Mouse Bladder Carcinogenicity of Certain Tryptophan

Metabolites and Other Aromatic Nitrogen CompoundsSuspended in Cholesterol*

GEORGET. BRYAN,! R- R- BROWN,t ANDJ. M. PRICE§(Division of Clinical Oncology, University of Wisconsin Medical School, Madison, Wisconsin)

SUMMARY

The carcinogenicity of seven metabolites of the essential amino acid tryptophanand nine other nitrogen-containing aromatic compounds was studied by the individual suspension of these chemicals in cholesterol pellets and the surgical implantationof the pellets into the bladders of mice. Pellets consisting of the 8-methyl ether of xan-thurenic acid, xanthurenic acid, 8-hydroxyquinaldic acid, 3-hydroxy-L-kynurenine,and 3-hydroxyanthranilic acid—all metabolites of tryptophan—and N-hydroxy^-acetylaminofluorene, Ar-acetyl-2-naphthylhydroxylamine, and 2-naphthylhydroxyl-

amine suspended in this medium induced a statistically greater number of bladdercarcinomas than did cholesterol pellets alone. Pellets of 4,8-quinolinediol, kynurenicacid, 8-hydroxyquinoline-A^-oxide, 2,8-quinolinediol, 2,6-quinolinediol, 2-naphthyl-acetamide, 2-nitronaphthalene, and 2-amino-l-naphthol hydrochloride mixed withcholesterol were not significantly more active than were the pellets of cholesterol.The significance of the data is discussed and related to the abnormalities of tryptophanmetabolism observed in some patients with urinary bladder cancer.

The suggestion (9) that the abnormal metabolism of theessential amino acid tryptophan in some patients withurinary bladder cancer might be of causal significance inthis disease has received considerable experimental support. Dunning, Curtis, and Maun (16) demonstrated ahigh incidence of bladder tumors in rats fed DL-tryptophanand 2-acetylaminofluorene, whereas no bladder tumorswere present in those animals fed only supplemental 2-acetylaminofluorene. Brown et al. (8, 9) reported the excretion of significantly elevated quantities of kynurenine,3-hydroxykynurenine, acetylkynurenine, and kynurenic

* Supported in part by Grant Nos. E-115A, E-202B, and E-116Efrom the American Cancer Society; by Grant Nos. A-1127 andA-1499 from the National Institutes of Arthritis and MetabolicDiseases ; by Grant Nos. C-3274 and CF-8245 from the NationalCancer Institute, United States Public Health Service; and by theElsa U. Pardee Foundation.

These results are taken from a thesis submitted by Dr. GeorgeT. Bryan to the University of Wisconsin in partial fulfillment ofthe requirements for the degree of Doctor of Philosophy.

A preliminary report of this work has been made (Proc. Am.Assoc. Cancer Res., 4: 8, 1963).

t Part of this investigation was carried out during the tenure ofa Special Fellowship from the National Cancer Institute, UnitedStates Public Health Service.

t Supported by Research Career Development Award (No.CA-K3-18,404) of the National Cancer Institute, United StatesPublic Health Service.

§American Cancer Society—Charles S. Hayden FoundationProfessor of Surgery in Cancer Research.

Received for publication August 6, 1963.

acid in the urine of about one-half of a group of patientswith spontaneous bladder cancer. Boyland and Williams(7) observed abnormally large quantities of 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and anthranilic acidin the urine of ten patients with bladder carcinomas, andAbul-Fadl and Khalafallah (1) found that 3-hydroxyanthranilic acid occurred in the urine of patients withbilharzial bladder cancer in 4 times the concentration thatit was present in the urine of control subjects. However,patients with industrial bladder cancer excreted normalquantities of urinary tryptophan metabolites (28).

