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Oxidoreductase Activities in Normal Rat Liver, Tumor-Bearing Rat Liver, and Hepatoma HC-2521

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Oxidoreductase Activities in Normal Rat Liver, Tumor-Bearing Rat Liver, and Hepatoma HC-2521 [CANCER RESEARCH 40, 4677-4681, December 1980] 0008-5472/80/0040-OOOOS02.00 Oxidoreductase Activities in Normal Rat Liver, Tumor-bearing Rat Liver, and Hepatoma HC-2521 Alexander S. Sun and Arthur I. Cederbaum Departments of Neoplastia Diseases [A. S. S.], and Biochemistry [A. I. C.], Mount Sinai School of Medicine City University of New York New York New York 10029 ABSTRACT mitochondria, microsomes, and other oxidases (2, 5, 7, 8, 10, 17, 27), the extent of oxygen toxicity in cells should depend Experiments were carried out to determine if the difference not only on the oxygen concentration in the environment but in rates of cell proliferation between normal and neoplastic also on the level of oxidative enzymes that convert the nontoxic cells may be related to altered levels of oxidative enzymes. oxygen to toxic oxygen radicals. If oxygen indeed contributes Assays were performed using homogenates from hepatocellu- to the control of cell proliferation in normal cells, differences in lar carcinoma HC-252, a rapidly growing and moderately well- these oxidase activities between rapidly proliferating "immor differentiated tumor; from normal liver; and from the liver of the tal" neoplastic cells and nonproliferating or slowly proliferating tumor-bearing ACI rat. Results of the mitochondrial enzymes normal cells may be expected. indicated that the activities of cytochrome oxidase and succi- The object of this report is to examine the activities of several nate dehydrogenase were 3-fold lower in tumor homogenates enzymes which are involved in pathways of oxygen utilization than in liver homogenates. Monoamine oxidase activity could in normal and neoplastic cells. The tumor used in this study not be detected in HC-252; mixing experiments indicated no was the transplantable hepatocellular carcinoma HC-252, a inhibitor was present in HC-252. Activities of the peroxisomal tumor well characterized (3, 4) and maintained in our laboratory enzymes, urate oxidase, o-amino acid oxidase, and L-a-hy- (6). The activity of some mitochondrial enzymes, peroxisomal droxy acid oxidase were either undetected in the tumor or were oxidases, and xanthine oxidase in homogenates of HC-252, of 12-fold lower than in liver homogenates. The activity of xan- TL,2 and of NL was evaluated. Portions of this work appeared thine oxidase, a cytoplasmic enzyme, was 5- to 6-fold lower in elsewhere in a preliminary form (34). the tumor. Catalase activity in the tumor was also lower than in liver; this may be indicative of a lower oxidative environment at MATERIALS AND METHODS the cellular level. These enzyme activities of the liver of tumor- bearing rats were in the same range as those of normal rat Origin of Tumor. Tumor HC-252 is moderately well differ liver, except that D-amino acid oxidase activity was slightly entiated, possesses approximately 44 chromosomes, and pro lower, and catalase activity was markedly lower and varied in duces no plasma proteins or a-fetoprotein (3, 4, 6). The tumor a wide range. was maintained in ACI rats by serial i.p. injection. In rats These results show an inverse correlation between the activ bearing these tumors, the liver is not infiltrated by tumor. Thus, ities of oxygen-utilizing enzymes and rates of proliferation of TL could be used to compare with tumor from the same rat and one tumor line and its control. The possible implications of with NL. Four weeks after i.p. inoculation, 5 to 15 g of tumor these results in neoplasia, cell proliferation, and cellular aging were harvested from each animal. are discussed. Preparation of Homogenate. Liver and tumor were homog enized (1 /10, w/v) in ice-cold 0.25 M sucrose; 0.001 M EDTA; and 0.1% ethanol with a Potter-Elvehjem homogenizer using a INTRODUCTION Teflon pestle speed of 1000 rpm. The homogenate was passed A major difference between normal and neoplastic cells is through 8 layers of cheesecloth and used for enzyme assays. that the former gradually lose their ability to proliferate after a Enzyme Assays. All the enzyme assays in this study were certain number of population doublings in vitro (11, 14, 19, carried out under substrate-saturating conditions and followed 28). However, the latter can proliferate continuously and in pseudo-zero order reaction kinetics; the rate of the reaction definitely and thus are considered as "immortal" cells. A was always a linear function of the protein concentration of the satisfactory explanation for the constraint on the ability of homogenate in the reaction mixture. normal cells to proliferate and the lack of this