Biochem. J. (1969) 111, 121Printed in Great Britain

Reaction of Urethane with Nucleic Acids in vivo

BY E. BOYLAND AND K. WILLIAMSChester Beatty Research In8titute, Institute of Cancer Research: Royal Cancer Hospital,

London, S.W. 3

(Received 16 July 1968)

1. [1-14C]Ethyl carbamate, ethyl [carboxy-14C]carbamate, [1-14C]ethanol andsodium hydrogen [14C]carbonate were injected intraperitoneally into C57 mice,and nucleic acids and proteins were separated from the liver and lungs with phenolas described by Kirby (1956). 2. Chromatographic analysis of the hydrolyticproducts of the urethane-labelled RNA showed the presence of a single radioactivecompound differing in behaviour from the major pyrimidine nucleotides andpurines. 3. The products from RNA labelled by [1-14C]ethyl carbamate or ethyl[carboxy-14C]carbamate appeared chromatographically identical but could not bedetected in the RNA of mice given [1-14C]ethanol or sodium hydrogen [14C]-carbonate. 4. The labelled product appeared to be the ethyl ester of cytosine-5-carboxylic acid formed by the reaction of urethane with RNA in vivo. 5. A directreaction between labelled urethane or the labelled metabolite of urethane, [1-3H]-ethyl N-hydroxycarbamate, and RNA was not detected.

Previous work using [1-'4C]ethyl carbamate andethyl [carboxy-14C]carbamate for investigation ofthe metabolism and mechanism of action ofurethane (Skipper et al. 1951; Berenblum, Haran-Ghera, Winnick & Winnick, 1958) showed no

marked localization in the mouse tissues. Over 90Oof the 14C left the body within 24hr. but a smallproportion, particularly of that from the [1-14C]-ethyl carbamate, remained. Comparison of in-corporation from urethane with that from [1-14C]-ethanol and sodium hydrogen [14C]carbonateindicated that most of this residue was due tometabolic incorporation of the breakdown productsof urethane into the tissues.Although Berenblum et al. (1958) determined the

subcellular distribution of labelled urethane theydid not investigate the labelling of the nucleic acids.There was evidence, however, that urethanecombines physically with nuclei and mitochondria.In the present work, use was made of the phenol

fractionation techniques developed by Kirby (1956)to determine the location of 14C in the liver andlungs ofC57 mice after the intraperitoneal injectionof ethyl [carboxy-14C]carbamate or [1-14C]ethylcarbamate.The carcinogenic action of urethane may be due

to modification of the nucleic acids, but to demon-strate a reaction in vivo it is necessary to isolatenucleic acids labelled by radioactive urethane andto show that a modified base has been labelled.Brookes & Lawley (1965) found that mustard gas[di-(2-chloroethyl) sulphide] preferentially alkylates

guanine, but the present results suggest thaturethane attacks the pyrimidine bases of thenucleic acid.

MATERIALS AND METHODS

Animals. Male and female C57 black mice weighing25-30g., approx. 3 months old, were maintained on a 20%-protein rat-cake diet for at least 48hr. before death. Theaddition of crushed maize to the diet was avoided because itmade the separation of the aqueous and interfacial layersduring the subsequent fractionation of the liver and lungsdifficult.

Chemicals. Cytosine, cytidine, cytidylic acid [cytidine-2'(3')-monophosphoric acid, mixed isomers], orotic acid(uracil-6-carboxylic acid), adenine and guanine were

obtained from British Drug Houses Ltd., Poole, Dorset.Cytosine-5-carboxylic acid and uracil-5-carboxylic acid(iso-orotic acid) were purchased from Sigma Chemical Co.,St Louis, Mo., U.S.A.; these acids and their ethyl esters(and that of orotic acid) were also made by the methodsoutlined by Wheeler, Johnson & Johns (1907) and Wheeler &Johns (1908). N4-Hydroxycytidine was prepared by themethod of Brown & Schell (1965) and N4-ethoxycarbonyl-cytosine (kindly supplied by Dr R. Nery) by the reactionof cytosine and ethyl chloroformate. 5-Methylcytosine was

obtained from Sigma Chemical Co.Enzymes. Purified uridine phosphorylase (uridine-

orthophosphate ribosyltransferase) was prepared fromfrozen Escherichia coli by DEAE-cellulose chromatography(Krenitsky, Barclay & Jacquez, 1964), the cells beingruptured by sonic vibration (Paege & Schlenk, 1952).Ribonuclease A and a suspension of alkaline phosphatasefrom E. coli in (NH4)2S04 soln. were obtained from SigmaChemical Co., deoxyribonuclease I(D) and Crotalus

121

E. BOYLAND AND K. WILLIAMSadamanteus venom phosphodiesterase were from Worthing-ton Biochemical Corp., Freehold, N.J., U.S.A., and crudeCrotalu.s adamanteus venom was from Koch-LightLaboratories Ltd., Colnbrook, Bucks.

