Urethan Carcinogenesis and Nucleic Acid Metabolism: in Vitro Interactions with Enzymes1

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[CANCER. RESEARCH 28, 1041-104Â«,June1968] Urethan Carcinogenesis and Nucleic Acid Metabolism: In Vitro Interactions with Enzymes1

Alvin M. Kaye2

Department of Experimental Biology, Isaac Woljson Building, The Weizmann Institute oÂ¡Science, Rehovoth, Israel

SUMMARY extensive biologic antagonism experiments, Rogers (35) con cluded in relation to urethan carcinogenesis that the "point of In the light of suggestions in the literature linking the car action of the carcinogen is on the pathway of nucleic acid syn cinogenic action of urethan (ethyl carbamate) with the inhibi thesis below orotic acid and perhaps at the level of ureido- tion of an enzymatic step in nucleic acid metabolism (par succinic acid." Elion et al. (IO, 11), from biologic antagonism ticularly pyrimidine synthesis), a series of enzymes has been experiments on urethan's carcinostatic activity against adeno- assayed in vitro at concentrations of urethan which are supra- carcinoma 755, suggested that urethan affects the formation of lethal in mice. These enzymes were: ornithine transcarbam- carbamyl aspartic acid from carbamyl phosphate and aspartic ylase; the enzymes leading to the synthesis of orotic acid, viz., acid, and the methylation and the animation of the uracil carbamyl phosphate synthetase, aspartate transcarbamylase, moiety. The previous paper in this series (24) reported in vivo dihydroorotase, and dihydroorotate dehydrogenase ; DNA and tests on possible reversal of urethan carcinogenesis by nucleic RNA methylase; DNA, RNA, and polyriboadenylate poly- acid pyrimidine precursors and possible potentiation of ure merase; alkaline and acid deoxyribonuclease ; alkaline and acid than's effect by aminopterin. In this paper, studies in vitro on phosphatase and snake venom phosphodiesterase. The ability enzyme systems which might be involved in the mechanism of of urethan to interfere with the induction of jS-galactosidase urethan carcinogenesis are described. in Escherichia coli was also investigated. In view of the reported linking of the biologic action of ure Nucleic acid methylases were found to be inhibited by ure than to the inhibition of an enzymatic step in pyrimidine syn than in high concentrations; the final urethan concentrations thesis (6, 10-12, 35) and the observations interpreted as lo necessary for 50% inhibition ranged from 0.07 to 0.25 M. No calizing this effect up to or including the level of orotic acid significant inhibition of any of the enzyme systems tested was in the pathway of pyrimidine biosynthesis, the enzymes in found when they were assayed at a final urethan concentration volved in orotic acid synthesis were individually tested for of 0.06 M. Since this concentration is more than double the their response to urethan. Some other enzymes which could lethal concentration for mice, it was concluded that no bio conceivably play a role in urethan carcinogenesis were also logically significant inhibition of nucleic acid metabolism by tested, and a survey of several enzymes participating in nucleic urethan has yet been demonstrated. acid synthesis and catabolism was made. None of the enzyme activities showed any significant alterations when they were INTRODUCTION assayed in concentrations of urethan which could be considered to have biologic significance. Although some organ-specific system must be invoked to ex The remainder of the orotic acid pathway of pyrimidine bio plain the mechanism of action of a carcinogen which shows synthesis as well as other possible sites of interaction with organ specificity, a general interaction, either directly with urethan were surveyed by in vivo tracer experiments using nucleic acids or with their metabolism or function, may be a orotic acid-14C and thymine. The experiments which have been necessary step in tumor formation. A link between urethan briefly described previously (19) and will be reported in full (ethyl carbamate) carcinogenesis and nucleic acid metabolism in the following paper of this series showed the same pattern was postulated two decades ago (see Ref. 24 for summary). of incorporation of pyrimidines into nucleic acid in the presence This idea became more specific with the elucidation of the and absence of urethan. pathway of pyrimidine biosynthesis (34). Following their dem A recent review by a member of our laboratory (31) on the onstration of the reversal by thymine (4) and thymidine (3) metabolism of urethan and W-hydroxyurethan describes pre of the chromosome-damaging effect of urethan, Boyland and vious work in the area of urethan carcinogenesis. Some of the Koller (4) suggested that urethan acted by inhibiting the results to be described in this paper have been reported briefly transmethylation of uridylic acid to thymidylic acid. From (19, 21).