Allen et al. (2) reported that 3-hydroxy-DL-kynurenine,3-hydroxyanthranilic acid, and 2-amino-3-hydroxyace-tophenone were carcinogenic for the mouse bladder whensuspended in cholesterol pellets and surgically implantedinto this organ. The combined incidence of benign andmalignant tumors present in groups composed of smallnumbers of animals was used to arrive at these conclusions.Clayson et al. (14) were unable to demonstrate a significantnumber of malignant bladder tumors following implantation of 3-hydroxyanthranilic acid suspended in cholesterol.Attempts to induce a significant incidence of mouse bladdercarcinomas with metabolites of tryptophan suspended inparaffin wax (2, 12) by the pellet implantation technic (23)were not successful. The in vivoevaluation of the rate ofelution of several tryptophan metabolites from a cholesterol vehicle (10) demonstrated that these compounds wereleached out of this vehicle much more rapidly than they

596

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

BKYANet al—Carcinogenicity of Tryptophan Metabolites 597

were from paraffin wax (12). Because this more rapidrate of exposure of the bladder mucosa to the test chemicalmight be more favorable to the induction of bladder neoplasia in this test system, and because it is consideredessential to ascertain whether these naturally occurringurinary metabolites possess oncogenic activity for theurinary bladder, it was decided to test a series of trypto-phan metabolites and several other aromatic nitrogen-containing compounds, for which elution data were available(10), for their mouse bladder carcinogenicity. These experiments are the subject of this report.

MATERIALS AND METHODS

The preparation of the chemicals (10,12) and the cholesterol pellets containing the test compounds (10) was previously described. The details of the animal selection andcare and of the surgical technic (2, 23) were presented (11).

Carcinogenicity studies.—Three separate experiments(Table 1) involving different groupings of the sixteen testcompounds and cholesterol were initiated. Pellets composed of cholesterol alone and pellets containing 2-nitro-naphthalene were included in all three experiments.AT-Hydroxy-2-acetylaminofluorene, JV-acetyl-2-naphthylhy-droxylamine, 2-naphthylhydroxylamine, 2-amino-l-naph-thol hydrochloride, and 2-naphthylacetamide were testedin Experiments 1 and 2. 2,6-Quinolinediol, 2,8-quino-linediol, 8-hydroxyquinoline-Af-oxide, 3-hydroxy-L-ky-nurenine, 3-hydroxyanthranilic acid, xanthurenic acid,8-hydroxyquinaldic acid, the 8-methyl ether of xanthurenicacid, 4,8-quinolinediol, and kynurenic acid were tested inExperiments 2 and 3. A group of animals in which a shamoperation was performed, consisting of (a) incision of thebladder, (6) momentary insertion of a pellet of pure cholesterol, (c) removal of the cholesterol pellet, and (d) closureof the bladder and suture of the abdomen, was included inExperiment 1. Six months elapsed between the completion of the surgical procedures on the animals included inExperiment 1 and the initiation of Experiment 2, and 3months passed between completion of the surgical procedures on the animals included in Experiment 2 and thebeginning of Experiment 3.

The following protocol was established prior to theinitiation of Experiment 1 and was utilized with the threeseparate experiments. A sufficiently large number of micewould be subjected to the surgical insertion of pellets containing either cholesterol alone or one additional compoundmixed with cholesterol so that a minimum of 30 animalswould survive longer than 175 days. The animals whosebladders contained pellets of the same composition wouldbe referred to as a group, and each compound would betested at least twice in separate experiments. A negativecontrol group of mice, given implants of pellets of purecholesterol, and a positive control group of mice, givenimplants of pellets of 2-amino-l-naphthol hydrochloride, abladder carcinogen for the mouse when implanted inparaffin wax pellets (4), were to be included in every experiment. However, because of the inability to obtain alarge enough number of survivors in the group exposed to2-amino-l-naphthol hydrochloride in Experiment 2 (Table1), and because of a scarcity of many of the other compounds, 2-nitronaphthalene was selected as the compound

to be tested in the three experiments. The animalswithin a group were allowed to survive until 455-490 daysfollowing surgery, or until approximately fifteen animalswere alive in any one group. Only animals surviving morethan 175 days were to be evaluated for the presence ofbladder carcinoma.

