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 37Â° 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.

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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 4Â°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 25Â°except contains the phenylhydrazone) were collected. Deionized water for KCN, which was kept at 0Â°.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 37Â°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 37Â°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 37Â° glycolate. One ml of reaction mixture containing various as described previously, and a molar extinction coefficient for amounts of homogenate was incubated at 37Â°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 0Â°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. The KCN stock solution (10 mM) was neutral was measured at 405 nm. ized with HCI and kept at 0Â°.The reaction was followed by the Urate Oxidase. The enzyme was assayed according to the change in absorbance at 290 nm at 37Â°,and a molar extinction method of MÃ¼llerand Malier (23). The reaction mixture con coefficient for uric acid of 1.21 x 104 was used to calculate tained 50 rriM glycine:NaOH buffer, pH 9.6; Triton X-100, 0.1 % enzyme activity (15). by volume; 1.0 mM EDTA; and 0.125 mM sodium urate. The Protein Determination. The method of Lowry ef al. (18) was enzyme activity was measured at 37Â°following the change in used. absorbance at 290 nm. A molar extinction coefficient for uric Statistical Method. Statistical analysis was performed by acid of 1.21 x 10" (15) was used to calculate enzyme activity. Student's f test. All values refer to mean Â±S.D. Results are o-Amino Acid Oxidase. The assay was modified from the from 3 to 4 experiments. method of Baudhuin ef al. (1). The reaction mixture contained (final concentrations) 20 HIM sodium pyrophosphate, pH 8.6; RESULTS 0.025 mM flavin adenine dinucleotide; 0.2% (v/v) Triton X- Mitochondria! Enzymes. The specific activity of cytochrome 100; and 50 mM o-alanine. One ml of the reaction mixture oxidase in the homogenate of NL was in the same range as containing various amounts of homogenate was incubated at that of TL (Table 1); the ratio of the former to the latter was 37Â°for 30 min. The reaction was stopped by adding 1.0 ml of 1.01 Â± 0.07. However, the specific activity of cytochrome 2 M sodium acetate at pH 5.0 and 1.0 ml of 0.1 % dinitrophen- oxidase in tumor homogenate (HC-252) was almost 3-fold

Table 1 Activity of mitochondrial and peroxisomal enzymes in homogenates from NL, TL. and tumor HC-252 Activity of the various enzymes was assayed as described in "Materials and Methods.'

(nmol/min/mgEnzymeCytochrome Specific activity

HC-25222.25.0 oxidase (3)Â° 8.0e Â±0.7eÂ± Succinate dehydrogenase (4) 67.8 Â± 6.2 71.5 22.8 ~0C Monoamine oxidase (4) 6.29 Â± 0.92 5.31 0.75 25.4e 2.82eÂ± CatalaseÃx 10 3) (4) 132.8 Â± 10.2 52.3 Â±Â±protein)Tumor19.58 0.08e Urate oxidase (4) 22.7 Â±Â±18.4Â°1.5 22.0 1.5 0.03 0.3d Â±0.35e D-Amino acid oxidase (3) 7.3 1.0 5.6 0.15 Â±0.38e L-a-Hydroxy acid oxidase (3)NL216.7 12.7Â± 1.3214.8 12.4TLÂ±Â± 0.377.3 1.12Â± Numbers in parentheses, number of experiments. 6 Mean Â±S.D. c Statistically different from that of NL (p < 0.001). d Statistically different from that of NL (p < 0.05).