Radioactive materials. [1-14C]Ethyl carbamate (specificradioactivity 0.5-2mc/m-mole) and ethyl [carboxy-14C]-carbamate (specific radioactivity 1-5 mc/m-mole) wereobtained from the New England Nuclear Corp., Boston,Mass., U.S.A. [1-14C]Ethanol (specific radioactivity5-20mclm-mole) and NaH14CO3 (specific radioactivity20-50mc/m-mole) were supplied by The RadiochemicalCentre, Amersham, Bucks. Each mouse was injectedintraperitoneally with 0-1 ml. of an aqueous solution of oneof these compounds, containing 0- mc of one of the firstthree compounds/ml. or lmc of NaH14C03/ml. The micewere given approx. 18-71mg. of [ethyl-1-14C]urethane/kg.or 7-36mg. of [carboxy-14C]urethane/kg. [1-3H]EthylN-hydroxycarbamate (0-4mc/m-mole) was made from[1-3H]ethanol and kindly supplied by Dr R. Nery.

Isolation of the nucleic acids and proteins. To obtainsufficient lung tissue 40 mice, of one sex, were used for eachexperiment. The animals were killed 24hr. after injection,and the liver and lungs were immediately dissected out andlaid on solid CO2.The isolation of the DNA, RNA and protein was achieved

as deseribed by Brookes & Lawley (1965), but the originalmethod ofKirby (1956) was used to remove polysaccharide.The frozen livers were homogenized in at least 15 parts(v/w) of 0.5% (w/v) sodium naphthalenedisulphonate witha Waring Blendor, and then stirred for lhr. with 15 vol. ofaq. 90% (w/v) phenol to which had been added 12% (v/v)of m-cresol and 0-1% (w/v) of 8-hydroxyquinoline. AnUltra-Turrax homogenizer was more effective than theWaring Blendor for breaking up the fibrous lungs. Somedifficulty was initially encountered in the subsequentdisruption of the protein-nucleic acid complexes, but thiswas overcome (as suggested by Dr Stanfield Rogers) bythe use of freshly distilled phenol for the Kirby (1956)phenol mixture and a selected batch of sodium dodecylsulphate.

Chromatography. Whatman no. 4 paper was used forascending chromatography with solvent (1) methanol-conc.HCl-water (7:2:1, by vol.) or (2) ethanol-aq. NHg soln.(sp.gr. 0.88)-water (40:1:9, by vol.). All-glass tanks werenecessary for the highly corrosive solvent (1). Whatman no.1 paper was used for descending chromatography in solvent(3) butan-l-ol-water (43:7, v/v), (4) butan-l-ol-aq. NH3soln.(sp.gr.0-88)-water(85:2:12,byvol.) or (5) butan-l-ol-acetic acid-water (2:1:1, by vol.).The papers were dried, examined under a u.v. lamp and

cut into 1 cm. strips. These strips were eluted with 0-1 N-HCland E260 of samples of these eluates was determined.

Detection of radioactivity. The positions of the u.v.-absorbing and radioactive spots were compared by addingother samples ofthe eluates to bottles for a Tri-Carb liquid-scintillation spectrometer (Packard Instrument Co. Inc.,Downers Grove, Ill., U.S.A.). The eluates were neutralizedwith NaHCO3 soln. to prevent acid quenching of thefluorescence of the scintillation fluid. To ensure that allthe radioactive material had been eluted and detected, insome cases the strips from the chromatograms were droppeddirectly into scintillation fluid.Only small amounts of 14C were present and a careful

check was therefore made for 3H contamination or variation

in the quenching of the scintillation fluid by use of thechannels-ratio facility on the Packard instrument.Assay of radioactive tissue component8. To prevent

precipitation, RNA and DNA solutions were first incubatedwith ribonuclease or deoxyribonuclease respectively, andproteins were dissolved in tetraethylammonium hydroxidebefore addition to the scintillation fluid, as described byRoberts & Warwick (1966). The scintillation fluid con-taining protein samples was kept in the dark for 10-12 hr.before counting to ensure the decay of the long-livedphosphorescence of alkaline scintillation fluid exposed tolight.

