In Vivo Tests for a Carcinogenic Metabolite in Plasma of

Animals Given Injections of Ethyl Carbamate (Urethan)

I. BERENBLUM,A. M. KAYE,ANDN. TRAININ

(Department of Experimental Biology, The Weizmann Institute of Science, Reliovcth, Israel)

SUMMARY

Plasma, obtained from rabbits given injections of urethan 2J hours previously, whenadministered intraperitoneally to Swiss mice, produced a significantly increased incidence of lung adenomas in these animals, as well as an increased incidence of skinpapillomas at the site of subsequent croton oil applications.

These effects were not obtained when the plasma was lyophilized or desiccated,with the consequent removal of the free urethan. The condensed vapors collected during the process of lyophilization, on the other hand, were carcinogenic.

Carbonyl-labeled urethan-C14 was used to determine the urethan content of the

plasmas and condensed vapors. The carcinogenic potencies for the lung and the initiating potencies for the skin were not significantly different from the effects of comparableamounts of free urethan.

The results obtained could, therefore, be entirely accounted for on the suppositionthat the carcinogenic effects were due to urethan itself, without the participation of ametabolite.

The possibility of the existence of such a metabolite is discussed in the light of theresults obtained.

The possibility that a specific carcinogenic metabolite might be involved in urethan carcinogene-sis arose from the observation by Rogers (15) thatmouse embryo lung hashings developed adenomasafter intramuscular implantation into adult micewhen the tissue was preincubated in plasma from arabbit that had previously been given an injectionof urethan, but not when pretreated with a mixtureof urethan and normal rabbit plasma. He concluded that it was not any residual, unchangedurethan in the plasma that was responsible for theneoplastic transformation of the lung tissue, but ametabolite, either of urethan itself or evoked bythe presence of urethan in the injected rabbit.

We attempted to extend these observations bymeans of an in vivo system of testing—i.e., by injecting plasma of urethan-treated rabbits intomice, and then observing (a) whether the tumorincidence would increase in the intact lungs, (6)whether subsequent croton oil applications to theskin of such treated mice would elicit skin papillomas (2), and (c) whether removal of free urethanfrom the plasma of urethan-injected rabbits (12)would affect its carcinogenicity.

Received for publication June 15, 1939.

In some preliminary experiments (with Ha-ran-Ghera), it was found that plasma obtainedfrom rabbits 5 hours after urethan injection, administered to mice, did raise the lung tumor incidence and also the incidence of skin tumors at thesite of subsequent croton oil applications, but thatthese effects were apparently prevented by desiccation of the plasma.

A more extensive series of experiments, reported in this communication, was then undertaken, not only to confirm these preliminary results, but also to explore the problem in an expanded series of in vivo tests of rabbit plasmas,withdrawn at intervals varying from 5 to 25 hoursfollowing the injection of urethan. The purpose ofthe latter tests was to examine more critically thepeak period of activity of the rabbit plasma and todetermine whether this peak of carcinogenic activity corresponded with, or differed from, the peakof maximum urethan concentration in the plasma.Such information, it was felt, might provide supplementary evidence for the existence of an activemetabolite. In addition, tests were made of thecarcinogenicity of the plasma of urethan-injected

rabbits, from which the free urethan had been re.

38

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BEREXBLUMet al.—Carcinogenic Metabolite of Urethan

moved by lyophilization followed by reconstitution of the plasma.

A different approach to the problem, reportedin the next communication (7), involved thechromatography of plasma from mice and rabbitsgiven injections of C14-labeled urethans, to followup the possible existence of an intermediate metabolite of urethan in the plasma or tissue by anindependent method.

MATERIALS AND METHODSMaterials.—The urethan was obtained from

British Drug Houses, Ltd., England; the ethyl-1-C14-urethan (1.3 mc/mmole) and the carbonyl-C14-urethan (1.8 mc/mmole) from the New EnglandNuclear Corporation, Boston, Mass., U.S.A.; andthe croton oil from Magnus, Mabee & Reynards,New York, N.Y., U.S.A.