Pathological examination.—At autopsy the bladderswere distended with Bouin's fixative inserted through theurethra. The bladders were bisected in the mid-sagittalIme and inspected grossly, sectioned at 5 n, and stainedwith hematoxylin and eosin. We are indebted to Mrs.Betty Decker and Mr. Jerald Armstrong for the preparation of these sections and to Dr. A. M. Pamukcu, Professorof Pathological Anatomy, Faculty of Veterinary Medicine,University of Ankara, Turkey, for the histological studyand review of many of these sections.

The bladders were inspected for the presence of squa-mous metaplasia, benign tumors, and carcinomas and wereevaluated according to the criteria of Bonser and Jull (5).Carcinomas which were not observed to invade musclewere classified as Grade I ; those which were seen to invademuscle or which demonstrated other evidence of malignancy such as local métastaseswere classified as Grade II.Those bladders containing a benign and malignant tumoror harboring two or more carcinomas were counted ashaving only one malignant tumor. Only the total incidence of carcinomas was used to assess carcinogenicity.Statistical comparison of the incidence of carcinomasrelated to the introduction of pellets of cholesterol containing a test chemical was made with the cholesterol alonegroup, and probabilities of statistical significance werecomputed by the exact method for 2 X 2 tables (19).

RESULTS

The number of animals subjected to the surgical implantation of pellets, the distribution of the deaths of the animals for 75-day periods beginning 175 days after surgery,the total number of animals surviving a minimum of 175days following surgery, and the average survival of theanimals in a group are tabulated in Table 1 for Experiments 1, 2, and 3. Thirty-eight, 33, and 36 per cent of themice in Experiments 1, 2, and 3, respectively, lived beyond175 days. This contrasts with a survival of 85 per cent ofthe mice whose bladders were implanted with paraffinpellets (12). Most of the animals in Experiments 1, 2,and 3 that did not live for 175 days following surgery diedwithin 30 days owing to impaction of the pellets into theurethra. A few animals were lost because of cannibalism.The average survival of the individual groups of animalsranged from 307 days (2-amino-l-naphthol hydrochloride,Experiment 2) to 489 days (cholesterol alone, Experiment1). Those animals that were exposed to test chemicalsand that demonstrated a significantly greater incidence ofbladder carcinomas than cholesterol alone (Table 2) lived,on the average, fewer days than did the cholesterol controlgroups. All animals living longer than 175 days withpellets in their bladders survived an average of 410 days.The total number of animals whose bladders could beevaluated in any one group varied from eight (2-amino-l-naphthol hydrochloride, Experiment 2) to 50 (3-hydroxyanthranilic acid, Experiment 3). In most groups at least

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

598 Cancer Research Vol. 24, May 1964

TABLE 1SURVIVALOP MICE LIVINGMORE THAN175 DAYSFOLLOWINGBLADDERIMPLANTATION,

ANDINCIDENCEOP CHANGESIN MOUSEBLADDERSWITHIMPLANTSOP COMPOUNDSSUSPENDEDIN CHOLESTEROL

COMPOUNDShamCholesterol

aloneJV-H

ydroxy-2-acetyl-aminofluoreneN-Acetyl-2-naphthylhy-droxylamine2-Naphthylhydroxyla-mine2-Naphthylacetamide2-Amino-l-naphthol

•HC12-Nitronaphthalene2

,6-Quinolinediol2

,8-Quinolinediol8-Hydroxyquinoline-JV-oxide3-Hydroxy-L-kynurenine3-HydroxyanthranilicacidXanthurenic

acid8-Hydroxyquinaldic

acid8-Methyl

ether ofxan-thurenicacid4

,8-QuinolinediolKynurenic

acidEXPERI

MENTNO.1123121212121212323232323232323232323No.MICEGIVENIM

PLANTS388214072666872475611260597811180764177728275758997585989889067929192849484101NO.

