[CANCER RESEARCH 30, 44-47, January 1970] Selected Enzymes of Pyrimidine Nucleotide Metabolism in Livers from Rats Fed a-Naphthylisothiocyanate1

Thomas W. Sneider, Edward L. Krawitt,2 and Van R. Potter

McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY 3- me

The effects of ingestion of a diet containing 0.1% by CDP1dCDPIt— UDP1dUDPIt-odUMP weight of the biliary hyperplastic agent a-naphlhylisolhio- cyanate on the activities of deoxycytidylate deaminase, Ihymidylate synthetase, thymidine kinase, cytidine kinase, and dCMP>ItCdR —»dTMPit rdTTP- »DNA-T thymine-degradative ability were studied in rat livers. In contrast to an earlier study on the effects of ingestion of the itïtRYTîtRU hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene, only »•»• deoxycytidylate deaminase activities were elevated during «^ ^* TIt a-naphthylisothiocyanate feeding. Considered in the light of '6UIB- •BAIB- previous histológica! and biochemical studies, the present DHT >C02 results bring into question the concept that 3'-methyl-4- Chart 1. Schematic representation of possible pathways of deoxy dimethylaminoazobenzene-induced liver tumors are derived thymidine triphosphate synthesis taken from an earlier paper from from bile duct epithelial cells. The data are discussed with this laboratory (15). The many feedback controls within and respect to alternative pathways of pyrimidine nucleotide affecting this scheme are too complex to be included in this chart or synthesis in liver cells at various stages of differentiation. discussed in the text. Abbreviations: CDP, UDP, dCDP, dUDP, and dTDP, the 5'-diphosphates of cytidine, undine, deoxycytidine, deoxyuridine, and deoxythymidine; dUMP, and 5-Me-dCMP, the INTRODUCTION 5'-monophosphates of deoxyuridine and 5-methvldeoxycytidine; dTTP, the 5 -triphosphate of deoxythymidine; DNA-T, thymine in Several lines of evidence suggest that among hepatomas of polymeric DNA; CdR and UdR, deoxycytidine and deoxyuridine; U the hepatocellular type there may be a high degree of and T, uracil and thymine; DHT, dihydro thy mine; BU1B and BAIB, diversity which is somehow related to the degree of /3-ureido- and (3-aminoisobutyric acids. dCMP deaminase catalyzes the differentiation of the normal liver cell in which the carcino deamination of dCMP to form dUMP; dTMP synthetase, the methyl- genic transformation occurred (10, 12, 15). It has been ation of dUMP to form dTMP; TdR kinase, the phosphorylation of suggested (9, 11, 15) that dCMP3 deaminase in particular, TdR to form dTMP. Thymine-catabolic activity is measured as the and possibly related enzymes of pyrimidine nucleotide ability to degrade thymine to CO2. metabolism, might be useful biochemical tools for unravel ling the relationship between hepatoma diversity and the week. Since the time course of changes in enzyme activity diversity in liver cell populations. A schematic representation correlated with histologically observed bile duct cell of the reactions catalyzed by these enzymes is shown in proliferation reported earlier by Price et al. (13), it was Chart 1. Earlier work of Pilot and Potter (9, 12) indicated concluded that dCMP deaminase alterations reflected changes that dCMP deaminase activity in livers from rats fed in cells other than hepatocytes. Although this conclusion was 3'-MDAB rose to high levels after the onset of dye feeding questioned by Emmelot et al. (3), who correctly pointed out and then fell to normal low levels of activity by the 6th that modulations in enzyme activity could theoretically occur in stem cells, bile duct cells, or hepatocytes, and by Reid (14), who could not demonstrate elevated dCMP deaminase activities in livers from 3'-MDAB-fed rats, recent 'This study was supported in part by American Cancer Society studies by Sneider and Potter (15) confirmed and extended Grant P-448 and by USPHS Grant T01-CA-5002 from the National the earlier work of Pilot and Potier. Harlman and Reid (4) Cancer Institute. 2USPHS Special Fellow (No. 5-F3-CA-35-234-02) from the National were also unable lo demonslrale alteralions in dCMP Cancer Institute. Present address: Department of Medicine, University deaminase activity in livers from rats fed a-naphthylisolhio- cyanale, a biliary hyperplaslic agenl (7, 8). This com- ofThe Vermont, abbreviations Burlington, used Vt. are: dCMP, deoxycytidine s'-monophos- municalion compares Ihe pattern of changes in the aclivities phate; 3 -MDAB, 3 -methyl-4-dimethylaminoazobenzene; ANIT, a-naphthylisothiocyanate; dTMP, deoxythymidine 5 -monophosphate; of certain enzymes of pyrimidine nucleotide melabolism in TdR, deoxythymidine; dCTP, deoxycytidine 5 -triphosphate. livers from rats fed ANIT with the unique patterns of changes seen during 3'-MDAB carcinogenesis and illustrales Received March 6, 1969; accepted May 7, 1969.