Nucleic acid hydrolysis. RNA was hydrolysed to thepurine bases and pyrimidine nucleotides, and DNA waspartially hydrolysed, by treatment with N-HCI for 1 hr. at1000 (Smith & Markham, 1950). Hydrolysis by formic acidin sealed ampoules for lhr. at 180° (Wyatt, 1951) wasfollowed by evaporation of the formic acid in a vacuumdesiccator containingNaOH pellets. The effect oftreatmentwith 20% (v/v) H2SO4 for 2hr. at 170° in sealed ampouleson the 14C labelling of the RNA was also determined.DNA was converted into nucleotides by treatment with

deoxyribonuclease, followed by Crotalus adamanteus venomphosphodiesterase. Nucleic acid (5mg.) was dissolved in1 ml. of 0-01 M-NH4HCO3 soln., pH7, and an equal volumeof 0-015M-MgCl2 was added. The mixture was incubatedat 370 for 1 hr. with 0-OlSml. of deoxyribonuclease solution(concn. 5mg./ml.), the pH was raised to 8-5 with aq. NH3soln., and incubation was continued for a further 2hr. with0-025ml. of a solution containing phosphodiesterase(5mg./ml.). This mixture was chromatographed on paper,or chromatographed on a column (10cm. x 1-5 cm.) ofWhatman PDE 10OF2 experimental fast-flow DEAE-cellulose developed with 500ml. of a 0-01-0-2M-NH4HCO3gradient.

Further treatment of the RNA hydrochloric acid hydrolysatebefore chromatography. (a) Periodate oxidation. Tanko,Zsindely & Berencsi (1967) showed that treatment withperiodate followed by lysine, under carefully controlledconditions, liberates bases from mononucleotides.Hydrochloric acid hydrolysates of labelled RNA treated inthis manner contained no specific compounds that could bedetected on chromatograms.

(b) Hydrolysis by alkaline phosphatase and nucleosidase.The labelled RNA was hydrolysed in u-HCI and the mixtureneutralized by the addition of aq. NH3 soln. and removal ofthe excess on a steam bath. To 0-1 ml. of the solution(containing approx. 5mg. of RNA) were added 0-4ml. ofO-Ol M-tris-acetate buffer, pH8, and O-lml. of the E. colialkaline phosphatase suspension. Incubation for 2hr. at 370was followed by the addition of 0-5ml. of O-lM-sodiumphosphate buffer, pH8, containing approx. 1 uridine-splitting unit of nucleosidase (Krenitsky et al. 1964), andincubation for another 15hr. The incubated material wasthen chromatographed.

(c) Hydrolysis by crude snake (Crotalus adamanteu8s)venom. This venom contains phosphodiesterase and5'-nucleotidase. Hydrochloric acid hydrolysate of RNA(containing about 5mg. in O-lml.) was neutralized andincubated overnight at 370 with 0-4ml. of O-Olm-tris-acetate buffer, pH 8, containing 1 mg. of venom. Theproduct was examined by paper chromatography.

(d) Reaction with hydrazine. This reagent reacts withpyrimidines to form pyrazoles or pyrazolones and urea. A

122 1969

URETHANE AND NUCLEIC ACIDmethod based on that of Baron & Brown (1955) was used.RNA (about 5mg./ml.) was dissolved in N-HCI and hydro-lysed by heating it on a steam bath for 1 hr. The hydrolysatewas cooled and neutralized with N-NaOH, and the mixturewas made 50% (v/v) with respect to hydrazine hydrate.This mixture was heated for a further 1 hr. at 70-75' andcompared chromatographically with known pyrimidinestreated with hydrazine, the position of the pyrazolone andpyrazole derivatives being detected by examination under au.v. lamp followed by spraying with FeCl3 soln. (15%, w/v,in 10%, v/v, HCI) or Ehrlich's reagent (Dihlmann, 1953).Ethyl acetate did not extract pyrazolone or pyrazolederivatives containing a carboxyl group from the reactionmixture.