1 This investigation was supported in part by USPHS Research MATERIALS AND METHODS Grant No. CA-05263 from the National Cancer Institute. 2 Herbert Sidebotham Research Associate. Chemicals. All reagents used were commercial preparations Received November 7, 1967; accepted February 14, 1968. with the exception of lithium carbamyl phosphate, which was

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1968 American Association for Cancer Research. Alvin M. Kaye synthesized by the method of Jones et cd. (18) and contained for carbamyl phosphate synthetase. The carbamyl phosphate >90% organic phosphate. formed by an enzyme from rat liver was measured by con Calf thymus DNA, methyl green DNA, crystalline bovine verting it to citrulline in the presence of excess ornithine plus pancreatic deoxyribonuclease, purified bovine spleen acid deoxy- ornithine transcarbamylase activity, and the citrulline was ribonuclease, snake venom phosphodiesterase (Crotalus ada- measured as above (32). Carbamyl phosphate synthesis in this manteus), and E. coli alkaline phosphatase were purchased system proceeded normally, even in the presence of 0.2 M ure from Worthington Biochemical Corp., and E. coli soluble RNA than (Chart IA), which resulted in a ratio of urethan to aden- from General Biochemicals Inc. Urethan (ethyl carbamate) osine of 40:1. was obtained from British Drug Houses Ltd. S-adenosyl-L- Asparlate Transcarbamylase. The crucial transcarbamyla- methionine-methyl-14C in H2SO4 was obtained from Tracerlab tion step in orotic acid biosynthesis was examined by using a Inc. Its specific activity ranged from 17.2 to 36.2 mc/mmole. partially purified enzyme from rat deciduoma. This source pro In the assay of carbamyl-amino acids, sulfuric acid (Analar vided an enzyme activity significantly higher than that found grade) obtained from British Drug Houses Ltd. was found to in comparable extracts of rat liver. A single ammonium sulfate give a high blank value. Baker's analytical grade sulfuric acid, precipitation step was sufficient to obtain an enzyme with a which gave low blank values, was therefore used in all assays. higher specific activity than the product of a 56-fold purifica Enzyme Assays. Enzyme assays were performed at two or tion from rat liver (7). In a preparation from fresh tissue, more concentrations of enzyme extract at which the rate of deciduoma extruded from the uterus of 3 rats 96 hours after enzymatic activity was linearly proportional to the concentra decidual induction by the method of Shelesnyak and Kraicer tion of protein present (as shown in Chart 1, A to D). Test (37) weighed 2.56 gm. A 10% homogenate in isotonic KC1 was concentrations of urethan in all cases included a concentration made in a glass homogenizer and the suspension was centrifuged of 0.06 M or higher, which was calculated to be more than for 5 minutes at 30,000 X g. To 18 ml of the supernatant solu double the lethal concentration in mice. All mice used for tion, 3.6 gm of ammonium sulfate was added to reach 36% enzyme preparations were from inbred C57BL and SWR strains saturation, and the suspension was centrifuged as above. The of this Institute. The animal sources of enzymes were mouse precipitate was resuspended in water and centrifuged again liver and urethan-induced transplantable thymic lymphosar- at 30,000 X g for 5 minutes to remove insoluble proteins. The coma from strain C57BL mice, normal and