constraint on Cytochrome Oxidase. Activity was determined at 37° by neoplastic cells has not yet been found. evaluating the changes in absorbance at 550 nm of reduced Recently, Packer and Fuehr (25) reported that a higher cytochrome c by an assay described previously (32, 35). The oxygen concentration in the environment than that in the air reaction mixture contained 100 ITIMTris-HCI, pH 7.0; 1.0 mw decreased, but a lower oxygen concentration increased, the EDTA; 0.05 mw cytochrome c [reduced as described previ total number of accumulated population doublings of normal ously (32, 35)]; and Emasol (Kao Atlas, Tokyo, Japan), 0.1% human embryonic lung fibroblasts in vitro. These data may by volume. A molar extinction coefficient of 1.92 x 104, re suggest that oxygen can affect the ability of the cell to prolif duced minus oxidized, was used to calculate enzyme activity. erate. Since molecular oxygen itself is not toxic but becomes Succinate Dehydrogenase. The enzyme was assayed ac toxic when it is reduced to oxygen radicals by endogenous cording to the method of Singer (30). The reaction mixture ' Supported in part by USPHS Grants AA-O4413 and 2K02-AA00003. 2 The abbreviations used are: TL, liver of the tumor-bearing ACI rat; NL. normal Received April 24, 1980; accepted September 12, 1980. rat liver; DCIP, 2,6-dichlorophenolindophenol. DECEMBER 1980 4677 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. A. S. Sun and A. I. Cederbaum contained 50 mw phosphate buffer, pH 7.6; 1.0 rriM KCN, pH ylhydrazine in 2 N HCI. After this was mixed and allowed to 7.6; Triton X-100, 0.1% by volume; 0.2 mw DCIP; 1.0 mM stand for 5 min, 2.0 ml of water-saturated toluene were added phenazine methosulfate; and 20 HIM sodium succinate. The to the mixture. The mixture was centrifuged at 4°at a speed of reaction mixture was made from freshly prepared stock solu 2000 x g for 10 min, and 1.5 ml of the toluene phase (which tions of each of the above compounds incubated at 25°except contains the phenylhydrazone) were collected. Deionized water for KCN, which was kept at 0°.The mixture was kept in the (0.5 ml) was added to the toluene fraction, mixed, and centri dark after addition of DCIP and phenazine methosulfate and fuged, and 1.0 ml of the toluene phase was collected. Na2COa then incubated at 37°for 2 min before adding the homogenate. (2.0 ml, 1 M) was mixed with the 1.0-ml toluene fraction and The reduction of DCIP by the enzyme was measured at 37°by centrifuged, and 1.0 ml of the aqueous phase was collected following the change in absorbance at 600 nm with a Gilford and mixed with 1.0 ml of 2 N NaOH. The absorbance of the 2400 spectrophotometer. A molar oxidation-reduction extinc phenylhydrazone complex was measured at 440 nm. Pyruvic tion coefficient for DCIP of 1.91 x 10" was used to calculate acid of known concentrations was used to construct a standard enzyme activity (30). curve. Monoamine Oxidase. The enzyme was assayed according L-a-Hydroxy Acid Oxidase. The assay was modified from to the method of Schnaitman ef al. (29). The reaction mixture the method of Baudhuin ef al. (1 ). The reaction mixture con contained 50 mw phosphate buffer, pH 7.6; Triton X-100, tained (final concentrations) 20 mM sodium pyrophosphate 0.1 % by volume; and 2.5 mM benzylamine. The enzyme activity buffer, pH 8.0; Triton X-100, 0.2% (v/v); and 25 HIM sodium was measured by the change in absorbance at 250 nm at 37° glycolate. One ml of reaction mixture containing various as described previously, and a molar extinction coefficient for amounts of homogenate was incubated at 37°for 30 min. The benzylamine of 1.34 x 10" was used to calculate enzyme reaction was stopped and then continued as described in the activity (29). assay method of o-amino acid oxidase, except that glyoxylic Catatase. The activity was measured according to a method acid of known concentrations was used as the standard. modified from Baudhuin ef al. (1). The reaction mixture con Xanthine Oxidase. The enzyme assay was modified from tained 20 HIM imidazole buffer, pH 7.2; 0.1% bovine serum the method of de Lamirande ef al. (9). The reaction mixture albumin; and 7.06 mM H2O2. The reaction mixture (2.0 ml) and contained (final concentrations) 50 mM phosphate buffer, pH 0.1 ml of 2% Triton X-100 (v/v) were incubated with 0.1 ml of 7.6; hypoxanthine, 1.0 mM; 0.1% Triton X-100; and 0.1 mM of homogenate at 0°for 30 min. The reaction was stopped by the potassium cyanide. The reaction mixture was made immedi addition of 2.0 ml of 28 mw titanium oxysulfate in 2 N H2SO4. ately before adding the homogenate. The stock solution of The absorbance of the yellow titanium peroxysulfate complex hypoxanthine (10 mM) was made by dissolving hypoxanthine formed from the remaining H2O2 and the titanium oxysulfate in 50 mM NaOH.
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