Mice.—Swiss mice, bred in this laboratory forover twenty generations by brother-sister matings,were employed in this study. In series I, youngadult mice, 47-55 days old, were used. Males ofthis age group ranged from 18 to 20 gm. in bodyweight; females weighed from 16 to 18 gm. Inseries II, 13-15-day-old mice were used, whichaveraged 6.5 gm. in weight. The mice were housedin an air-conditioned room at 21°-23°C. and fed

on Purina Laboratory Chow (supplemented withbarley and sunflower seeds) and water ad libitum.

Rabbits.—These were obtained from severalsources and were therefore genetically heterogeneous. Young adults, both male and female, wereused, ranging in weight from 1.7 to 4.1 kg. Theywere given intraperitoneal injections of 10 per centurethan in distilled water at a dose rate of 1.5gm/kg. Blood, obtained at various times followinginjection, was withdrawn in a heparinized syringeby cardiac puncture under sterile conditions. Toprevent loss of volatile components (urethan or ahypothetical metabolite), the separation of theplasma, by centrifugation of the blood at 30,000 Xg for 5 minutes and subsequent décantation,wascarried out at 2°-4°C. One-mi, samples were im

mediately injected into mice and aliquots takenfor urethan analysis. The urethan was dissolved ineither normal rabbit plasma or 0.15 M NaCl forinjection into mice.

Lyophilization and reconstitution of plasma.—Portions of plasma were lyophilized and the volatile components collected in glass traps immersedin liquid air. Analysis for C14, from carbonyl-labeled urethan added to the original urethan solution injected into rabbits, showed that the radioactivity of the lyophilized plasma plus the collected vapors was equal to the radioactivity of the

whole plasma. However, not all the volatile urethan could be removed from the plasma solids by asingle lyophilization. Therefore, for the experiments in series II, the dried plasma, followinglyophilization, was ground into a fine powder,spread on a watch glass, and allowed to remain atroom temperature for 12-48 hours. The plasmawas then reconstituted by addition of appropriateamounts of sterile twice-distilled water and filteredthrough pyrex wool. The pH of the reconstitutedplasma was adjusted to neutrality where necessary, and its tonicity was checked on mouse redblood corpuscles.

Urethan analysis.—In the 10 per cent urethansolution used for injection into the rabbits, ure-than-C14 (labeled in the carbonyl position) was included at a dose rate of 1-3 /ic/kg. By comparisonof an appropriate dilution of this solution with 0.5-ml. aliquots of plasma, taken from rabbits at various times following injection, by the "alkali labile"

method described previously (3), it was possible tocalculate the urethan concentration. Since parallelpaper Chromatographie studies of rabbit plasma,with both carbonyl and méthylènecarbon-labeledurethan, failed to demonstrate any other migrating C14-containing component and negligible incorporation of C14in nonmigrating protein and nucleic acid during the first few hours following injection (7), the observed radioactivity was expressedas mg urethan/ml.

Promoting action of croton oil.—Twice weeklypaintings with 5 per cent croton oil in liquid paraffin, as described previously (2), were continued for20 weeks, starting 1 day following urethan treatment, in series I, after which the animals werekilled. However, in the 13-15-day-old mousegroups (series II), 6 additional weeks for growth ofthe young mice were permitted before the 20-weekcroton oil treatment was begun. Tumors were recorded as previously described (2). The lungs wereexamined with a low-power dissecting microscope,and only tumors larger than 0.2 mm. were counted.In the earlier groups, all the adenomas were confirmed histologically; in later groups, only questionable adenomas were so tested.

Ninety-five per cent confidence intervals formean values were calculated by the "t" distribu

tion for small sample sizes (6).

RESULTSSeries I.—The incidence of lung adenomas and

of skin papillomas in mice painted with croton oilafter an initial injection of "U" plasma (rabbit

plasma obtained at various intervals after urethaninjection) is presented in Table 1. Values for aver-

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40 Cancer Research Vol. 20, January, I960

age urethan content of the plasmas, estimatedfrom aliquots as described above, are included inTable 1 for some of the time intervals.