regenerating rat clear supernatant solution had a specific activity of 5.15 /Â¿moles liver (kindly supplied by Dr. N. Trainin of this department), of carbamyl aspartate synthesized per mg protein in 15 min rat deciduoma (artificially induced maternal placenta obtained utes at 37Â°C.From a similar preparation made from a frozen through the kindness of Prof. M. C. Shelesnyak, of the De dried KC1 extract of deciduoma, a specific activity of 8.28 was partment of Biodynamics, Weizmann Institute of Science), obtained. The Koritz and Cohen (26) method was used to de and human hypertrophie prostate glands obtained through the termine the carbamyl aspartate formed in the reaction. In the courtesy of a local hospital. Escherichia coli and Azotobacter presence of 0.134 M urethan (ratio of urethan to carbamyl cells were grown by the Bacteriological Service of this Institute. phosphate 45:1 ), the synthesis of carbamyl aspartate proceeded Protein concentrations were determined using the biuret re at the same rate as in the absence of urethan (Chart IB). action (14). Dihydroorotase. The conversion of carbamyl aspartic acid (ureidosuccinic acid) to dihydroorotic acid was measured by the method of Yates and Pardee (41), in which the dihydro RESULTS orotic acid formed was determined by virtue of its loss of Ornithine Transcarbamylase absorbancy in the ultraviolet region upon alkaline hydrolysis. The enzyme source was a frozen dried KC1 extract of rat In order to test for a general effect of urethan on transcar- deciduoma. The rate of dihydroorotic acid synthesis in this bamylation (17), the ornithine transcarbamylase activity, in a system was unaffected by the presence of 0.067 M urethan dialyzed supernatant fraction of an isotonic KC1 extract of (Chart 1C), which provided a 33:1 molar ratio of inhibitor to rat liver, was measured in high concentrations of urethan, substrate. reaching a molar ratio of urethan to carbamyl phosphase of Dihydroorotate Dehydrogenase. This last enzyme in the 45:1 when the urethan concentration was 0.134 M. The rate of pathway of orotic acid synthesis (Chart 1) was assayed in the citrulline synthesis was determined by the method of Oginsky system described in Wu and Wilson (40), the orotic acid syn (32). In addition, the inorganic phosphate production in the thesized being determined spectrophotometrically (41). Since reaction was measured by the Fiske-SubbaRow procedure (30). this determination does not distinguish orotic acid from sub Both methods gave identical results and showed that the rate sequent products in nucleic acid synthesis which contain the of ornithine transcarbamylase activity was unaffected by ure same pyrimidine ring, the results are expressed as the increase than in vitro. of absorbancy at 290 m/t, a method which measures pyrimidine ring formation. Regenerating rat liver obtained 44 hours after Biosynthesis of Orotic Acid partial hepatectomy was the source of the enzyme used for the Carbamyl Phosphate Synthetase. Combined Action of experiments reported in Chart ID. The final urethan concen Carbamyl Phosphate Synthetase and Ornithine Transcar tration tested in this case was 0.275 M, which gave a urethan bamylase. The finding that urethan did not affect ornithine to substrate ratio of 66:1. In incubations with regenerating rat transcarbamylase activity made possible a convenient assay liver brei (Chart ID) or where deciduoma, regenerating rat