MICE WHICH DIED ORWERE KILLED(DAYS)175-

2500111120242213400015201211124213500324251-3257320124523202141210108502353412324424326-4003574633154444604902255531313351723451342401-47551151614891510461435413915131583091334010101861429131634>47611229191481814171912111710918101215Total2632342122343022252730252583033152522323732492317285032232626332134232444AVER

AGESUR

VIVAL(DAYS)417489434411399451461330366411434438392307467457413438373429454401428386382454412399335443374398417430367415408SQUA-MOUSMETAPLASIA0000322021425020001000012161120111000BE

NIGNTU

MORS1942667132345154142212521342226501034CARCINOMASGradeI0103625111132121214041422263332383201GradeII0021221594412051011041644092332341226Total01248466105544172225082106621556646124427

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

BRYANet al—Carcinogenicity of Tryptophan Metabolites 599

twenty animals survived long enough to have their bladders subjected to microscopic inspection.

The incidence of microscopic changes in the mousebladders implanted with compounds suspended in cholesterol is listed in Table 1. No bladder carcinomas were observed in the group of animals in which a sham operationwas performed. The incidence of carcinomas noted forthe replicate groups of animals was in some cases strikinglyuniform—e.g., JV-acetyl-2-naphthylhydroxylamine (6/30—20 per cent, 6/22—27 per cent), 2-naphthylacetamide(5/30—17 per cent, 4/25—16 per cent), 3-hydroxy-L-kynurenine (6/23—26 per cent, 6/17—35 per cent).However, in other cases the duplication of tumor incidences among replicate groups of animals was poor—e.g.,3-hydroxyanthranilic acid (2/28—7 per cent, 15/50—30per cent), 2,8-quinolinediol (0/32—0 per cent, 8/37—22per cent), 2-nitronaphthalene (7/30—23 per cent, 2/33—6per cent, 2/15—13 per cent). Since most of the bladdersof the mice were inspected after the mice had surviveda definite period of time, no data were collected to determine the average time at which carcinomas werenoted. Some carcinomas were observed in the bladdersof animals that died prior to the termination of the experiment and those compounds that were associatedwith a significant incidence of carcinomas (Table 2) werealso associated with the early appearance of carcinomas—e.g., 3-hydroxy-L-kynurenine, 185 days; 2-naphthyl-hydroxylamine, 214 days; JV-acetyl-2-naphthylhydroxyl-amine, 301 days; and the 8-methyl ether of xanthurenicacid, 313 days following surgery.

The group demonstrating the greatest number of benigntumors was the cholesterol alone group in Experiment 1.However, this group on the average survived longer thanany other group, and most of the benign lesions were observed in the bladders of the oldest mice in this group.Other groups showed a comparably low incidence of benigntumors which did not parallel the distribution of carcinomas. A small number of bladders showed mucosalsquamous metaplasia.

The incidence of carcinomas and the probabilities ofstatistical significance are summarized for the combined,replicate groups of animals in Table 2. Only animalssurviving more than 175 days have been included in thisevaluation in order that the results obtained may be compared to the incidence of these lesions reported by otherworkers (2-4, 12, 14). Seven carcinomas were found inthe bladders of 87 mice exposed to the presence of cholesterol alone, a tumor incidence of 8 per cent.

Af-Hydroxy-2-acetylaminofluorene was the only amino-fluorene derivative tested in this series. The twelve carcinomas observed in the bladders of the 56 mice examined(21 per cent) were considered to be significant. A highlysignificant incidence of 29 per cent bladder carcinomaswas observed for the mice exposed to 2-naphthylhydroxyl-amine, and a significant incidence of 23 per cent bladdercarcinomas was found in those animals subjected to pelletscontaining JV-acetyl-2-naphthylhydroxylamine. 2-Naph-thylacetamide and 2-amino-l-naphthol, urinary metabolites of 2-naphthylamine (15, 35), and 2-nitronaphthalenewere found to be inactive when implanted in cholesterol.