Control experiment8. Ethyl [carboxy-14C]carbamate(0-025mc) was added to the sodium naphthalenedisul-phonate solution used during the homogenization of about25g. ofnormal mouse liver. The tissue was then fractionatedin the normal manner. Ethyl [carboxy-14C]carbamate(0-025mc) was also added to unlabelled liver RNA (2mg.in 1 ml.) just before HCI hydrolysis and the radioactivity onthe subsequent chromatograms was compared with that ofurethane treated alone in the same way.

Experiments in vitro. [1-14C]Ethyl carbamate or ethyl[carboxy-14C]carbamate (0-01 mc of either) or 0-5mc of[1-3H]ethyl N-hydroxycarbamate (0-4mc/m-mole) wasadded to 100mg. of normal liver RNA dissolved in 5ml. of0-2M-tris-acetate buffer, pH6. After incubation for lhr.at 370 the RNA was precipitated by making the mixture70% (v/v) with respect to ethanol. The RNA was then freedfrom excess of urethane by redissolving and reprecipitatingit twice, and the radioactivity of the washed RNA wasdetermined.

Protein. The proteins ('cytoplasmic' and 'nuclear')isolated from the livers of mice given labelled urethane werealso heated at 1000 in N-HCI for 1 hr. and chromatographedon paper.

RESULTSTable 1 shows the labelling associated with the

liver and lung proteins precipitated from thephenol used in the first extraction ('cytoplasmic'),with the proteins liberated from the interfacial

layer ('nuclear') and with the RNA and DNA 24 hr.after injection ofeither ofthe two labelled urethanes,labelled sodium hydrogen carbonate or labelledethanol. All four compounds labelled the proteinsand RNA but only the urethanes definitely labelledthe DNA.For chromatography the labelled RNA was

hydrolysed with N-hydrochloric acid for 1 hr. at1000; this treatment caused little loss of radioactivematerial. The radioactiVe component in DNA andRNA labelled by either urethane was found,however, to be unstable to more vigorous hydro-lysis; after heating in formic acid at 1800 for 1 hr.and evaporation over sodium hydroxide pellets in avacuum desiccator most of the 14C was lost. Theradioactivity was also greatly decreased by heatingthe nucleic acids in 20% (v/v) sulphuric acid insealed ampoules at 170° for 2hr. before placingthem in the desiccator.Chromatography of the urethane-labelled liver

RNA hydrochloric acid hydrolysate gave a singleradioactive spot associated with, but differingslightly from, the normal pyrimidine nucleotidesin four solvents, but in solvent (2) 14C was locatedclose to adenine (Table 2 and Fig. 1). The chromato-graphic mobilities of the radioactive materials inthe RNA labelled by ethyl [carboxy-14C]carbamateand by [1-14C]ethyl carbamate were identical(Table 2). About 80% of the 14C from the urethane-labelled RNA was found in this single spot, but nodefinite radioactive region could be detected onchromatograms of the hydrochloric acid hydro-lysates of RNA labelled by [1-14C]ethanol orsodium hydrogen [14C]carbonate developed withsolvent (1). However, this may be partly due toincorporation causing the small amount of 14C tobe divided between the four or five (including ribose)spots of the normal components ofRNA.Treatment of the hydrochloric acid hydrolysate

Table 1. Labelling of mouse liver and lung tis8uefraction8 24 hr. after intraperitoneal injection of 14C-containingcompound8

The radioactivity is expressed as counts/min./mg. corrected to 100% efficiency. When NaH'4CO3 was used tentimes as much 14C was given (see the text). The values given are the mean oftwo separate experiments; variationwas within 20%. Each sample contained 1-25mg. of protein or nucleic acid. The overall counting efficiency,with quenching of the scintillation fluid by the sample solutions, was 70-80%.

Radioactivity (counts/min./mg.)

Liver

Labelling compound[1-14C]Ethyl carbamateEthyl [carboxy-14C]carbamate[1-14C]EthanolNaH'4CO3

Cytoplasmicprotein330200400630

Nuclearprotein320190350730

Lung

Cytoplasmic NuclearRNA DNA protein protein500 410 210 160600 320 80 80430 12 320 330200 8 180 140

RNA DNA410 320130 100230 14160 10

Vol. ill 123

E. BOYLAND AND K. WILLIAMS

Table 2. RF value8 of 8Ome purine and pyrimidine derivative8 on paper chromatogram8

Composition of the solvents and other details are given in the text.