44 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1970 American Association for Cancer Research. Pyrimidine Synthesis in ANIT-fed Rats the importance of considering the role of shifting cell pyrimidine deoxyribonucleotides are shown in Chart 3. populations and alternative metabolic pathways in studies on Thymidine kinase, thymidylate synthetase, and thymine- hepatocarcinogenesis. degradative activities of livers from ANIT-fed rats showed no marked alterations (elevations or depressions) from control MATERIALS AND METHODS levels. Cytidine kinase activities tended toward a slow increase over the 5-week ANIT feeding period. When livers from ANIT-fed rats were homogenized in buffered medium Animals. Male rats weighing 150 to 200 g, obtained from Holtzman Rat Company, Madison, Wis., were housed in not containing dCTP, no elevations in dCMP deaminase individual cages in a temperature- and humidity-controlled activity in liver extracts were observed. If the homogenizing room in which lighting was regulated to provide 12 hr of medium contained 0.5 mM dCTP, pronounced elevations in dCMP deaminase activities were noted with ANIT feeding. darkness (9 a.m. to 9 p.m.) and 12 hr of light (9 p.m. to 9 The effect of 3'-MDAB feeding on the activities of these a.m.). Animals were fed from 9 a.m. to 5 p.m. but were allowed water ad libitum. The basal and 0.05% 3'-MDAB enzymes in rat livers has been noted previously (15) with the exception of cytidine kinase. The effects of feeding 0.05% diets were described in an earlier report (15). Diet containing 3'-MDAB on the activity of this enzyme in rat liver are AN1T was identical with the basal diet except that 10 g ANIT/10 kg diet were added in the cerulose component. shown in Chart 4. The elevations in activity of this enzyme closely paralleled the changes noted in both dCMP deaminase Upon receipt from the supplier, the rats were placed on and TdR kinase activities during 3'-MDAB feeding (15). basal diet until their average weight reached 200 to 225 g. At that time, the animals were randomly assigned to their respective experimental groups and feeding with ANIT- or 3'-MDAB-containing diets was begun. Three rats were killed at LIVER aCMP DEAMINASE (+dCTP! the onset of ANIT or 3'-MDAB feeding and at each weekly interval thereafter. The body weight changes and mortality of rats on ANIT diet are shown in Chart 2. Similar data for rats fed 0.05% 3'-MDAB can be found in an earlier paper from this laboratory (15).

ANIT"^^_i DIET +0.1% LIVER dCUP DEAMINASE (-ÃŒCTP) TMP SYNTHETASE

È 300 oÕ

—-"iBASAL S«" 200 ov zf 100 25 oS fe ^V* •i*• i iosoio n-•

THYMINE DEGRADATION ANIT BEGUN DAYS ON DIET

Chart 2. Body weight changes and mortality of rats fed diet

containing 0.1% ANIT. Circles along the abscissa, no. of rats that o x died on any given day (see right ordinate).