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mouse lymphosarcoma fractions for RNA methylase activity. The rate of incorporation of methyl-14C groups from S- adenosyl-L-methionine-methyl-HC into an acid-insoluble form was measured by a filtration method described previously (22). The rate of methylation in all four systems tested was found to be decreased by urethan (Chart 3). The final urethan con centration necessary for 50% inhibition ranged from 0.07 to 0.25 M. This result makes the biologic significance of the gen eral inhibition of methylase enzymes by urethan questionable, since carcinogenic concentrations of urethan are 0.01 M and below and concentrations of urethan over 0.025 M are lethal for mice. The Induction of /3-Galactosidase Synthesis As a check on a general interference by urethan with the synthesis or function of messenger RNA, we (in collaboration with Dr. L. Lewin of the Biochemistry Section of the Weizmann Institute of Science) turned to a bacterial system. In order to OS 1.0 150 OK) O 20 test for a general inhibition of enzyme induction by urethan, PROTEIN (mg) the synthesis of /8-galactosidase (38) induced by 2 X 10~3 M thiomethyl-y8-(/-galactoside in E. coli B was measured in the presence of growth-inhibiting concentrations of urethan. It was Chart 1. Assay of enzymes involved in orotic acid biosynthesis in the presence and absence of urethan. Open circles, controls; first established that the addition of 1% urethan to a culture in closed circles, the presence of 200 /umoles of urethan. Incubations lag phase resulted in a signifiant inhibition of subsequent bac were carried out for 15 minutes. terial growth (Chart 2). This concentration is within the range A. Carbamyl phosphate synthetase. The final concentration of reported by previous authors (39) for growth inhibition in urethan tested was 0.200 M. The mixture incubated at 37Â°C this system. contained (in AmÃ³les) JV-aeetyl-L-glutamine, 5; adenosine tri- The rate of hydrolysis of o-nitrophenol-/8-d-galactopyranoside phosphate, 5; (NH4)2CO3> 50; MgSO4, 10; L-ornithine, 5; all by whole cells or by a spheroplast suspension, as followed adjusted to pH 6.8 with CO2; and rat liver (0.1% cetyltri- colorometrically, was not affected by the presence of 1% ure methylammonium bromide extract) ; in a total volume of than during the induction period (Chart 2). 1.0ml. B. Aspartate transcarbamylase. The final concentration of urethan tested was 0.134 M. The mixture incubated at 37Â°Ccontained Survey of Other Enzymes Related to Nuleic Acid Metabolism (in /tmoles) diethanolamine buffer (pH 9.2), 200; aspartic acid, Other enzymes involved in nucleic acid synthesis or catab- 10.5; lithium carbamyl phosphate, 4.5; and a fraction of an olism were surveyed for their activity in concentrations of ure isotonic KC1 extract of rat deciduoma precipitating at 36% than equal to or greater than 0.06 M. saturation with ammonium sulfate; in a total volume of 2.0 ml. Enzymes catalyzing the polymerization of nucleoside tri- C. Dihydroorotase. The final concentration of urethan tested was 0.067 M. The mixture incubated at 37Â°Ccontained (in /tmoles) phosphates into DNA and RNA were assayed in the presence acetate buffer (pH 5.5), 30; carbamyl aspartic acid, 6; and a and absence of urethan by measuring the rate of incorporation solution made from frozen-dried rat deciduoma; in a total of an appropriate radioactively labeled nucleoside triphosphate volume of 3.0 ml. into an acid-insoluble polymer. In collaboration with Lucy D. Dihydroorotate dehydrogenase. The final concentration of ure Shapiro and Malcolm Gefter (Dept. of Molecular Biology, than tested was 0.275 M. The mixture incubated at 32Â°Ccon Albert Einstein College of Medicine, New York, N. Y.) we tained 3 /miÃ³les dihydroorotic acid and regenerating rat liver tested the effect of urethan on the rate of DNA (29) and RNA brei in Schulman's solution (0.035 M sodium phosphate buffer (16) polymerase activity in extracts of E. coli. No significant pH 6.8, 0.13 M KC1, 0.04 M KHC03, 0.01 M MgCL.) plus addi tional Schulman's solution to make 0.7 ml. urethan effect was found on these enzymes with concentrations of urethan which are below supralethal levels in living systems. Urethan was also shown to have no effect in vitro on poly- kidney or normal rat liver were enzyme sources, urethan riboadenylate polymerase of E. coli. caused no change in the rate of orotic acid synthesis. Phosphatases and those enzymes capable of degrading DNA were tested in vitro for their activity in the presence of urethan ; DNA and RNA Methylases deoxyribonuclease activity in mouse spleen following injection The methylases, enzymes capable of adding methyl groups of urethan to the animal was also measured. Human prostatic to specific sites on nucleic acid polymers, were assayed (in col acid phosphatase was partially purified according to the pro laboration with Bertold Fridlender) in the presence of high cedure of Davidson and Fishman (8). Its rate of liberation of concentrations of urethan. Both DNA and RNA methylases inorganic phosphate (30) from adenosine monophosphate was were tested (13, 22) using ammonium sulfate fractions of E. unaffected by urethan. Crotalus adamanteus venom phospho-- coli and Azotobacter for DNA methylase, and E. coli and diesterase showed the same rate of splitting of calcium bis-