CompoundCytosineCytidineCytidylic acidCytidylic acid5-MethylcytosineCytosine-5-carboxylic acidCytosine-5-carboxylic acidEthyl cytosine-5-carboxylate

Ethyl cytosine-5-carboxylateN4-EthoxycarbonylcytosineN4-HydroxycytidineUracilUridineUridylic acidUridylic acidUracil-6-carboxylic acid (orotic acid)Uracil-6-carboxylic acid (orotic acid)Ethyl uracil-6-carboxylateUracil-5-carboxylic acid (iso-orotic acid)Uracil-5-carboxylic acid (iso-orotic acid)

Ethyl uracil-5-carboxylateEthyl uracil-5-carboxylateAdenineGuanine

Further treatmentSolvent ... (1)

None 0-54None 0-56None 0-67Hydrazine 0-62None 0-65None 0 44Hydrazine 0-44None 0-65

Hydrazine { 0-330-61f

None 0-74None 0-75None 0-66None 0-70None 0-83Hydrazine 0-81None 0-58Hydrazine 0-77None 0-85None 0-54Hydrazine 0-78

NoneHydrazineNoneNone

0-730-570-430-25

Rp

(2) (3)0-51 0-120-46 0-080-10 00-65 0-390-54 0-230-31 0-010-25 0-010-54 0-56

0-24 0

0-59 0-200-58 0-270-58 0-300-57 0-130-13 0-100-57 0-160-41 0-120-20 0-030-52 0-400-12 0-270-52 0

0-240-49 0-490-30 0-010-53 0-110-30 0-04

Radioactive spots from liver RNA hydrolysed with HCI

Labelling urethaneEthyl [carboxy-14C]carbamateEthyl [carboxy-14C]carbamateEthyl [carboxy-14C]carbamate[1-14C]Ethyl carbamate

Further treatmentSolvent ... (1)

None 0-79

Phosphatase 007.5*Hydrazine tNone 0-80

RP

(2) (3)0-55 0-05

00-17*

0-22 0-050-54 0-04

* Two spots: the other value is due to unchanged material.t Not detectable.

of urethane-labelled liver RNA with alkalinephosphatase moved the position of the radioactivespots from the nucleotide to the nucleoside regionof paper chromatograms developed in solvents (1)and (3) (Table 2), but the addition of nucleosidaseafter the phosphatase had no further effect.Heating the neutralized hydrochloric acid hydro-

lysate of liver RNA, labelled in vivo by ethyl[carboxy-14C]carbamate, with hydrazine hydratealtered the Rp values of the 14C-containing spot insome ofthe solvents used (Table 2). No radioactivitycould be detected when solvent (1) was used todevelop the chromatogram.

Chromatography of hydrolysates of lung RNA,labelled by [1-14C]ethyl carbamate, in solvent (1)or (2) showed the presence of a radioactive com-

ponent similar to that found in liver RNA. Thelimited amount of material available prevented theuse of other solvents. The 14C content of lung RNAlabelled by ethyl [carboxy-14C]carbamate was onlyone-third ofthat labelled by [1-14C]ethyl carbamate,and could not be clearly detected on the paper

chromatograms.After partial hydrolysis of urethane-labelled liver

DNA with hydrochloric acid and chromatographyin solvents (1) or (2), the radioactivity remained

124 1969

(4)0-170-0900-380-220-100-020-32

0

0-200-250-300-2000-150-030-010-36000-240-120-050-270-05

(5)0-380-330-200-660-440-000-70

0-60

0-760-510-580-460-320-740-390-600-780-430-73

0-700-350-350-26

(4) (5)0-05 0-21

0-05 0-580-07 0-21

URETHANE AND NUCLEIC ACID

0-51 (a)

(b)

0-55

00 10 30

.-I

Fo

50 i0

Ca

10

0P.

o

0.

e

50 10

C)

Ca0

Distance from origin (cm.)

Fig. 1. Eluates with 0-1N-HCl from paper chromatograms of HCI hydrolysates ofRNA labelled in vivo by ethyl[carboxy-14C]carbamate. (a) Chromatogram run in solvent (1), methanol-conc. HCl-water (7:2:1, by vol.); (b) runin solvent (2), ethanol-aq. NH3 soln. (sp.gr. 0.88)-water (40:1:9, by vol.). The radioactive peaks are shownhatched.

associated with the apurinic acid residue on theorigins. Enzymic hydrolysis with deoxyribonucleaseand phosphodiesterase followed by chromatographyon paper or on DEAE-cellulose columns also didnot separate the 14C from the undegraded DNAresidue.