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Oxidoreductases in Liver and Hepatoma lower than that found in homogenates of NL and TL (Table 1). dase or by the possibility that the rate of reduction was faster Similar results were obtained with succinate dehydrogenase, than the rate of oxidation of the substrate in the tumor homog another enzyme localized in the mitochondrial inner membrane; enate. whereas specific activities in homogenates of NL and TL were Peroxisomal Enzymes. Four peroxisomal enzymes were in the same range, activity in homogenates of HC-252 was 3- measured; 3 enzymes utilized oxygen, whereas the fourth, fold lower (Table 1). catalase, catalyzes the decomposition of hydrogen peroxide. The specific activity of monoamine oxidase in NL was slightly The specific activity of catalase in homogenates of NL was but not significantly higher than that in TL. Monoamine oxidase about 6.8-fold higher than that in tumor homogenates (Table activity in HC-252, however, was undetectable (Table 1). With 1). The presence of the tumor in ACI rats showed a depressive liver homogenates, as little as 0.05 mg of protein was sufficient effect on the catalase specific activity in TL (Table 1). to give measurable changes in absorbance at 250 nm. With The specific activity of o-amino acid oxidase in homogenates HC-252, no activity was detected even at protein concentra of NL was slightly higher than that in homogenates of TL (Table tions as high as 4.8 mg. The undetectable activity of mono 1). However, this activity was undetectable in the tumor ho amine oxidase in HC-252 could be due to the presence of an mogenate even when 6.46 mg of HC-252 protein per sample inhibitor for the enzyme or the possibility that the rate of were used (usually 0.1 mg of protein of normal liver homoge reduction of substrate is faster than the rate of oxidation of the nate was sufficient for the enzyme assay). The specific activity substrate. These possibilities would require monoamine oxi of L-a-hydroxy acid oxidase in homogenates of NL was com dase activity in NL and in TL to be inhibited in the presence of parable to that in homogenates of TL (Table 1). These activities, HC-252. Mixing experiments were therefore carried out. As however, were 12-fold higher than that found in the tumor shown in Table 2, the sum of (NL) and (TL), the respective rate homogenate (Table 1). of the sample that contained either homogenate of NL or TL, The specific activity of urate oxidase in NL homogenates was almost the same as (NL + TL), the rate of the sample that was comparable to that in homogenates from TL (Table 1). contained both homogenates with the same protein concentra However, urate oxidase activity in homogenates of HC-252 tions. The mean value of the ratio (NL) + (TL)/(NL + TL) was was undetectable even when 2.6 mg of HC-252 homogenate 1.083 Â±0.184. Therefore, monoamine oxidase activity in NL protein per sample were used (usually 0.1 mg of protein of NL and TL can be considered independent of each other. The ratio homogenate was sufficient for the enzyme assay) (Table 1). (NL) + (HC-252)/(NL + HC-252) and the ratio (TL) + (HC- Mixing experiments similar to those described for monoamine 252)/(TL + HC-252) were all about 1.0 when homogenates of oxidase showed that no inhibitor of urate oxidase appeared to NL or of TL were mixed with that of HC-252 (Table 2). Thus, be present in HC-252, nor was the rate of reduction faster than the lack of monoamine oxidase activity in HC-252 cannot be the rate of oxidation of the substrate in the tumor homogenate explained by the presence of an inhibitor of monoamine oxi (Table 2).

Table 2 Rate of monoamine oxidase reaction and urate oxidase reaction in mixture of homogenates of NL. TL. and tumor HC-252 All the numbers in the table refer to rate of the reaction in nmol/min except for the numbers in Column 4. (NL) indicates the rate of reaction of samples containing 0.22 and 0.55 mg (monoamine oxidase) or 0.12 and 0.24 mg (urate oxidase) of protein of homogenate of NL. (TL) refers to the rate of reaction of samples containing 0.20 and 0.51 mg (monoamine oxidase) or 0.10 and 0.20 mg (urate oxidase) of protein of homogenate of TL. (HC-252) refers to the rate of reaction of samples containing 0.35 and 1.38 mg (monoamine oxidase) or 0.14 and 0.35 mg (urate oxidase) of protein of homogenate from tumor HC-252. (NL + TL), (NL + HC-252), and (TL + HC-252) refer to the rate of reaction of samples containing the homogenate of both NL and TL. of NL and tumor, and of TL and tumor, respectively. The amount of protein per sample of the mixture was the same as that applied for the individual sample. oxidaseRate Monoamine oxidaseRate

(nmol/min)NL1.455of the reaction (nmol/min)NL3.072of the reaction rateExperimental rateExperimental +TL2.015 rate(NL) +TL5.380 rate(NL)

1.455 2.591 3.918 +(TL)(NL 3.072 4.183 7.053 +(TL)(NL 3.090 1.276 4.455 + TL) 5.106 2.423 8.092 + TL) 1.083 Â±0.184a(NL) 1.044 Â±0.043Â°(NL) 3.090NL1.4552.591HC-252-0.336â€”5.888NL 5.106NL3.0724.183HC-252-0.1448.928NL

+ + HC-2521.455 HC-2523.635

+(HC-252)(NL +(HC-252)(NL 1.455 0.896 0.537 3.072 0.288 3.231 + HC-252) + HC-252) 3.090 -0.336 3.090 1.023 Â±0.065a(TU 5.106 -0.144 5.827 0.921 Â±0.109a(TL) 3.090TL1.276-0.896HC-252-0.366-0.3662.127TL 5.106TL2.4230.288HC-252-0.1445.827TL

+ + HC-2520.784 HC-2522.625

+(HC-252)(TL +(HC-252)(TL 1.276 0.425 2.423 0.288 2.452 + HC-252) + HC-252) 2.591 2.396 1.096 Â±0.217aUrate 4.183 -0.144 4.298 0.962 Â±0.101a 2.591TL1.276-0.896NL 1.254Calculate 4.183TL2.423 0.288NL 4.788Calculate * Mean Â±S.D. The mean is not statistically significantly different from the true mean 1.0 (p > 0.05).

