tase. Superoxide Dismutase Analysis. Superoxide dismutase was determined in terms of its ability to catalyze the disproportion ation of O2-~ in alkaline aqueous solution. The disproportiona tion was directly studied in a spectrophotometer, essentially as described before (16), the difference being that both CuZn Superoxide dismutase and Mn Superoxide dismutase were 1Supported in part by the Swedish Medical Research Council (O4761) and the Lions Research Foundation. Department of Oncology. University of Umeâ, Urnea. Sweden. Received September 12. 1980; accepted April 15. 1981. 2 The abbreviations used are: CuZn Superoxide dismutase, copperand zinccontaining Superoxide dismutase: Mn Superoxide dismutase. managanese-containing Superoxide dismutase. 2962 CANCER RESEARCH VOL. 41 on June 7, 2017. © 1981 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Superoxide Dismutase Tumors assayed at pH 9.50. One unit in the assay is defined as the activity that brings about a decay in O2~ concentration at a rate of 0.1 s~1 in 3 ml buffer. It corresponds to 8.3 ng human and to 4.1 ng bovine CuZn Superoxide dismutase and 65 ng bovine Mn Superoxide dismutase. The pure human enzyme has not been investigated with this assay, but its specific activity is probably similar to that of the bovine enzyme. The xanthine oxidase-cytochrome c assay for Superoxide dismutase works at physiological conditions, neutral pH and low O2-~concentra tion (20). When bovine and human enzymes are analyzed, one unit in the present assay corresponds to 0.024 units CuZn Superoxide dismutase and 0.24 units Mn Superoxide dismu tase, respectively, in the "xanthine oxidase" assay. The pres ent assay is thus about 10 times more sensitive for CuZn Superoxide dismutase activity than for Mn Superoxide dismu tase activity. Staining for Superoxide dismutase in agarose gel plates was performed with slight modifications of the methods of Beauchamp and Fridovich (2) and Bohnenkamp and Weser (4). Protein Analysis. For protein analysis, Coomassie Brilliant Blue G-250 was used (6). Human serum albumin was used for standardization. This sensitive convenient method was com pared with the more established technique of Lowry ef al. (15). Human tissue homogenates (pancreas, lymphatic node, mus cle, lung, heart, renal cortex, renal medulla, thyroid gland, liver, brain white matter, brain gray matter, adipose tissue, and spleen) were analyzed with both methods standardized the same way. The results were very similar, the ratio between the results of Lowry ef al. and the results of the present method being 1.12 ±0.14 (S.D) (range, 0.98 to 1.42). RESULTS Table 1 collects the results of CuZn Superoxide dismutase and Mn Superoxide dismutase analysis in human tissues. Table 2 presents the results of analysis on tumors. As seen, the activity of Mn Superoxide dismutase was in general almost as large as that of CuZn Superoxide dismutase if one takes into account the fact that the method used was 10 times less sensitive for Mn Superoxide dismutase. The enzymic activities were also studied after electrophoresis in agarose gel plates and subsequent staining for Superoxide dismutase. The results of the semiquantitative evaluation of the plates agreed very well with the results of the direct enzymic analysis pre sented in Tables 1 and 2. DISCUSSION The tumors in general appear to possess less CuZn superoxide dismutase than do tissues, at least when compared with metabolically active organs such as liver, kidney, and adrenal gland. However, there ¡sa large overlap between the levels of tissues and tumors. Only in a few cases can tissues and tumors derived from the tissue be compared. There is a large differ ence between kidney and the renal carcinoma and between testis and the testis embryonal carcinoma. On the other hand, lymphatic nodes and spleen do not possess more Superoxide dismutase than do corresponding tumors. When making this type of comparison, one must take into consideration the fact that the tumors in general are derived from one cell type, whereas the tissues are composed of several cell types. The Table 1 Superoxide dismutase in human tissues The tissues were analyzed as described in Materials and Methods.' Note that the activity figures for Mn Superoxide dismutase should be multiplied by 10 in order to make them comparable with the CuZn Superoxide dismutase figures on a physiological activity basis. CuZn Superoxide dis mutaseTissueLiverRenal