Cytoplasmic and nuclear proteins labelled byurethane did not give any detectable radioactivespots on the chromatograms when treated in thesame way as nucleic acid.The experiments with RNA in vitro did not

give any detectable radioactive reaction productsof normal RNA and labelled urethane or hydroxy-urethane.

Orotic acid, uracil-5-carboxylic acid and cytosine-5-carboxylic acid were found, by chromatography,to be stable in N-hydrochloric acid at 1000 for 1 hr.Under these conditions about 70-80% of the ethylesters of these compounds were hydrolysed to theacids, but in 0-1N-hydrochloric acid only 10-20%hydrolysis occurred. Oroticacidwas stable to formicacid at 180° for 1 hr., but the pyrimidine-5-carboxylic acids were converted into the bases. N4-Ethoxycarbonylcytosine was completely hydro-lysed to uracil and a little cytosine in less than10min. at 1000 in N-hydrochloric acid, but N4-hydroxycytidine was stable for at least 1 hr.The Rp values of the pyrazolone and pyrazole

derivatives obtained by the reaction of pyrimidineswith hydrazine are shown in Table 2. Ferricchloride solution was the better reagent for thedetection of these pyrazolones and pyrazoles onpaper, as Ehrlich's reagent gave only faint colours.

The pyrazole obtained from the ethyl ester ofcytosine-5-carboxylic acid was unstable to develop-ment in the strongly acid solvent (1): two brownstreaky spots appeared when the chromatogramwas sprayed with ferric chloride solution insteadof the usual single discrete blue-green spot.

DISCUSSION

These results indicate that after the injection of[14C]urethane a labelled pyrimidine-5-carboxylicacid was present in RNA. This was not due to theincorporation of metabolic breakdown products ofurethane into a normal constituent since labelledethanol or sodium hydrogen carbonate did not givethe same result.The same labelled compound was formed with

both [1-14C]ethyl carbamate and ethyl [carboxy-14C]carbamate, showing that the ethoxycarbonylgroup had combined with the nucleic acids. Partialhydrolysis of urethane-labelled DNA with hydro-chloric acid liberated the purines but left the 14Cassociated with the apurinic acid residue. Thechromatographic behaviour of the radioactivecomponent in hydrochloric acid hydrolysates ofurethane-labelled liverRNA suggested a pyrimidinenucleotide, and this view was supported by theapparent formation of a nucleoside after treatmentwith alkaline phosphatase. The resistance of thesuspected nucleoside to attack by a nucleosidasemay be due to enzyme specificity.The radioactive component of urethane-labelled

RNA was stable to N-hydrochloric acid for 1 hr. at

0

Vol. 110 125;

4C

0eD03

E. BOYLAND AND K. WILLIAMS1000 but not to formic acid at 1800, whereasN4-ethoxycarbonylcytosine was completely con-verted into uracil (and a little cytosine) after 10min.in hydrochloric acid at 1000. On the other hand,orotic acid (uracil-6-carboxylic acid) was stable tothe formic acid treatment. Iso-orotic acid (uracil-5-carboxylic acid) and cytosine-5-carboxylic acidformed uracil and cytosine respectively on heatingwith formic acid but were resistant to hydrolysis byhydrochloric acid. The carboxy-14C-labelled RNAlost radioactivity when heated in 20% (v/v)sulphuric acid at 1700 for 2hr. Wheeler et al. (1907)found that orotic acid was not altered by thistreatment but iso-orotic acid was quantitativelyconverted into uracil.The ethyl esters of these 5-carboxylic acids were

partially hydrolysed by hydrochloric acid whereasthe labelling of RNA from mice given [1-14C]ethylcarbamate was stable to this treatment. The esterofthe 5-carboxylic acid nucleotide, however, shouldbe more stable to acid hydrolysis than the ester ofthe free base as the pyrimidine in the nucleotidewould have the keto structure.The reaction with hydrazine was also in agreement