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pared with extracts of normal spleens for deoxyribonuclease activity. The rate of liberation of acid-soluble deoxyribonucleo- tide compounds from calf thymus DNA (9) was the same in extracts made from normal and from urethan-treated mice.

DISCUSSION

Parallel with our in vivo studies (24) we have utilized in vitro enzyme assays to investigate the suggestions listed in the intro duction of this paper (4, 6, 10-12, 35) for a site of action of urethan in the inhibition of pyrimidine biosynthesis. The ex periments described in this communication, together with the in vivo studies reported previously (24) and the isotopie tracer studies (19) to be presented in full in the subsequent paper of this series, comprise direct tests for each of the above suggestions. Our work has shown that none of these sites of action is significantly affected by concentrations of urethan which are more than double the lethal level for mice. Rogers lias recently confirmed his suggestion (personal com munication) that the discrepancy between these results (24) and his findings might be due to a nutritional difference in the mice. Mice fed a diet of Purina Laboratory Chow (which is enriched in folie acid) did not show the reversal of urethan- induced adenoma formation which Rogers had obtained in mice fed a diet poorer in folie acid (35). Particular attention in our experiments was paid to the cru cial enzyme aspartate transcarbamylase, which occurs at the URETHAN(M) branch point of the biosynthetic pathway taking aspartic acid Chart 2. The effect of urethan on nucleic acid methylase activity. either to proteins or nucleic acid. Aspartate transcarbamylase The mixture incubated at 37Â°C for 30 minutes contained (in was partially purified from rat deciduoma to yield a product /ttmoles) s-adenosyl-methionine-methyl-14C, 5; Tris buffer pH 8.0, with a specific activity which is greater than the activity of 20; mercaptoethanol, 2; MgCl2, 1; in a total volume of 0.5 ml. this enzyme reported after partial purification from rat liver In the assays for DNA methylase activity (circles), 2 tig heated RNase and 50 /Â¿gcalf thymus DNA were added. In RNA methyl (7). Bresnik (6) reported that at great excess urethan was ase assays (triangles), RNase was omitted and 50 ^g of E. coli able to inhibit aspartate transcarbamylase activity in a super soluble RNA was used as an acceptor of methyl groups. E. coli natant fraction obtained by centrifugation at 600 X g of son- enzyme activity (closed symbols) was from a 30-60% saturation ically disrupted Ehrlich ascites cells. Recently, Giri and ammonium sulfate fraction (22) which contributed 0.65 mg pro Bhide (12) have claimed an inhibition of the activity of tein. Azotobacter enzyme activity (open circles) was from a 0- aspartate transcarbamylase in crude homogenates of mouse 60% saturation ammonium sulfate fraction (22) which contributed organs from mice given urethan 6 hours previously. However, 0.71 mg protein. Urethan-induced transplantable thymic lympho- as has been pointed out (6), in crude homogenates, effects on sarcoma enzyme activity (open triangles) was from a 35-75% the stability of carbamyl phosphate must first be ruled out. saturation ammonium sulfate fraction which contributed 0.70 mg Mary Ellen Jones (personal communication) has found no protein. significant effect of urethan on transcarbamylation in vitro, in accord with the results presented in this paper (Chart 1). nitrophenol phosphate (25) in the presence or absence of The recent discovery by Borek of methylation of nucleic urethan. Assays by the method of Heppel et al. (15) showed acids at the polynucleotide level by the naturally occurring no effect of urethan on E. coli alkaline phosphatase. Crystalline nucleic acid methylating enzymes and his suggestion that bovine pancreatic alkaline deoxyribonuclease and purified bo "some methylating enzyme complex may be a naturally oc vine spleen acid deoxyribonuclease were assayed in vitro, using curring carcinogen" (2) led us to carry out direct tests of the methyl green DNA methods of Kurnick (27, 28), without nucleic acid methylase activity in the presence of urethan. The revealing any effect of urethan on the rates of DNA degrada present results (Chart 2) indicate that urethan inhibits over tion. all nucleic acid methylase activity only at levels (approx. Since an increase in intracellular deoxyribonuclease activity 0.1 M) which are supralethal in mice. Specific loci of inhibition, following urethan administration may result in damage to DNA however, may be important at lower concentrations of urethan. related to carcinogenesis, the in vivo action of urethan on The lack of effect of urethan on the induction of j8-galactosi- deoxyribonuclease levels in mouse spleen was tested. Mice were dase in E. coli (Chart 3) indicates that the various steps in given 1.25 mg urethan/gm body weight, and 30 hours later volved in the induction of an enzyme, namely, interaction of extracts of the atrophie spleens were prepared (27) and com inducer with repressor, synthesis of messenger RNA, and syn-