Seven metabolites of tryptophan were tested for bladder

TABLE 2COMPARISONOF THE INCIDENCEOF CARCINOMASOBSERVED,

WITHTHE ELUTIONHALF-LIFE (T%) OF COMPOUNDS,FROMCHOLESTEROL

COMPOUNDCholesterol

aloneActive:*8-Methyl

ether of xanthurenicacid3-Hydroxy-L-kynurenine3-Hydroxyanthranilic

acid8-Hydroxyquinaldicacid./V-Acetyl-2-naphthylhydroxyl-amineXanthurenic

acid2-Naphthylhydroxylamine-V-Hydroxy-2-acetylaminofluoreneIn

active:f4,8-QuinolinediolKynurenic

acid8-Hydroxyquinoline-N-oxide2-Naphthylacetamide2

,8-Quinolinediol2,6-Quinolinediol2-Nitronaphthalene2-Amino-l

-naphthol hydrochlorideT»(DAYS)—0.75.25.36.19.147541074.26.39.923333697?CAKCINOUASNumber/

no.mice7/8718/5412/4017/7810/5212/5211/5515/5212/568/579/6812/819/558/697/4711/785/33Percent833302219232029211413151612151315Pvalue—0.00020.0020.010.050.030.040.0020.030.190.210.120.100.300.170.150.20

* P < 0.05.

t P > 0.05.

carcinogenic activity. The 8-methyl ether of xanthurenicacid was the most active of all the compounds tested inthese experiments, inducing eighteen bladder carcinomasin the 54 animals examined (33 per cent). 3-Hydroxy-L-kynurenine and 3-hydroxyanthranilic acid were found toproduce a highly significant incidence, whereas xanthurenicacid and 8-hydroxyquinaldic acid were noted to induce asignificant incidence of bladder carcinoma when comparedwith cholesterol alone. This represents the first demonstration of the bladder carcinogenicity of the 8-methylether of xanththurenic acid, the natural L-isomer of 3-hy-droxykynurenine, xanthurenic acid, and 8-hydroxyquinaldic acid. 3-Hydroxy-DL-kynurenine and 3-hydroxyanthranilic acid were reported by Allen et al. (2) tobe active as bladder carcinogens on the basis of the numberof both benign and malignant lesions observed. 4,8-Quinolinediol and kynurenic acid were inactive as mousebladder carcinogens in the present studies.

Three substituted quinoline compounds were includedin this study for comparison with the naturally occurringquinoline metabolites of tryptophan. 2,6-Quinolinediol,2,8-quinolinediol, and 8-hydroxyquinoline-N-oxide werenot found to be active as bladder carcinogens.

DISCUSSION

A low incidence of 8 per cent carcinomas was found whenpellets of cholesterol alone were implanted into mousebladders. This incidence is comparable to the 6.5 (3) and

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

600 Cancer Research Vol. 24, May 1964

9.0 (14) per cent of bladder carcinomas observed with thisvehicle by other workers. Hieger and Orr (21) demonstrated the weak sarcomagenic properties of cholesterolfollowing repeated subcutaneous injections into mice.Thus, though cholesterol possesses the advantages of beingreadily compressed into pellets without heating and ofallowing the test compounds to diffuse into the urine at afairly rapid rate (10), the low but constant incidence ofbladder carcinomas induced by this vehicle precludes thechoice of this substance as an ideal suspending media.However, no known vehicle is free from this objection, sincepellets made of molten (4) or crushed paraffin (3) wax orstearic acid (3) alone, inserted into mouse bladders, alsoinduced a small number of vesical carcinomas. The mechanism of the induction of bladder carcinomas by thesethree vehicles alone, paraffin, stearic acid, and cholesterol,is unknown.