Preparation of Cell Extracts and Enzyme Assays. High speed supernatant fractions (150,000 X g for 1 hr) of liver homogenates were prepared as noted in an earlier paper (15). 012345 These fractions were assayed for dCMP deaminase, dTMP WEEKS ON ANIT WEEKS ON ANIT synthetase, TdR kinase, thymine-catabolic activity, and cytidine kinase activity as previously described (6, 15, 16). Chart 3. Alterations in activities of enzymes of pyrimidine nucleotide The requirement for dCTP in the homogenizing medium for biosynthesis in livers from rats fed 0.1% ANIT for 5 weeks. Note demonstration of dCMP deaminase activity has been described differing magnitudes for various ordinales. For dCMP deaminase assays, samples of liver tissue were either homogenized in the in more detail elsewhere (15). presence of 0.5 mM dCTP (top left graph) or homogenized minus dCTP but assayed in presence of equivalent amounts of dCTP (middle RESULTS left graph). AU assays were performed in duplicate. Each point represents the average value for 3 rats. Abbreviations: dUMP, The effects of ANIT feeding on théability of extracts of deoxyuridine 5 -monophosphate; T, thymidine; dCTP, deoxycytidine rat liver to catalyze several reactions undergone by 5 -triphosphate; TMP, thymidine 5 -monophosphate; CR, cytidine.

JANUARY 1970 45

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1970 American Association for Cancer Research. T. W. Sneider, E. L. Krawitt, and V. R. Potter

of bile duct cells, then the different patterns of enzyme alterations evoked by them might reflect the utilization of different pathways to thymidine triphosphate synthesis in the same cell type, the pathway used being dependent upon the agent inducing proliferation. This possibility cannot be ruled out on the basis of available data. A case can be made for the temporary increase in the activity of dCMP deami nase (via modulation or induction followed by decay) in bile duct cells and/or other hepatic cell types as Emmelot et al. (3) pointed out. The finding (15) that dCTP is a stabilizing factor which prevents the irreversible decay of dCMP deaminase in vitro may be an important clue as to how the enzyme could be modulated and made to disappear when no longer needed. Future studies would be greatly O l 2 l IO II facilitated by suitable methodology for the cytochemical ONOYE GROUPnGROUPm GROUPH WEEKS OFF DYE ON OYE localization of the enzyme in fetal, newborn, and precarcino- AGAIN genic livers. It would be of great interest in connection with Chart 4. Cytidine (CR) kinase in livers from rats fed diet containing the concept of blocked ontogeny (10) if certain hepatoma 0.05% S'-MDAB. All rats in this experiment were started on the cells retained the enzyme while morphologically similar cells dye-containing diet at Week 0. At Week 3, animals in Group II were showed only a transient increase in the enzyme. taken off the dye diet and fed basal diet for the next 2 weeks, after The different patterns of enzyme alterations in the livers of which time they were replaced on the 3'-MDAB diet. Group III ANIT- and 3'-MDAB-fed rat, might, however, reflect prolifera animals were fed basal diet after the 3rd week of dye diet. Rats in Group I were fed the 3'-MDAB diet over the entire course of the tion of 2 cell types which, although morphologically similar, differ in their cell origin. The observation by Price et al. (13) experiment. Each point represents the average value for 3 rats. that "bile duct cell" proliferation in livers from 3'-MDAB-feed rats led to occupation of 80% of the lobular area by DISCUSSION "bile duct cells" with no disruption of the cellular architec McLean and Rees (8) and Lopez and Mazzanti (7) reported ture of