1044 CANCER RESEARCH VOL. 28

1.0 B 0.8 75 5 E ÃœJ o 0.6 CE CM 50 >- 0.4 LÃ¼ 25 g 0.2

2 4 60 IO 20 30 TIME (hours) TIME(minutes)

Chart 3. Induction of 0-galactosidase in E. coli B in the presence of urethan. A. Inhibition by urethan of growth of E. coli B: arrow indicates time of addition of urethan to a final concentration of 1% (closed circles) ; controls, open circles. B. /3-galactosidase assay. Open circles, control; closed circles, E. coli B spheroplasts which had been incubated in 1% urethan during the induction period. The /3-galactosidase assay reagent consisted of 4 AmÃ³lesof o-nitrophenyl-/3-d-galactopyranoside, 0.4 mmoles of sodium phosphate buffer pH 7.5, and 0.16 ml of an extract of a 25% suspension of baker's yeast, in. a total volume of 4.0 ml. Readings were taken using a Number 42 filter.

thesis of protein, are not general sites of inhibition by urethan. Esther Yatom for technical assistance, and Prof. I. Berenblum for Nucleic acid polymerases and hydrolyses were also tested in his advice and encouragement. this investigation without revealing a site of urethan inhibition. The effect of urethan on a variety of other enzyme systems has REFERENCES been tested as well (see Refs. 5, 23). The conclusion was reached that an interaction of urethan with an enzyme in vitro. 1. Bieber, S., and Hitchings, G. H. Effects of Growth Inhibitors on Amphibian Tail Blastema. Cancer Res., 19: 112-115, 1959. which may indicate the importance of that enzyme in the 2. Borek, E. The Methylation of Transfer RNA: Mechanism and in vivo process of carcinogenesis, is still to be demonstrated. Function. Cold Spring Harbor Symp. Quant. Biol., 28: 139- In one case in which we showed that concentrations of urethan 148, 1963. which could be attained in a living animal (approximately 3. Boyland, E. In: G. E. W. Wolstenholme and M. O'Connor 0.01 M) were capable of inhibiting Â«-chymotrypsin, the almost (eds.), Ciba Foundation Symposium on Carcinogenesis, noncarcinogenic homolog of urethan, n-propyl carbamate, also Mechanisms of Action, p. 79. London: J. & A. Churchill Ltd., showed the same activity (23). 1959. The possible usefulness of urethan's ability to inhibit chymo- 4. Boyland, E., and Koller, P. C. Effects of Urethane on Mitosis trypsin and thrombin as a model system for possible interac in the Walker Rat Carcinoma. Brit. J. Cancer, 8: 677-684, tions with protein has been pointed out in the preceding paper 1954. 