It is possible that chemical decomposition of the cholesterol in the pellets occurs in situ in the bladder, giving riseto small amounts of relatively strong carcinogens or largequantities of relatively weak carcinogens. If this decomposition were effected only by the urine—i.e., if the addedpresence of the test chemical did not influence this hypothetical alteration of the cholesterol, then one would expectto observe the same incidence of tumors from group togroup. Clearly, this is not the case, since some groups ofanimals had a significantly greater number of bladder carcinomas than the cholesterol alone group (Table 2). Furthermore, no spectrophotometric or Chromatographieevidence of the interaction of organic urinary constituentswith cholesterol pellets could be detected (10). Alternatively, it is possible that the presence of high concentrations of test chemicals in the cholesterol might facilitatethe chemical decomposition of this substance. Underthese circumstances it might be anticipated that thechemical alteration of the cholesterol would be associatedwith a simultaneous chemical alteration of the test chemical, because few organic chemical reactions are knownwhere the interacting chemical species do not bothundergo chemical alteration, with the possible exception oforganic catalysts. It appeared probable that some of themore reactive and labile test compounds would demonstrate evidence of decomposition that might be detectedwith the methods used. Yet for most of the compoundsno evidence could be found, by Chromatographie or spectrophotometric methods of analysis, for the chemical alteration of the test chemicals either before or after theirimplantation into the mouse bladders (10). On the otherhand, the obviously extensive decomposition of 2-amino-l-naphthol hydrochloride in the cholesterol pellets implantedinto the bladders of mice (10) was not associated with asignificant incidence of neoplasms. Thus, there does notappear to be good evidence for a relationship between decomposition of the chemicals in the pellets and the incidence of carcinomas obtained.

The biological data indicate that the test compoundplays a major role in the induction of the bladder carcinomas observed and that the cholesterol vehicle plays aminor, if any, part in this process. 20-Methylcholan-threne administered locally to the skin (30), subcutaneoustissues, mammary gland, striated muscle, peritoneum,

spleen, lung, uterus, thyroid gland, kidney, liver (18),prostate gland (22), and a number of other organs in avariety of animals (20) induced a high incidence of malignant tumors. Chapman (13) reported that this compoundsuspended in mineral oil induced 70-80 per cent bladdercarcinomas when the bladder was implanted as a subcutaneous bladder cyst. The additional presence of a paraffinforeign body within the cyst did not have any effect on theincidence or the tune of appearance of these transplantabletumors. Bonser and co-workers (3) demonstrated a 49and 58 per cent incidence of bladder carcinomas followingthe introduction of paraffin wax pellets containing 20-methylcholanthrene into mouse bladders. Though thisexperiment was terminated after 40 weeks, it appears thatthe criticism (13, 26) that the yield of carcinomas obtainedby this technic is low with potent carcinogens is no longera serious one.

2-Naphthylhydroxylamine was demonstrated by Boy-land et al. (6) to induce a high incidence of peritonealsarcomas following repeated intraperitoneal injections inrats. This compound has been shown concurrently andindependently by three different groups of workers toinduce a significant incidence of 21 (3), 29, and 33 (3) percent bladder carcinomas when suspended in pellets ofcrushed paraffin wax (3), cholesterol (reported here), andstearic acid (3), respectively. It was reported (3) that thestearic acid pellets containing this compound disintegratedafter 2-3 weeks in situ. Thus, the period of exposure ofthe bladder to either the test chemical or the vehicle wasless than 10 per cent of the time that the test animals wereobserved. One-half of the 2-naphthylhydroxylaminesuspended in the cholesterol pellets disappeared in situafter 54 days (10), and, therefore, the exposure of thebladder mucosa to this compound was longer when suspended in cholesterol than when suspended in stearic acid.It is not known how long this compound remained in thecrushed paraffin vehicle. It is difficult to conceive of acommon carcinogenic decomposition product resultingfrom the interaction of 2-naphthylhydroxylamine withcholesterol, paraffin, and stearic acid. The only commondenominator in the three independent experiments wasthe presence in pellets of 2-naphthylhydroxylamine, andone must conclude that this compound played a most important role in the genesis of the bladder carcinomas thatwere observed in significant numbers. 2-Naphthylhydroxylamine has been isolated as a urinary metabolite of2-naphthylamine in man and the dog (32), and it is possiblethat it may be a proximate carcinogen of this industriallyhazardous compound.

