Kinetic Properties of Alcohol Dehydrogenase in Hepatocellular Carcinoma and Normal Tissues of Rat

[CANCER RESEARCH 36, 2274-2277, July 1976] Kinetic Properties of Alcohol Dehydrogenase in Hepatocellular Carcinoma and Normal Tissues of Rat

Arthur I. Cederbaum 2 and Emanuel Rubin

Department of Pathology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029

SUMMARY catalytic properties, and substrate specificity (5). It appears that the liver and tumor enzymes are different isozymes (5). It was previously reported that the properties of alcohol Neoplastic transformation is often associated with the dehydrogenase of a rat hepatocellular carcinoma (Becker appearance of isozymes that are different from those of the H-252), a tumor of intermediate growth rate, were different parent tissues (for reviews, see Refs. 6, 15, and 16). In slow- from those of the liver enzyme, suggesting different iso- growing, highly differentiated hepatocellular carcinomas, zymes. To determine whether the degree of differentiation the isozyme pattern is nearly identical with that of the adult affected the isozyme of alcohol dehydrogenase, a fast- liver, whereas with decreased differentiation and increased growing, poorly differentiated tumor and one that is well growth rate, there is a loss of the liver isozymes, which are differentiated and of intermediate growth rate were studied. replaced by isozymes that are of low activity or absent in the Alcohol dehydrogenase from Morris hepatoma 7288ctc, a normal adult liver (15, 16). It was therefore of interest to fast-growing, poorly differentiated tumor, had properties determine whether the degree of differentiation of hepato- similar to those found with the Becker H-252 tumor, includ- cellular carcinoma correlates with the presence of this iso- ing a high Kr, for ethanol and acetaldehyde and the absence zyme of alcohol dehydrogenase. of substrate inhibition. By contrast, alcohol dehydrogenase The properties of alcohol dehydrogenase from rat stom- from the well-differentiated Morris hepatoma 5123C had ach of male ACI rats are identical with those of the enzyme properties similar to those of the liver enzyme. Thus, alco- in Becker H-252 hepatocellular carcinoma (5). Therefore, hol dehydrogenase is another example of an enzyme the we surveyed kinetic properties of alcohol dehydrogenase in isozyme composition of which changes with neoplastic de- several different tissues of ACI rats to determine whether differentiation. Further studies, including gel electrophore- the liver or tumor isozyme was present. sis, substrate specificity patterns, and interaction with antibodies to alcohol dehydrogenase, are required to determine the factors responsible for the biochemical defect that oc- MATERIALS AND METHODS curs at the molecular level during carcinogenesis and whether the alcohol dehydrogenase isozymes in the Becker Tumors. Morris hepatomas 7288ctc, a poorly differen- H-252 and Morris 7288ctc hepatomas are identical. A survey tiated rapid-growing transplanted hepatocellular carcinoma of several normal rat tissues revealed that only the stomach (3) and 5123C, a well-differentiated intermediate growing contains this unique isozyme of alcohol dehydrogenase. tumor (7, 12), were kindly provided by Dr. E. Farber. The tumors were transplanted into male Buffalo rats by s.c. or i.p. injection and were allowed to grow at these sites for 2 to INTRODUCTION 3 weeks (7288ctc) or 8 weeks (5123C). Since these tumors do not involve the norrnal liver, tumor and liver supernatant The properties of alcohol dehydrogenase in the 100,000 • fractions may be prepared from the same rat, the latter g supernatant fraction of Becker H-252 rat hepatocellular serving as a control for the former. carcinoma, a moderately well-differentiated tumor of inter- Preparations. Rats were decapitated and the livers were mediate growth rate induced in ACI rats (1,14) are different removed, care being taken to avoid any contamination with from those of the liver (5). Whereas liver alcohol dehydro- the i.p. tumor. Homogenates of liver and tumor (1:4) were genase has a Km for ethanol of about 0.3 mM and is subject prepared in 0.25 M sucrose-O.01 M Tris-HCI (pH 7.4)-0.001 M to substrate inhibition, the K~n of the tumor enzyme is about EDTA. The homogenates were centrifuged at 10,000 • g for 220 mM, and no substrate inhibition is observed (5). Alcohol 10 min. The resulting supernatant fluid was then centri- dehydrogenase activity in the Becker H-252 tumor is NAD § fuged at 100,000 • g for 60 rain. This supernatant fraction dependent, is inhibited by pyrazole, and is also inhibited by was used as the source of liver or tumor alcohol dehydro- an antibody to pure horse liver alcohol dehydrogenase (5). genase. Similar procedures were used to prepare superna- The liver and tumor enzymes differ in electrophoretic mobil- tant fractions of stomach, kidney, testes, intestine, spleen, ity, susceptibility to heat treatment, pH optimum, some and pancreas from male ACI rats. In the case of stomach, only the glandular portion of the stomach was removed. The This work was supported in part by USPHS Grant AA00287. mucosa was not separated from submucosa and muscle for 2 Recipient of Career Development Award 1 K02 AA00003-01 from the National Institute on Alcohol Abuse and Alcoholism. these experiments, hence the activity of alcohol dehydro- Received December 11, 1975; accepted March 17, 1976. genase in the mucosa of the stomach is obviously much