It will be noted that a significant increase inlung tumor incidence was demonstrable only in thetwo large groups of animals, corresponding to "U"

plasmas at £and 2^ hours, and a significant increase in skin papilloma production only in the2^-hour group. While the peak tumor incidence (atabout 2j hours) corresponded approximately tothe highest average content of urethan in the plasma, the correlation can be considered no more thansuggestive in view of the wide range of variations,

both in urethan content and in tumor incidence.On the other hand, lyophilization of the 2^-hour"U" plasma, which led to a considerable reduction

in urethan content, led to a reduction in tumor incidence to within the range of the control values(Table 2). It will also be noted that the tumor incidence of the unlyophilized 2|-hour "U" plasma

group was similar to that obtained with a dose ofurethan, injected directly into the mice, comparable to the urethan content of the "U" plasma

(Table 2).The results of this series are, therefore, com

patible with the view that the biological activity of

TABLE1LUNGANDSKINTUMORSIN47-55-DAY-OLDMICERECEIVINGASINGLEINJECTIONOF

l ML."U" PLASMAFOLLOWEDBYREPEATEDCROTONOILAPPLICATIONSTOSKIN

Influence of time of blood withdrawal.

TlME OF BLOOD WITH

DRAWAL (HH. AFTER

UKETHANINJECTION)Controls

(nourethan)è

i2Ì4

51025Av.

URETHAN CON

TENT OF RABBIT

PLASMA(MO/ML)*0.0

1.7(0.9-2.4)

1.9(1.5-2.4)

1.8(1.5-2 0)

1.5(0.8-2.2)LUNG

ADENOMASMice

bearingtumors6/19313/97

5/3223/1304/343/331/190/18Tumors

permouse*0.03(0.01-0.05)0.13(0.06-0.20)

0.16(0.02-0.30)0.18(0.11-0.25)0.12(0.00-0.24)0.09(0.00-0.19)0.05(0.00-0.16)0.00SKDÕ

PAPILLOMAsMice

bearingtumors10/1937/97

5/3218/1304/342/331/191/18Tumore

permouse*0.05(0.03-0.07)0.07(0.02-0.12)

0.16(0.02-0.30)0.14(0.08-0.20)0.12(0.00-0.24)0.06(0.00-0.15)0.05(0.00-0.16)0.06(0.00-0.18)

"U" Plasma = plasma from rabbits injected with 1.5 gm/kg of urethan.Croton oil = 5 per cent solution in liquid paraffin, twice weekly for 20 weeks.* Figures in parentheses give 95 per cent confidence interval.

N.B. In all mice included in this table only a single lung and/or skin tumor was found.

TABLE 2

LUNGAMDSKINTUMORSIN 47-55-DAY-OLDMICERECEIVINGASINGLEINJECTIONOFl ML."U" PLASMAFOLLOWEDBYREPEATEDCROTONOILAPPLICATIONSTOSKIN

Kffect of (a) lyophilization of plasma (removal of volatile urethan), (b) various levels of urethan in place of "U"plasma.

MATKRIALINJECTEDINTOMICE"U"

plasma at 2}hr."U"pksma at 2|hr.lyophilizedControls

(nourethan)UrethanAMT.

or UBETHAN(MG.)1.9(1.5-2.4)0.20.01.12.24.4UHETHANDOSE(>IG/GM

MOUSE)~100<

11061123245LUNG

ADENOMASMicebearingtumore23/1303/796/1931/299/737/28Tumors

permouse0.18(0.11-0.25)0.04(0.00-0.08)0.03(0.01-0.05)0.03(0.00-0.10)0.12(0.04-0.20)0.25(0.08-0.42)SKIN

PAPILLOMASMicebearingtumore18/1308/7910/1931/295/732/28Tumors

permouse0.14(0.08-0.20)0.10(0.03-0.17)0.05(0.03-0.07)0.03(0.

00-0.io)0.07(0.01-0.13)0.07(0.00-0

16)

N.B. In all mice included in this table only a single lung and/or skin tumor was found.