DECEMBER 1980 4679

XanthJne Oxidase. The specific activity of xanthine oxidase eration and extents of differentiation will be of interest to in the NL homogenate was comparable to that in the homoge- determine the generality of our observation. For example, urate nateof TL(1.142 Â±0.096 and 1.034 Â±0.200 nmol/min/mg oxidase and o-amino acid oxidase activities could not be de of protein, respectively; means of 3 independent experiments). tected in the rapidly growing HC and HW hepatomas but were Xanthine oxidase activity in the tumor homogenate was 0.238 detectable in slowly growing Morris hepatomas (36). The activ Â± 0.047, a value 4- to 5-fold lower than that found in the ity of monoamine oxidase in slowly and intermediately growing homogenate of NL and TL. Morris hepatomas is lower than in normal liver and undetectable in rapidly growing Morris hepatomas (26). Therefore, it is DISCUSSION possible that the differences in these oxidase activities may be observed between resting normal liver and rapidly proliferating The 3-fold lower activity of cytochrome oxidase and succi- tumors such as HC-252 but not between normal liver and some nate dehydrogenase in the tumor homogenate than in liver slowly growing hepatomas (26, 36). The implications of the homogenate is consistent with our previous observation that difference in activities of these oxidases in normal and neo- oxygen uptake by mitochondria (using ascorbate or Buccinate plastic cells would be better understood if the toxic products of as the substrate) in the homogenate of NL is about 3-fold these enzymes could be measured. Unfortunately, the in vivo higher than that of the tumor homogenate (6). However, similar concentration of the Superoxide radical and H2O2 produced by rates of respiration were observed between mitochondria iso these enzymes is difficult, if not impossible, to measure be lated from HC-252 and from NL (6). These results suggest that cause of the short half-life of these radicals; the presence of enzyme ratios and respiration in HC-252 and liver are qualita catalase, Superoxide dismutase, and other oxidases; as well tively similar but quantitatively altered. The activity of mono- as the numerous unspecific oxidative reactions (or damages) amine oxidase, an enzyme localized in the mitochondrial outer initiated by these radicals. As a whole, our data and those of membrane (29), could not be detected in HC-252. This appar others support the hypothesis that the activities of these oxi ent absence of monoamine oxidase in HC-252 suggests that dases and possibly the Superoxide radical and H2O2 produced there is a qualitative alteration, namely, a loss of an important by these enzymes (especially when activities are apparently component, in the mitochondrial outer membranes of this tumor absent) are much lower in rapidly growing tumors than in line. Further study of other enzymes of the mitochondrial outer resting liver. membrane on HC-252 would be of interest to extend this Additional studies will be required to determine if the lower suggestion. oxidative environment at the subcellular level contributes to the Since Cv and H2O2 are produced during mitochondrial rapid proliferation of certain tumors. It is of interest that free respiration (5, 17), the lower rate of mitochondrial respiration radicals, the major source of which is oxygen radicals (27), and the apparent absence of monoamine oxidase activity in have been implicated as a potential cause for aging (13), and HC-252 may suggest the possibility of a lower Cv- and H2O2- aging may be considered as a gradual loss of the ability of the generating capacity in the tumor. In a similar manner, the normal cell to proliferate (14). Since this loss is absent in apparent absence of the activities of urate oxidase and o-amino "immortal" neoplastic cells, the latter becomes a useful control acid oxidase as well as the lower L-a-hydroxy acid oxidase for study on the mechanism of aging at the cellular level (33). activity, all of which produce H2O2, may also be suggestive of The present study suggests that lower activities of oxidative a lower concentration of H2O2 produced in the tumor. Xanthine enzymes in tumor may generate a lower oxidative environment oxidase is an enzyme that produces Cv and H2O2 (21, 22). at the cellular level and may provide a more agreeable condition Moreover, hydroxyl radical production has been observed to for rapid cell proliferation of neoplastic cells. It also implies that occur from the interaction of O2~ and H2O2 produced during higher activities of oxidative enzymes in normal cells may the xanthine oxidase reaction (2). In vitamin E-deficient rats, a generate a higher oxidative environment at the cellular level higher level of lipid peroxidation in liver was associated with an and thus may lead to a decrease in the ability of the normal cell increased level of xanthine oxidase activity (20); whether this to proliferate, which is considered as aging at the cellular level. relationship also occurs in HC-252 is of considerable interest. In a previous study (6), the activity of various microsomal oxidative enzymes, e.g., NADPH oxidase, microsomal oxygen ACKNOWLEDGMENTS uptake, and microsomal drug metabolism, was also found to The authors are grateful for the invaluable advice and encouragement of Dr. be much lower in HC-252 than in the host liver, which is James F. Holland. consistent with the present findings. Of interest is the finding that the activity of another peroxi- REFERENCES somal enzyme, catalase, which catalyzes the decomposition of 1. Baudhuin. P.. Beaufay, H., Raham-Li. Y., Sellinger. O. Z.. Wattiaux. R.. H2O2 (8), was also lower in HC-252 than in liver. Moreover, Jacques, P., and de Duve. C. Tissue fractionation studies, 17. Intracellular catalase is an inducible enzyme; the level of catalase in vivo is distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase. D-amino acid oxidase and catalase in rat-liver tissue. controlled by the oxygen concentration in the environment (31, Biochem. J.. 92. 179-184, 1964. 37). In view of the decreased level of various oxidative en 2. Beauchamp, C., and Fridovich. I. A mechanism for the production of ethylene zymes, the lower level of catalase in HC-252 may be indicative from methional: the generation of the hydroxyl radical by xanthine oxidase. J. Biol. Chem., 245. 4641-4646, 1970. of a lower oxidative environment at the cellular level. The lower 3. Becker, F. F., Klein, K. M., Wolman, S. R., Asofsky. R., and Sell. S. catalase activity in the liver of tumor-bearing animals compared Characterization of primary hepatocellular carcinoma and initial transplant to normal animals has also been found in humans (24) and has generations. Cancer Res., 33. 3330-3338, 1973. 4. Becker, F. F.. Wolman, S. R.. Asofsky, R., and Sell, S. Sequential analysis been discussed elsewhere (12, 16). of transplantable hepatocellular carcinomas. Cancer Res., 35. 3021-3026, Additional studies utilizing tumors of varying rates of prolif 1975.