with the product being a pyrimidine-5-carboxylicacid derivative. Hydrazine replaces the ureamoiety of pyrimidines or their nucleotides andwould be expected to react with the ethyl ester ofcytosine-5-carboxylic acid as shown in Scheme 1.The chromatographic behaviour of this reactionproduct was similar to that of the radioactivecomponent from hydrochloric acid hydrolysates ofethyl [carboxy-14C]carbamate-labelled RNA treatedwith hydrazine.Urethane itself did not appear to react with RNA

in vitro under physiological conditions but this wasnot unexpected in view of other evidence that a

metabolite is responsible for the carcinogenic actionof urethane. The carcinogenic action of urethaneoccurs at sites remote from that of application(Haddow, 11963; Liebelt, Yoshida & Gray, 1961;Trainin, Precerutti & Law, 1964). It is inactive intest systems in which metabolism of foreigncompounds does not proceed (see Nery, 1968).A possible reaction of the urethane metabolite

N-hydroxyurethane could be as shown in Scheme 2,by analogy with the reversible reaction of hydroxyl-amine with the C-5-C-6 double bond of cytosine(Brown & Schell, 1965; Verwoerd, Zillig & Kohlhage,1963), but this direct reaction was not detected.Nery (1968) has suggested that the S-ethoxy-carbonyl derivatives formed in vivo by urethane andN-hydroxyurethane (Boyland & Nery, 1965) aredue to the reaction of metabolically generatedethoxycarbonyl radicals. The chemical oxidationof N-hydroxyurethane also forms these radicals(Boyland & Nery, 1966) and the carcinogen N-methyl-N-nitrosourethane S-ethoxycarbonylatescysteine in vitro (Schoental & Rive, 1965). Whetherthe formation of the ethyl ester of cytosine-5-carboxylic acid plays a part in carcinogenesis isunknown, but Berenblum et al. (1959), after testingurethane derivatives for skin initiating action andlung carcinogenesis, suggested that the carcinogenicactivity was dependent on the intact ethoxy-carbonyl group of urethane.

[1-14C]Ethyl carbamate and ethyl [carboxy-14C]-carbamate labelled the liver fractions to a similarextent, but the former gave better labelling of thelung components (Table 1). Comparison with theresults obtained with [1-14C]ethanol and sodiumhydrogen [14C]carbonate showed that this differ-ence was mainly due to metabolic incorporation ofthe 14C, but the results with the two labelled

NH2

I C02.H NH CNHa H2N C02.C2H5

0 N'. Scheme 1HR

Scheme 1.

NH2

N~ C02CO2H5

Oj AN NH.OH

NH2

-N2 CO2 .C2H5

IR

Scheme 2.

NH2

O N

R

CO2 . C2H5+

NH.OH+ NH2.OH

126 1969

Vol. 111 URETHANE AND NUCLEIC ACID 127urethanes were noteworthy, as in the adult mousethe lungs were more susceptible than the liver to thecarcinogenic action of urethane (see Mirvish, 1969).

Unlike the alkylating agent studies by Brookes &Lawley (1965) urethane did not appear to label thepurines. The total labelling of the nucleic acid wassmall, however, the radioactive spot formed on thechromatograms giving counts only two to threetimes that ofthe background, and any other labelledcomponents containing significantly less 14C wouldnot have been detected. The extent of the reactionof urethane with RNA was only approximately oneurethane molecule/7 x 103-6 x 104 base residues.Farber et al. (1967) found that administration of

labelled ethionine (a compound that induces livertumours in rats) preferentially labelled the RNA, inparticular the soluble RNA, of the liver, but theradioactive components formed were not finallyidentified.

Thanks are due to Dr R. Nery for helpful discussion andto Mr E. Nice for valuable technical assistance. Thisinvestigation was supported by grants to the Chester BeattyResearch Institute (Institute of Cancer Research: RoyalCancer Hospital) from the Medical Research Council andthe British Empire Cancer Campaign for Research, and byPublic Health Service Research Grant no. CA-03188-10from the National Cancer Institute, U.S. Public HealthService.

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Boyland, E. & Nery, R. (1965). Biochem. J. 94, 198.Boyland, E. & Nery, R. (1966). J. chem. Soc. p. 354.Brookes, P. & Lawley, P. D. (1965). Isotopes in ExperimentalPharmacology, p. 403. Chicago: University of ChicagoPress.

Brown, D. M. & Schell, P. (1965). J. chem. Soc. p. 208.Dihlmann, W. (1953). NaturWissenschaften, 19, 510.Farber, E., McConomy, J., Franzen, B., Marroquin, F.,

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