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

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Urethan Carcinogenesis and Nucleic Acid Metabolism: in Vitro Interactions with Enzymes1 [CANCER. RESEARCH 28, 1041-104«,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 o¡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 JUNE 1968 1041 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 /¿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 37°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.
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