2274 CANCER RESEARCH VOL. 36

higher than the activity reported here for the whole stom- this tumor enzyme was apparent. The K,~ value for ethanol ach. Tissues were rinsed with 0.9% NaCI solution, sliced, for tumor 7288ctc was about 285 mM, a value comparable to and gently blotted before homogenization. that previously found for the Becker H-252 tumor enzyme Assays. All enzyme assays were performed in a cuvet [220 mM (5)]. Ethanol oxidation by the supernatant fraction with a final volume of 3.0 ml at 25 ~ Changes in absorbance of hepatoma 7288ctc was NAD § dependent and sensitive to were monitered at 340 nm with a Gilford recording spectro- pyrazole, an inhibitor of alcohol dehydrogenase. photometer. The reaction was initiated by addition of en- To support these results, alcohol dehydrogenase activity zyme (supernatant). In the oxidative direction, the assay was assayed in the reverse direction, using acetaldehyde system contained 75 mM sodium pyrophosphate buffer (pH plus NADH as substrates. It was previously observed with 9.6), 75 mM semicarbazide, 25 mM glycine, 1.5 mM NAD +, supernatant fractions of liver that the rate of reduction of and varying concentrations of ethanol. In the reductive di- acetaldehyde did not vary as the concentration of acetalde- rection, the assay system contained 100 mM sodium phos- hyde increased from 0.6 to 12 mM (5). By contrast, with the phate buffer (pH 7.0), 0.3 mM NADH, and varying concentra- Becker H-252 hepatoma under these conditions, the rate of tions of acetaldehyde or m-nitrobenzaldehyde. All rates reduction of acetaldehyde increased from 75 to 300% (5). were corrected for blanks, which contained all components Using livers of rats implanted with Morris hepatoma except the substrate. Protein was determined by the 7288ctc, the rate of reduction of acetaldehye also did not method of Lowry et ai. (10) using bovine serum albumin as vary as the concentration of acetaldehyde increased 20-fold the standard. The variability between experiments was less (Table 2). However, with Morris hepatoma 7288ctc, the rate than 20%. of reduction of acetaldehyde depended on the acetaldehyde concentration, the activity increasing at elevated levels of acetaldehyde (Table 2). No such increase in activity was RESULTS observed with Morris hepatoma 5123C. Alcohol Dehydrogenase Activity in Supernatant Frac- Alcohol Dehydrogenase Activity in Morris Hepatomas tions of Rat Tissues. In previous experiments with ACI rats, and Liver. Supernatant fractions of livers from rats im- the kinetic and electrophoretic data suggested that the al- planted with the 2 Morris hepatomas showed no increase in cohol dehydrogenases of stomach and Becker hepatoma H- alcohol dehydrogenase activity when the concentration of 252 were identical (5). Previous experiments showed that ethanol was raised from 5 to 500 mM (Table 1). Some de- striking differences in activity between liver and tumor or crease in activity was observed at higher ethanol concentra- stomach supernatants were obtained with m-nitrobenzalde- tions, indicating substrate inhibition of the enzyme (5). The hyde as the substrate, the rate with the tumor was 32 times K,, for ethanol for the liver enzyme was about 0.3 to 0.5 mM. greater than that with the liver (5). Therefore, both acetalde- In a similar manner, alcohol dehydrogenase activity of the hyde and m-nitrobenzaldehyde were used as substrates in a well-differentiated Morris hepatoma 5123C also did not in- survey of rat tissues. As shown in Table 3, unlike the stom- crease with an increasing concentration of ethanol (Table ach enzyme, the rate of acetaldehyde reduction by liver, 1). The K,, for ethanol for the tumor enzyme was about 0.6 kidney, intestine, and testes was not changed as the con- mM. However, in the less differentiated Morris hepatoma centration of acetaldehyde was raised. Similarly, whereas 7288ctc, alcohol dehydrogenase activity was dependent on m-nitrobenzaldehyde reductase activity in the stomach was the concentration of ethanol, with striking increases in the increased as the concentration of m-nitrobenzaldehyde was rates of ethanol oxidation at higher concentrations of elevated, no changes were observed in liver, kidney, testes, ethanol (Table 1). I~o evidence for substrate inhibition of or intestine (Table 3). m-Nitrobenzaldehyde reductase activity in the stomach was virtually abolished by pyrazole, indi- Table 1 cating the involvement of alcohol dehydrogenase. Similar Effect of ethanol concentration on alcohol dehydrogenase activity results were obtained when ethanol and NAD + were used as in 100,000 x g supernatant fractions of liver and substrates, although activity in some tissues was low and Morris hepatomas 7288ctc and 5123C difficult to assay. Under these conditions, alcohol dehydro- Results are means of 2 to 3 different preparations. genase activity was not detected in the spleen or pancreas. Activity (nmoles NADH formed/min/mg protein) Table 2 Ethanol con- 5123C 7288ctc Acetaldehyde reduction by supernatant fractions of liver and centration of Morris hepatoma 7288ctc (mM) Liver Tumor Liver Tumor Results are means of 3 different preparations. 0.5 31.4 8.6 1.5 36.2 9.7 Activity 5.1 38.4 16.7 41.0 12.1 (nmoles NADH oxidized/min/mg protein) 15.4 39.1 16.4 41.0 21.7 Acetaldehyde con- 25.5 41.7 31.4 centration (mM) Liver Tumor 51.0 34.7 15.8 40.4 55.5 0.6 125.4 48.2 102.0 31.6 15.4 39.2 103.7 1.2 130.2 96.5 154.0 36.6 147.4 1.8 130.2 120.6 255.0 27.4 13.7 34.3 200.2 3.0 142.3 168.8 357.0 31.2 229.0 6.0 135.0 337.6 510.0 25.4 13.0 30.2 227.3 12.0 149.5