5. Boyland, E., and Williams-Ashman, H. G. The Influence of of this series (24). The lack of a significant effect of urethan in Urethanes on the Enzymic Activity of Normal and Malignant physiologic concentrations on enzymes of nucleic acid metabo Tissues. Acta UniÃ³ Intern. Contra Cancrum., 7: 432-434, 1951. lism, on short-term incorporation of thymidine and uridine 6. Bresnick, E. Effect of Urethane upon Pyrimidine Biosynthesis. into nucleic acids (42), on DNA itself in vitro (20, 43), and Federation Proc., 19: 201, 1960. on such fundamental biologic processes as regeneration (1, 36) 7. Bresnick, E. Inhibition by Pyrimidines of Aspartate Trans- focuses attention on the alternate hypothesis that a specific carbamylase Partially Purified from Rat Liver. Biochim. Bio- interaction of urethan with a protein or proteins (rather than phys. Acta, 67: 425-434, 1963. a nucleic acid) may be involved in the carcinogenic action of 8. Davidson, H. M., and Fishman, W. H. A Simplified Procedure urethan. for Human Prostatic Acid Phosphatase Based on pH and Ammonium Sulfate Fractionation. J. Biol. Chem., 2$4: 526- ACKNOWLEDGMENTS 528, 1959. 9. Diesche, Z. Color Reactions of Nucleic Acid Components. In: The author wishes to thank L. Lewin, Lucy Shapiro, M. Gefter, E. Chargaff and J. N. Davidson (eds.), The Nucleic Acids, and B. Fridlender for their collaboration in part of the present Vol. 1, pp. 285-305. New York: Academic Press, 1955. work, Dr. S. Rogers for his information concerning the influence 10. Elion, G. B., Bieber, S., and Hitchings, G. H. Studies on the of dietary folie acid on inhibition of urethan carcinogenesis, Miss Mechanism of Action of Urethane on Mammary Adenocar-

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cinoma 755. Acta UniÃ³ Intern. Contra Cancrum, 16: 605-608, 27. Kurnick, N. B., Massey, B. W., and Sandeen, G. The Effect of 1960. Radiation on Tissue Deoxyribonuclease. Radiation Res., 11: 11. Elion, G. B., Bieber, S., Nathan, H., and Hitchings, G. H. 101-114, 1959. Uracil Antagonism and Inhibition of Mammary Adenocar- 28. Kurnick, N. B., and Sandeen, G. Acid Desoxyribonuclease cinoma 755. Cancer Res., 18: 802-817, 1958. Assay by the Methyl Green Method. Biochim. Biophys. Acta, 12. Giri, C. P., and Bhide, S. U. Metabolic Studies on the Mecha 39: 226-231, 1960. nism of Urethan Action. II. Age and Tissue Specificity. J. 29. Lehman, I. R. DNA Synthesis (Bacterial). In: S. P. Colowick Nati. Cancer Inst., 39: 579-584, 1967. and N. 0. Kaplan (eds.), Methods in Enzymology, Vol. 6, pp. 13. Gold, M., Hurwitz, J., and Anders, M. The Enzymatic Methyl- 34-39. New York: Academic Press, 1963. ation of RNA and DNA. Biochem. Biophys. Res. Commun., 30. Leloir, L., and Cardini, C. E. 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1046 CANCER RESEARCH VOL. 28

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1968 American Association for Cancer Research. Urethan Carcinogenesis and Nucleic Acid Metabolism: In Vitro Interactions with Enzymes

Alvin M. Kaye

Cancer Res 1968;28:1041-1046.

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