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BERENBLUMet al.—Carcinogenic Metabolite of Urethan 41

"U" plasma (both for skin initiating action and

lung carcinogenesis) is due to the presence of avolatile product—presumably unchanged urethan.However, in view of the wide range of variations inthe results, an attempt was made to repeat theexperiment under more sensitive conditions.

Series II.—Advantage was taken, in the following experiment, of the observations by Rogers (13,14) that very young mice were more responsivethan adults to urethan lung carcinogenesis. Agroup of young mice of various ages was thereforetested for their tolerance to intraperitoneal injection of as large an amount as 1 ml. of rabbit plasma, and the youngest age group that withstoodsuch treatment was found to be 13-15-dav-old

urethan content. Lyophilization of the "U" plas

ma and subsequent prolonged exposure to air notonly reduced its urethan content to negligibleamounts, but also led to a reduction of its capacityto induce lung adenomas to a level within therange of normal plasma. The recovered vapors, onthe other hand, produced a tumor incidence corresponding to their urethan content.

The skin papilloma incidence showed qualitatively the same picture as that obtained for lungadenomas. However, because of the high controltumor incidence (due to background carcinogenic-ity of the croton oil itself), the statistical significance of the skin tumor data in this series was lessthan that for lung adenomas.

TABLE3LUNGANDSKINTUMORSIN 13-15-DAY-OLDMICERECEIVINGASINGLEINJECTIONOF

l ML."U" PLASMAFOLLOWEDBYREPEATEDCROTONOILAPPLICATIONSTOSKIN

MA L lA I. EC ED L' 0 MCEControls

(nonna Iplasma)Normal

plasma+urethan"U"

plasma, 2Jhr."U"plasma, 5hr."U"plasmalyophilizedVapors

from "U"plasmalyophilizationAMT.

offun

i(MC.)0.00.52.04.01.91.80.0030.6U

RETHANDOSE(MO/G

M MOUS K)0773086162932770.0593LÃœNGMice

beilringtumorsPer

No.cent1/37

38/322514/285024/278921/326610/29353/31107/38

18ADENOMASlumors

permouse0.03(0.00-0

08)0.25(0.09-041)0.54(0.32-0.76)1.88(1.10-2.66)0.85(0.56-1.14)0.41(0.16-0.63)0.10(0.00-0.20)0.23(0.05-0.41)SKINMice

heuringtumors..

Per.So.cent6/37

167/32229/29

3115/27569/32

2817/30573/31

1011/38

29PAPILLOMAS1

umors permouse0.16(0.03-0.29)0.22(0.07-0.37)0.41(0.15-0.67)0.85(0.45-1.25)0.28(0.11-0.45)0

60(029-0.91)0.10(000-0.20)0.29(0.14-0.44)

mice, weighing about 6.5 gm. each. A preliminarytest was then performed on eighteen such mice todetermine their response to urethan alone. Aftereach mouse received 0.6-1.0 mg. of urethan, theaverage lung tumor incidence was found to be 2.6adenomas per mouse—i.e., about double the incidence in young adults receiving such a dose (2).

In the experiment proper, 300 mice, 13-15 daysold, were given injections intraperitoneally of 1ml. of rabbit plasma (normal plasma, 2j-hour "U"plasma, 5-hour "U" plasma, lyophilized and reconstituted "U" plasma, and vapors from "U" plasma

lyophilization), followed, after an interval of 6weeks, by standard croton oil paintings to the skin.Of these 300 mice, 254 survived the half-yearcourse of the experiment.

As shown in Table 3, the incidence of lung tumors in both the 2f- and 5-hour "U" plasma

groups was significantly higher than that in thenormal plasma control, but not significantlyhigher than the values to be expected from the

DISCUSSIONRogers (15) reported that mouse embryo lung

bashings developed tumors after implantation intoadult mice when pretreated with plasma from arabbit that had previously been given an injectionof urethan, but not when pretreated with a mixtureof urethan and normal plasma. He concluded thata metabolite was involved, detectable in the bloodof urethan-injected animals.