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Table 3 Alcohol dehydrogenase activity in rat tissues Acetaldehyde Activity (/~.moles m-Nitrobenzalde- Activity (/~moles concentration NADH oxidized/ hyde concentra- NADHoxidized/ Tissue (mM) min/g tissue) tion (mM) min/g tissue) Liver 0.3 2.82 0.17 9.84 0.6 2.97 0.33 9.56 1.8 3.11 1.0 10.13 3.0 3.33 1.67 8.54 6.0 3.10 3.33 8.82

Stomach 0.3 0.087 0.17 5.06 0.6 0.131 0.33 10.43 1.8 0.167 1.0 16.20 3.0 0.203 3.33 24.30 6.0 0.289 6.67 30.09 2.0 0.434

Kidney 0.6 0.218 0.33 0.62 1.8 0.218 1.0 0.72 3.0 0.203 1.67 0.58 6.0 0.182 3.33 0.57 2.0 0.188

Testes 0.6 0.131 0.33 0.87 1.8 0.146 1.0 0.81 3.0 0.131 1.67 0.93 6.0 0.126 3.33 0.96 2.0 0.152

Intestine 0.6 0.146 0.33 0.48 1.8 0.146 1.0 0.41 3.0 0.131 1.67 0.58 6.0 0.123 3.33 0.54