2V-Hydroxy-2-acetylaminofluourene was reported byMiller, Miller, and Hartmann (25) as capable of inducinga wide variety of malignant tumors in the rat when administered by injection or in the diet, and was suggested to bea proximate carcinogen of 2-acetylaminofluorene. JV-Hy-droxy-2-acetylaminofluorene also induced a significantnumber of bladder carcinomas when tested in cholesterol.2-Aminofluorene, 2-amino-l-fluorenol, 2-amino-3-fluorenol,and 2-amino-7-fluorenol were inactive as bladder carcinogens when implanted in cholesterol (3, 14) and paraffin(3, 24), whereas 2-amino-5-fluorenol was inactive whensuspended in paraffin (24). All these fluorene derivatives

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

BRYAN et al—Carcinogenicity of Tryptophan Metabolites 601

have been found to be urinary metabolites of 2-acetyl-aminofluorene when this carcinogen is administered sys-temically to the rat (25, 33, 34). AT-Acetyl-2-naphthyl-hydroxlyamine was the third AT-hydroxy derivative todemonstrate bladder carcinogenicity when suspended incholesterol. This compound was demonstrated as aurinary metabolite of 2-naphthylamine in man (31). It ispossible that the JV-hydroxy amine metabolites of aromaticamines may be potent bladder carcinogens if presented tothe bladder mucosa in sufficiently high concentration.

3-Hydroxyanthranilic acid, a urinary metabolite oftryptophan, was shown capable of inducing a high incidence of myelogenous leukemia and one bladder carcinomawhen administered by repeated injection to mice (17).The data obtained in the present experiments indicate thatcertain metabolites of tryptophan including the 8-methylether of xanthurenic acid, 3-hydroxy-L-kynurenine, 3-hy-droxyanthranilic acid, xanthurenic acid, and 8-hydroxy-quinaldic acid are as potent as mouse bladder carcinogensin this test system as are some of the carcinogenic metabolites of 2-naphthylamine and 2-acetylaminofluorene.When considered in connection with the reports of alteredexcretion patterns of tryptophan metabolites observed tooccur in about one-half of a group of patients with spontaneous bladder cancer (8, 9, 28, 29) and the elevatedlevels of excretion of 3-hydroxyanthranilic acid by patientswith bilharzial bladder cancer (1), the data on the incidenceof carcinomas induced in mouse bladders with these tryptophan metabolites take on added interest. Furthermore,patients with industrial bladder tumors excreted normalquantities of these metabolites (28). If the increasedlevel of urinary tryptophan metabolites excreted bypatients with spontaneous bladder tumors (7-9, 29) wasthe result of the disease, patients with the clinically andhistologically similar industrial lesions might be expectedto excrete similar quantities of these same metabolites(28). In addition, data have been presented (11, 27) suggesting that Turkish cows with spontaneous bladdercarcinomas have quantitative abnormalities in the urinaryexcretion of tryptophan metabolites. The observationsreported in this study provide supporting circumstantialevidence suggesting that the urinary excretion of elevatedquantities of some of these, or possibly related, metabolitesmay be of causal significance in the etiology of human andbovine bladder cancer.

A comparison of the 50 per cent elution time (Tj) (10)and the incidence observed for the carcinogenicity of thesignificantly active and inactive compounds is presentedin Table 2. The most active compound, the 8-methylether of xanthurenic acid, was also the one most rapidlyeluted from the cholesterol pellets. On the other hand,JV-hydroxy-2-acetylaminofluorene was eluted from thepellets more slowly than any other compound; yet it tooinduced a significant incidence of bladder carcinomas.3-Hydroxy-L-kynurenine, 3-hydroxyanthranilic acid, and8-hydroxyquinaldic acid were active carcinogens and wereeluted with about the same T} value as 4,8-quinolinedioland kynurenic acid, which were not significantly active.In a similar manner, Ar-acetyl-2-naphthylhydroxylamineand 8-hydroxyquinoline-Ar-oxide were eluted at a comparable rate, yet the former was active as a carcinogen whereas

the latter was not. It appears, contrary to an earlier suggestion (23), that prolonged, uniform exposure of thebladder to the test compound is not a prerequisite for theinduction of a significant number of bladder carcinomas,at least when cholesterol and stearic acid (3) are used asthe vehicles. It is possible that the tumor incidencemight be significantly altered by a change of vehicle withan associated alteration of the elution half-life.