4680 CANCER RESEARCH VOL. 40

5. Boveris, A., and Chance, B. The mitochondrial generation of hydrogen 22. McCord, J. M., and Fridovich, l. Superoxide dismutase: an enzymic function peroxide. Biochem. J., 134: 707-716, 1973. for erythrocuprein (hemocuprein). J. Biol. Chem., 244: 6049-6055. 1969. 6. Cederbaum, A. I., Becker, F. F.. and Rubin, E. Ethanol metabolism by a 23. MÃ¼ller,M., and Moller. K. M. Urate oxidase and its association with peroxi- transplantable hepatocellular carcinoma: role of microsomes and mitochon somes in Acanthamoeba sp. Eur. J. Biochem., 9. 424-430. 1969. dria. J. Biol. Chem., 251: 5366-5374, 1976. 24. Ohnuma. T.. Maldia, G., and Holland, J. F. Hepatic catalase activity in 7. Cohen, G.. and Cederbaum, A. I. Chemical evidence for production of advanced human cancer. Cancer Res., 26. 1806-1818, 1966. hydroxyl radicals during microsomal electron transfer. Science (Wash. D. 25. Packer, L., and Fuehr, K. Low oxygen concentration extends the lifespan of C.), 204: 66-68, 1979. cultured human diploid cells. Nature (Lond.), 267: 423-425, 1977. 8. de Duve, C.. and Bauhduin, P. Peroxisomes (microbodies and related 26. Petersen. P. L., Greenawalt. J. W., Chan, T. L., and Morris, H. P. A particles). Physiol. Rev., 46: 323-357, 1966. comparison of some ultrastructural and biochemical properties of mitochon 9. de Lamirande, G.. Allard, C., and Cantero, A. Purine-metabolizing enzymes dria from Morris hepatomas 9618A, 7800. and 3924A. Cancer Res.. 30' in normal rat liver and Novikoff hepatomas. Cancer Res., 18: 952-958, 2620-2626, 1970. 1958. 27. Pryor, W. A. The role of free radical reactions in biological systems. In: W. 10. Fridovich, l. Superoxide dismutases. Annu. Rev. Biochem., 44: 147-159, A. Pryor (ed.). Free Radical in Biology, 1-49. New York: Academic Press, 1975. Inc., 1976. 11. Goldstein, S., Littlefield. J. W . and Soeldner, J. S. Diabetes mellitus and 28. Rafferty, K. A., Jr. Epithelial cells: growth in culture of normal and neoplastic aging. Diminished plating efficiency of cultured human fibroblasta. Proc. forms. Adv. Cancer Res.. 21: 249-272, 1975. Nati. Acad. Sei. U. S. A., 64: 155-161, 1969. 29. Schnaitman, C., Erwin, V. G., and Greenawalt, J. W. The submitochondrial 12. Hargreaves, A. B., and Deutsch, H. F. The in vitro inhibition of catalase by location of monoamine oxidase. J. Cell Biol., 32: 719-735. 1967. a tumor factor. Cancer Res., / ): 720-727, 1952. 30. Singer, T. P. Determination of the activity of Buccinate, NADH, choline, and 13. Harman, D. Prolongation of life: role of free radical reactions in aging. J. o-glycerophosphate dehydrogenases. Methods Biochem. Anal.. 22: 123- Am. Geriatr. Soc., 17: 721-735. 1969. 173, 1974. 