Unfortunately, by error, the control incubationmixture in the experiment by Rogers was 1 percent urethan in plasma, instead of 0.1 per cent, theintended concentration, based on an analysis ofrabbit plasma following urethan injection made bySkipper et al. (16). (From our own data, Table 1,the concentration of urethan in rabbit plasma atthe 2|-5-hour period after ¡njestionwas approximately 0.2 per cent.) The possibility arises that a1 per cent concentration of urethan might act as aninhibitor of some cellular processes, preventing tumor induction, while a 0.2 per cent concentration

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Cancer Research Vol. 20, January, 1960

might induce tumors. An example of an enzymesystem which behaves in this way is given byKeith and Dinning (8), who found that in vitro inhibition of rat liver choline oxidase was 4 per centwith 0.16 per cent urethan, 27 per cent with 0.31per cent urethan, and 78 per cent with 1.56 percent urethan. This was confirmed by Boyland andWilliams-Ashman (5), who reported a 50 per centinhibition with 1 per cent urethan.

The possibility of in vitro carcinogenesis by freeurethan in appropriate concentrations cannot,therefore, be ruled out on the basis of the experiments reported by Rogers (15).

Our attempts to find independent confirmationfor the existence of a carcinogenic metabolite, byin vivo testing of "U" plasma, failed.

The results of series I could mean that the postulated metabolite was present in too low a concentration to be detected by the methods used. However, the active agent was found to be volatile.Therefore, Chromatographie systems were devised,capable of detecting urethan or any other component with the same order of magnitude of volatility, as reported in the following communication(7). A Chromatographie analysis was made of "U"

plasma from rabbits and mice given injections ofcarbonyl-C14- or methylene-C14-labeled urethan,with a variety of solvent systems for the development of the chromatograms, yet no evidence couldbe found for any migrating component other thanthat of free urethan.

In series II of the present communication, amore sensitive in vivo test system was used, withadditional tests for the collected volatile components of the plasma (which also included free urethan). The results obtained could be completelyaccounted for on the supposition that the carcinogenic effects were due to urethan itself, without theparticipation of a metabolite of urethan or oneevoked by its presence in the body.

There still exists, however, the possibility of ametabolite being implicated, but one which is rapidly destroyed or one which is required in additionto free urethan. The latter possibility, though remote, cannot be ruled out by the present experiments.

Obtaining chemical evidence for the presence ofan intermediate metabolite of urethan is beset withdifficulties, as demonstrated by the work ofSkipper et al. (4,12,17) and the subsequent resultsreported from this laboratory (2). Experimentswith C14-labeled urethan have shown (a) that inmice urethan is almost completely eliminated fromthe body within 24 hours, in contrast to its veryslow catabolism in rabbits; (6) that the C14is uniformly distributed throughout the body; (c) that

its metabolism proceeds rapidly to complete breakdown into ammonia, carbon dioxide, and ethanol;(d) that the residual C14activity remaining in thebody after 5.5 hours is still mainly in the form offree urethan, most of which is present in the supernatant fraction of cell homogenates; and (e) thatbound C14activity can be accounted for by nonspecific incorporation of C14O2or C'^HjOH fromthe normal catabolic pathways of urethan.

Reference has already been made above to thedifficulty of interpreting negative results, in viewof the possibility that the postulated metabolitemight exist but be present in too low a concentration to be detected by the present methods of testing. It should be pointed out, however, that thestrongly positive results of the in vitro experimentsof Rogers (15) are in striking contrast to the negative results of the in vivo tests described here andto the equally negative results of the Chromatographie studies reported in the following paper (7).

It should also be pointed out that the negativeresults refer only to the question of a metabolitecirculating in plasma from urethan-injected animals. They do not exclude the possibility that acarcinogenic metabolite might develop and operatein situ in the tissues on which the urethan actscarcinogenically.

A different approach to the problem of the possible existence of carcinogenic intermediate metabolites of urethan was by the biological testing ofanalogs, possible degradation products, and substituted derivatives of the compound (see Larsen[9-11], Berenblum et al. [1]). No evidence of such ametabolite could be determined from these studies.