Similar results were also obtained with male Sprague-Daw- differentiated tumors (Morris 7288ctc, Becker H-252) is as- ley rats; only the stomach appeared to contain this kineti- sociated with an isozyme that has different properties from cally unique isozyme of alcohol dehydrogenase. the normal adult liver enzyme, whereas the properties of alcohol dehydrogenase in the more highly differentiated tumor (5123C) resemble those of adult liver. A more detailed DISCUSSION study of the properties of alcohol dehydrogenase in Morris hepatoma 7288ctc, including gel electrophoresis, pH op- In dedifferentiation of the liver, normal liver isozymes are tima, substrate specificity patterns, kinetic analysis of pyra- often replaced by other isozymes not found to any great zole inhibition, stability to heat treatment, and possible extent in the normal adult liver (15, 16). For example, the cross-reactivity with antibodies to alcohol dehydrogenase is normal liver isozymes of hexokinase, glucokinase, aldolase, required to understand the factors responsible for the bio- pyruvate kinase, and glycogen phosphorylase are replaced chemical defect occurring at the molecular level in carcino- by isozymes normally not present to any significant extent genesis. Such studies may also indicate whether the alco- in rapidly growing, poorly differentiated tumors, whereas in hol dehydrogenase isozymes in Becker H-252 and Morris slow-growing, highly differentiated hepatocellular carcino- hepatoma 7288ctc are identical. A similar study with Becker mas, the isozyme pattern resembles that of adult liver (for H-252 transplantable hepatocellular carcinoma revealed review, see Refs. 15 and 16). that the tumor enzyme differed from the liver enzyme in its We previously observed that the properties of alcohol migration pattern on 2 different gels, pH optima, substrate dehydrogenase in Becker H-252 transplantable hepatocel- specificity pattern, stability to heat treatment, and Ki for lular carcinoma, a tumor of intermediate differentiation, are inhibition by pyrazole (5). conspicuously different from those of the liver enzyme (5). Alcohol dehydrogenase from the stomach has kinetic and Similar results were obtained with Becker H-242, a tumor electrophoretic properties similar to those in less differenJ similar to Becker H-252. Morris hepatomas 5123C and tiated tumors (2, 5). Other rat tissues (e.g., kidney, testes; 7288ctc are well characterized, the former being a well- and intestine) possess alcohol dehydrogenase activity with differentiated tumor (3, 7, 12) whereas the latter is less kinetic properties similar to those of the liver enzyme, rather differentiated (3). The data presented here demonstrate that than that of the stomach. In adults, the isozyme pattern in the isozyme pattern of alcohol dehydrogenase varies with lung or kidney is electrophoretically similar to that of liver the differentiation of the tumor, and this enzyme can now be (13), whereas the enzyme from the retina is reported to be included in the group undergoing isozyme alterations dur- electrophoretically distinct (8). Since the major function of ing neoplasia. The activity of alcohol dehydrogenase in less alcohol dehydrogenase in the retina may be the oxidation of