the lobule, coupled with the distinctly different marked bile duct proliferation in livers of rats after 5 to 6 histológica! alterations attendant upon ANIT feeding (7, 8), weeks of ingestion of diet containing 0.1% ANIT. Price et al. supports the viewpoint that at least 2 different cell types are (13) found that almost 80% of the lobular area in livers involved in the responses to 3'-MDAB and ANIT feeding. As from rats fed 0.06% 3'-MDAB for 35 days was occupied by discussed more fully in a recent paper by Sneider and Potter cells that morphologically resembled bile duct cells. The (15), the cellular proliferation noted in the initial stages of ingestion of either 3'-MDAB or ANIT was said to have had 3'-MDAB feeding may not be bile duct cell proliferation as no effect on dCMP deaminase activities in rat livers (14) suggested earlier (9) but might represent the dediffer- contrary to the earlier reports of Pitot and Potter (9, 12) entiation and proliferation of hepatic parenchymal cells who found elevations in the activity of this enzyme in livers and/or the proUferation of extant primitive or protostem from 3'-MDAB-fed rats. The observations of Pitot and Potter cells of the hepatic cell line. The "repopulation" of the liver were recently confirmed by Sneider and Potter (15) who lobule by parenchymal cells reported by Price et al. after 35 also found that 3'-MDAB feeding led to (a) increased liver to 40 days of 3'-MDAB feeding was notable for the relative TdR kinase activities that almost perfectly paralleled the absence of mitotic figures. If the initial cellular proliferation elevations of dCMP deaminase, (b) no alterations from the in livers from 3'-MDAB-fed rats resulted from dedifferenti- low adult level of dTMP synthetase activity, and (c) an ation and proliferation of parenchymal cells, the subsequent initial severe depression of hepatic thymine-degradative "amitotic" repopulation of the lobule by parenchymal cells ability which returned to high adult liver levels after the 4th might be due to redifferentiation of protostem cells of the week of continuous 3'-MDAB feeding. As shown in Chart 4, parenchymal cell line. Studies of the incorporation of various 3'-MDAB ingestion is accompanied by elevations in liver labeled pyrimidine deoxyribonucleoside precursors into the cytidine kinase activities that parallel the changes in dCMP DNA of different classes (2) of hepatic nuclei from 3'-MDAB-fed rats are in progress to test this hypothesis. The deaminase and TdR kinase activities. The effects of ANIT feeding on the activities of these same various stages of differentiation may be characterized by the enzymes in rat liver differ. There is almost no effect of utilization of alternative pathways for the synthesis of deoxythymidine 5'-triphosphate (15) or by possession of ANIT on the activities of dTMP synthetase, TdR kinase, cytidine kinase, and thymine-catabolic activities. However, isozymes with varying physicochemical characteristics on this dCMP deaminase activities became elevated after 7 days of pathway (1, 5, 15).4 The differing patterns of enzyme ANIT feeding and remained so over the 5-week experimental period. Although both ANIT and 3'-MDAB give rise to what is Further analyses of these possibilities should be made with newly developed techniques for the separation of different cell types within termed bile duct cell proliferation, the patterns of alterations given tissues during differentiation (personal communication, G. in the activities of enzymes associated with cell proliferation Miller and R. A. Phillips, University of Toronto, Separation of Cells are markedly different. If both agents lead to proliferation by Velocity Sedimentation).