14. Hayflick, L., and Moorhead, P. S. The serial cultivation of human diploid cell 31. Stevens, J. B., and Author, A. P. Oxygen-induced synthesis of Superoxide strains. Exp. Cell Res., 25. 585-621, 1961. dismutase and catalase in pulmonary macrophages of neonatal rats. Int. 15. Kalckar, H. M. Differential spectrophotometry of purine compounds by Acad. Pathol. Monogr.. 37: 470-478, 1977. means of specific enzymes. I. Determination of hydroxypurine compounds. 32. Sun, A. S., Aggarwal, B. B., and Packer. L. Enzyme levels of normal human J. Biol. Chem., 167: 429-443, 1947. cells: aging in culture. Arch. Biochem. Biophys., 170: 1-11, 1975. 16. Kampschmidt, R. F. Mechanism of liver catalase depression in tumor-bearing 33. Sun, A. S., Alvarez, L. J., Reinach, P. S., and Rubin, E. 5'-Nucleotidase animals: a review. Cancer Res., 25. 34-45, 1965. levels in normal and virus-transformed cells: implications for cellular aging 17. Loschen, G., Azzi, A., Richter, C., and Flohe, L. Superoxide radicals as in vitro. Lab. Invest.. 41: 1-4. 1979. precursors of mitochondrial hydrogen peroxide. FEBS Lett., 42: 68-72, 34. Sun, A. S., and Cederbaum, A. I. Oxidative enzymes in transplantable 1974. hepatocellular carcinoma HC-252, tumor-bearing rat liver and normal rat 18. Lowry, O. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. Protein liver: implication for cellular aging. J. Cell Biol., 83: 38a, 1979. measurement with the Folin phenol reagent. J. Biol. Chem., (93: 265-275. 35. Sun, A. S., and Poole. B. Fractionation of rat fibroblasts in a zonal rotor by 1951. means of viscosity barrier. Anal. Biochem., 68: 260-273, 1975. 19. Martin, G. M., Sprague, C. A., and Epstein, C. J. Replicative lifespan of 36. Wattiaux. R., Wattiaux-DE, S. C., van Dijck, J. M., and Morris, H. P. cultivated human cells. Lab. Invest., 23: 86-92, 1970. Subcellular particles in tumors. III. Peroxisomal enzymes in hepatomas HC 20. Masugi, R., and Nakamura, T. Effect of vitamin E deficiency on the level of and Morris hepatomas 7794A. 7794B. 5123A, and 7316A. Eur. J. Cancer, Superoxide dismutase, glutathione peroxidase, catalase and lipid peroxide 6:261-268. 1970. in rat liver. Int. J. Vitam. Nutr. Res., 46: 187-191, 1976. 37. Zimniak, P., Hartter, E., and Ruis. H. Biosynthesis of catalase T during 21. McCord, J. M., and Fridovich, I. The reduction of cytochrome c by milk oxygen adaptation of Saccharomyces cerevisiae. FEBS Lett.. 59: 300-304, xanthine oxidase. J. Biol. Chem., 243 5753-5760, 1968. 1975.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Oxidoreductase Activities in Normal Rat Liver, Tumor-bearing Rat Liver, and Hepatoma HC-252

Alexander S. Sun and Arthur I. Cederbaum

Cancer Res 1980;40:4677-4681.

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