2276 CANCER RESEARCH VOL. 36

vitamin A alcohol to the aldehyde, an isozyme different from alcohol dehydrogenase activity or alcohol metabolism at that of the liver enzyme may have evolved. only 1 substrate concentration may be misleading. The physiological function of stomach alcohol dehydrogenase is not known. Although endogenous ethanol was ACKNOWLEDGMENTS found in all sections of the alimentary tract of the rat, We thank M. Imam for expert technical assistance. particularly in the stomach (9), the high Km of stomach alcohol dehydrogenase for ethanol probably precludes a REFERENCES significant function of the stomach enzyme in endogenous ethanol metabolism. The stomach enzyme also shows good 1. Becker, F. F., Klein, K. M., Wolman, S. R., Asofsky, R., and Sell, S. Characterization of Primary Hepatocellular Carcinomas and Initial Trans- activity with long-chain, unsaturated, and aromatic alco- plant Generations. Cancer Res., 33: 3330-3338, 1973. hols; these may represent substrates for the enzyme. It is 2. Bertolotti, R., and Weiss, M. C. Expression of Differential Functions in possible that the stomach enzyme may play a role in metab- Hepatoma Cell Hybrids. VI. Extinction and Re-expression of Liver Alco- hol Dehydrogenase. Biochimie, 54: 195-201, 1972. olizing exogenously administered alcohol. In rats given 10% 3. Blocl1-Frankenthal, L., Langan, J., Morris, H. P., and Weinhouse, S. ethanol in their drinking water, the concentration of ethanol Fatty Acid Oxidation and Ketogenesis in Transplantable Liver Tumors. Cancer Res., 25: 732-736, 1965. in the stomach after 1 hr is about 0.24 M (9). At this concen- 4. Carter, E. A., and Isselbacher, K. J. Metabolism of Ethanol to CO2 by tration, ethanol is a good substrate for stomach alcohol Stomach and Small Intestinal Slices. Proc. Soc. Exptl. Biol. Med., 138: dehydrogenase. Indeed, it has been reported that rat stom- 817-819, 1971. 5. Cederbaum, A. I., Pietruszko, R., Hempel, J., Becker, F. F., and Rubin, ach slices can oxidize ['4C]ethanol to '4CO,~ (4). The availa- E. Characterization of a Nonhepatic Alcohol Dehydrogenase from Rat bility of NAD ~, mitochondrial activity, and the capacity of Hepatocellular Carcinoma and Stomach. Arch. Biochem. Biophys., 171: substrate shuttles may all represent rate-limiting factors for 348-361, 1975 6. Criss, W. E. A Review of Isozymes in Cancer. Cancer Res., 31: 1523- the oxidation of ethanol in the stomach. 1542, 1971. The different Km values for ethanol for isozymes present in 7. Elwood, J. C., Lin, Y. C., Cristofalo, V. J., Weinhouse, S., and Morris, H. P. Glucose Utilization in Homogenates of the Morris hepatoma 5123 and stomach and liver explain why the reaction velocity of the Related Tumors. Cancer Res., 23: 906-913, 1963. stomach enzyme was reported to be twice that of the liver 8. Koen, A. L., and Shaw, C. R. Retinol and Alcohol Dehydrogenase in enzyme (11), since the assays were performed at an ethanol Retina and Liver. Biochim. Biophys. Acta, 128: 48-54, 1966. 9. Krebs, H. A., and Perkins, J. R. The Physiological Role of Alcohol concentration of 533 mM, a level that leads to substrate Dehydrogenase. Biochem. J., 118: 635-644, 1970. inhibition of the liver enzyme, but not of the stomach en- 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein zyme (5.) Ethanol oxidation by stomach slices was reported Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- 275, 1951. to be only 20% that of liver slices, whereas alcohol dehydro- 11. Mistilis, S. P., and Birchall, A. Induction of Alcohol Dehydrogenase in genase activity of the stomach was 3 times greater than that the Rat. Nature, 223: 199-200, 1969. 12. Ohe, K., Morris, H. P., and Weinhouse, S. /3-Hydroxybutyrate Dehydro- of the liver (4). In that study, the concentration of ethanol genase Activity in Liver and Liver Tumors. Cancer Res., 27: 1360-1371, was 533 mM. Thus the stomach enzyme showed high activ- 1967. ity, whereas the liver enzyme displayed substrate inhibition. 13. Smith, M., Hopkinson, D. A., and Harris, H. Developmental Changes and Polymorphism in Human Alcohol Dehydrogenase. Ann. Hum. Genet., 34: However, in experiments with tissue slices, the final con- 251-271,1971. centration of ethanol was 2.6 mM, a level well below the Kr, 14. Teebor, G. W., and Becker, F. F. Regression and Persistance of Hyper- plastic Hepatic Nodules Induced by N-2 Fluorenylacetamide and Their of the stomach enzyme for ethanol but greater than the Kr, Relationship to Hepatocarcinogenesis. Cancer Res., 31: 1-3, 1971. of the liver enzyme. Hence, rates of CO2 production from 15. Weinhouse, S. Glycolysis, Respiration and Anomalous Gene Expression ethanol would be much lower for stomach than those for in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture. Cancer Res., 32: 2007-2016, 1972. liver. The finding that different isozymes of alcohol dehy- 16. Weinhouse, S. Metabolism and Isozyme Alterations in Experimental drogenase exist in normal rat tissues suggests that assaying Hepatomas. Federation Proc., 32: 2162-2167, 1973.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Kinetic Properties of Alcohol Dehydrogenase in Hepatocellular Carcinoma and Normal Tissues of Rat

Arthur I. Cederbaum and Emanuel Rubin

Cancer Res 1976;36:2274-2277.

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