46 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1970 American Association for Cancer Research. Pyrimidine Synthesis in ANIT-fed Rats activities of the pyrimidine deoxyribonucleotide pathway 5. Klemperer, H. G., and Haynes, G. R. Thymidine Kinase in Rat noted between livers from 3'-MDAB-fed rats and regenerating Liver during Development. Biochem. J., 108: 541-546, 1968. or neonatal rat liver (15) and established, highly differ 6. Krawitt, E. L., Betel, !.. and Potter, V. R. A Study of the entiated, slowly growing rat hepatomas (16) point to the Cytidine Kinase Pathway of Nucleotide Biosynthesis in Regen erating Rat Liver. Biochim. Biophys. Acta, 174: 763-765, 1969. importance, from the mechanistic and chemotherapeutic standpoints, of changes in cell populations in hepatocar- 7. Lopez, M., and Mazzanti, L. Experimental Investigations on a-Naphthylisothiocyanate as a Hypcrplastic Agent of the Biliary cinogenesis. Ducts in the Rat. J. Pathol. Bacteriol.. 69: 243-250, 1955. ADDENDUM 8. McLean, M. R., and Rees, K. R. Hyperplasia of Bile Duct Cells Induced by a-Naphthylisothiocyanate: Experimental Biliary Cirrhosis Free from Biliary Obstruction. J. Pathol. Bacteriol., 76: Relevant to the above discussion is an overlooked earlier paper on rats treated with 3'-MDAB. The authors propose that 175-188, 1958. "It would seem that the onset of a neoplastic process in the 9. Pilot, H. C., and Potter, V. R. An Enzyme Study of the Cellular Origin of the Dunning and the Novikoff Hepatomas in the Rat. adult hepatic cell results in the de novo synthesis, by a recall Biochim. Biophys. Acta, 40: 537-539, 1960. or 'derepression' mechanism, of a constituent which that organ 10. Potter, V. R. Recent Trends in Cancer Biochemistry: The synthesized during its fetal stage of development" (Ref. 18, p. Importance of Studies on Fetal Tissue. Can. Cancer Conf.. 8: 1995). In view of the technical difficulties caused by the 9-30, 1969. instability of dCMP deaminase, which may explain conflicting 11. Potter, V. R., Pilot, H. C., Ono, T., and Morris, H. P. The reports from different laboratories, it also seems important to Comparative Enzymology and Cell Origin of Ral Hepalomas. I. cite a very recent report on a technique in which ethylene Deoxycytidylate Deaminase and Thymine Degradation. Cancer Res., 20: 1255-1261, 1960. glycol is used to stabilize the enzyme from the moment of 12. Potter, V. R., and Watanabe, M. Some Biochemical Essentials of homogenization, in comparison with dCTP (17). Malignancy: The Challenge of Diversity. In: Proceedings of Inler- ACKNOWLEDGMENTS national Conference on Leukemia-Lymphoma, pp. 33-46. Phila delphia: Lea & Febiger, 1968. The excellent technical assistance of Mrs. Lynn Dean is gratefully 13. Price, J. M., Harman, J. W., Miller, E. C., and Miller, J. A. acknowledged. We also thank Drs. E. C. Miller and J. A. Miller for Progressive Microscopic Allerations in the Livers of Rats Fed the generously supplying the ANIT and 3 -MDAB diets. Hepatic Carcinogens 3 -Methyl-4-dimethylaminoazobenzene and 4(-Fluoro-4-dimethylaminoazobenzene. Cancer Res.,2/: 192-200, REFERENCES 1952. 14. Reid, E. Biochemical Approaches to Cancer, pp. 114-115. New 1. Bresnick, E., Thompson, U. B., Morris, H. P., and Liebelt, A. G. York: Pergamon Press, Inc., 1965. Inhibition of Thymidine Kinase Activity in Livers and Hepatomas 15. Sneider, T. W., and Potter, V. R. Deoxycytidylate Deaminase and by TTP and dCTP. Biochem. Biophys. Res. Commun., 16: Related Enzymes of Thymidine Triphosphate Metabolism in 278-284, 1964. Hepatomas and Precancerous Rat Livers. Advan. Enzyme 2. Bushnell, D. E., Whittle, E. D., and Potter, V. R. Differential Régulât.,7:375-394, 1969. Utilization of Pyrimidines for RNA Synthesis in Two Classes of 16. Sneider, T. W., Potter, V. R., and Morris, H. P. Enzymes of Rat Liver Nuclei. Biochim. Biophys. Acta, 779. 497-499, 1969. Thymidine Triphosphate Synthesis in Selected Morris Hepatomas. 3. Emmelot, P.. Hampe, J. F., Bos, C. J.. and Reyers, I. H. M. Cancer Res., 29: 40-54, 1969. Deoxycytidylate Deaminase and Thymine Catabolic Activity of 17. Sonoda, S., and Endo, H. Stabilization of Deoxycytidylate Transplanted Mouse and Rat Hepatomas and Their Histology. Deaminase from Mammalian Tissues. Biochim. Biophys. Acta, Brit. J. Cancer, 25. 138-145, 1961. 4. Hartman, G. C., and Reid, E. Is Hepatic Deoxycytidine- 191: 425-429, 1969. 18. Stanislawski-Birencwajg, M., Uriel, J., and Grabar, P. Association 5 -monophosphate Deaminase Confined to Bile Duct Cells? of Embryonic Antigens with Experimentally Induced Hepatic Biochem. J., 92: 28P, 1964. Lesions in the Rat. Cancer Res., 27: 1990-1997, 1967.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1970 American Association for Cancer Research. Selected Enzymes of Pyrimidine Nucleotide Metabolism in Livers from Rats Fed α-Naphthylisothiocyanate

Thomas W. Sneider, Edward L. Krawitt and Van R. Potter

Cancer Res 1970;30:44-47.

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