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DOT,A.; SIMON,E.; and TRAININ,N. Skin Initiating Actionand Lung Carcinogenesis by Derivatives of Urethane(Ethyl Carbamate) and Related Compounds. Biochem.Pharmacol., 2:168-76, 1959.

2. BERENBLUM,I., and HARAN-GHERA,N. A QuantitativeStudy of the Systemic Initiating Action of Urethane(Ethyl Carbamate) in Mouse Skin Carcinogenesis. Brit. J.Cancer, 11:77-84, 1957.

3. BERENBLUM,I.; HARAN-GHEBA,N.; WINNICK,R.; andWINNICK,T. Distribution of C'Mabeled Urethans in Tissues of the Mouse and Subcellular Localization in Lung andLiver. Cancer Research, 18:181-85, 1958.

4. BRYAN,C. E.; SKIPPER,H. E.; and WHITE, L., JR. Car-bamates in the Chemotherapy of Leucemia. IV. The Distribution of Radioactivity in Tissues of Mice Following Injection of Carbonyl-labeled Urethane. J. Biol. Chem.,177:941-50, 1949.

5. BOYLAND,E., and WILLIAMS-ASHMAN,H. G. The Influenceof Urethanes on the Enzymic Activity of Normal andMalignant Tissues. Acta Unióinternat, contra cancrum,7:43-2-85, 1951.

6. FISHER,R. A., and YATES,F. Statistical Tables for Bio-

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7. KATE, A. M. Chromatographie Studies in Relation toEthyl Carbamate (Urethan) Carcinogenesis Using C14-labeled Compounds. Cancer Research, 20:44^49, 1960.

8. KEITH, C. K., and DINNING,J. S. Inhibition of Rat LiverCholine Oxidase by Urethane in vitro. Fed. Proc., 9:189,1950.

9. LABSEN,C. D. Evaluation of the Carcinogenicity of aSeries of Esters of Carbamic Acid. J. Nat. Cancer Inst.,8:99-101, 1947.

10. . Pulmonary Tumor Induction with AlkylatedUrethans. Ibid., 9:35-37, 1948.

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12. MITCHELL,J. H., JR.; HUTCHISON,0. S.; SKIPPER,H. E.;and BRYAN,C. E. Carbamates in the Chemotherapy ofLeucemia. VII. The Rate of Catabolism of Urethane inNormal and Neoplastic Mice. J. Biol. Chem., 180:675-80,1949.

13. ROGERS,S. Age of the Host and Other Factors Affectingthe Production with Urethane of Pulmonary Adenomas inMice. J. Exper. Med., 93:427-49, 1951.

14. . Host Factors and Initiation of Pulmonary Adenomas in Mice with Urethane. Fed. Proc., 12:400-101, 1953.

15. . Studies of the Mechanism of Action of Urethan inInitiating Pulmonary Adenomas in Mice. I. The IndirectNature of Its Oncogenic Influence. J. Nat. Cancer Inst.»16:1675-83, 1955.

16. SKIPPER,H. E., et al. Free Urethane in the Blood of Experimental Animals Following Administration of an Anesthetic Level. Progress Report on Carbamates in the Chemotherapy of Leukemia. Birmingham, Alabama: SouthernRes. Inst., 1948, pp. 98 (quoted by Rogers [15]).

17. SKIPPER, H. E.; BENNETT,L. L., JR.; BRYAN,C. E.;WHITE,L., JR.; NEWTON,M. A.; and SIMPSON,L. Carbamates in the Chemotherapy of Leukemia. VIII. Over-allTracer Studies on Carbonyl-labeled Urethan, Methylene-labeled Urethan, and Methylene-labeled Ethyl Alcohol.Cancer Research, 11:46-51, 1951.

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1960;20:38-43. Cancer Res   I. Berenblum, A. M. Kaye and N. Trainin  Animals Given Injections of Ethyl Carbamate (Urethan)

Tests for a Carcinogenic Metabolite in Plasma ofIn Vivo

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