2-Amino-l-naphthol hydrochloride failed to inducebladder tumors when suspended in cholesterol. Theapparent decomposition of this compound when suspendedin cholesterol and exposed to urine has been described (10)and may have influenced the low incidence of tumors observed. The carcinogenicity (4) of this compound for theurinary bladder of the mouse has again been reported (3)when suspended in crushed paraffin pellets made by compression.

The technic of implantation into mouse bladders ofvehicles containing test compounds as an assay system forthe carcinogenicity of urinary substances is of value. Itrequires the evaluation of large numbers of animals thatare exposed to the same compound and are observed forprolonged periods of time. Statistical methods of analysismust be used to assess results, and it seems most reasonableto use the number of carcinomas as the sole criterion ofactivity of a compound. Little is known about the manyfactors that must play a role in the induction of tumors bythis method. However, some estimate of the probabledose of compound and the duration of exposure of thebladder to the chemical can be obtained by a study of thein vivo elution of chemicals from the pellets.

ACKNOWLEDGMENTS

The assistance of Mr. Carl Morris, Mr. John Drye, Miss NormaYess, Mrs. Betty Decker, Mrs. Paula White, and Mrs. MaryBryan with the surgical procedure was greatly appreciated. Mr.Anthony Fuss and Miss Sharon Boehm provided valuable helpwith the inspection, care, and maintenance of the animals.

REFERENCES

1. ABUL-FADL, M. A. M., ANDKHALAFALLAH,A. S. Studies onthe Urinary Excretion of Certain Tryptophan Metabolites inBilharziasis and Its Possible Relation to Bladder Cancer inEgypt. Brit. J. Cancer, 16:479-82, 1961.

2. ALLEN, M. J.; BOYLAND,E.; DUKES, C. E.; HORNING, E. S.;ANDWATSON,J. G. Cancer of the Urinary Bladder Induced inMice with Metabolites of Aromatic Amines and Tryptophan.Brit. J. Cancer, 11:212-28, 1957.

3. BONSER, G. M.; BOYLAND,E.; BUSBY, E. R.; CLAYSON,D. B.;GKOVER,P. L.; ANDJULL, J. W. A Further Study of BladderImplantation in the Mouse as a Means of Detecting Carcinogenic Activity: Use of Crushed Paraffin Wax or Stearic Acidas the Vehicle. Brit. J. Cancer, 17:127-36, 1963.

4. BONSBR, G. M.; BHADSHAW,L.; CLAYSON,D. B.; ANDJULL,J. W. A Further Study of the Carcinogenic Properties ofOrtho Hydroxy-Amines and Related Compounds by BladderImplantation in the Mouse. Brit. J. Cancer, 10:539-46, 1956.

5. BONSER, G. M., ANDJULL, J. W. The Histological Changes inthe Mouse Bladder Following Surgical Implantation of Paraffin Wax Pellets Containing Various Chemicals. J. Pathol.Bacterio!., 72:489-95, 1956.

6. BOYLAND,E.; DUKES, C. E.; ANDGROVER,P. L. Carcinogenicity of 2-Naphthylhydroxylamine and 2-Naphthylamine.Brit. J. Cancer, 17:79-84, 1963.

7. BOYLAND,E., ANDWILLIAMS,D. C. The Metabolism of Tryptophan. 2. The Metabolism of Tryptophan in Patients Suffering from Cancer of the Bladder. Biochem. J., 64:578-82, 1956.

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

602 Cancer Research Vol. 24, May 1964

8. BROWN,R. R.; PRICE, J. M.; SATTER, E. J.; ANDWEAR, J. B.The Metabolism of Tryptophan in Patients with BladderCancer. Acta Unió Intern. Contra Cancrum, 16:299-303, 1960.

9. BROWN,R. R.; PRICE, J. M.; ANDWEAR, J. B. The Metabolismof Tryptophan in Bladder Tumor Patients. Proc. Am. Assoc.Cancer Res., 2:7, 1955.

10. BRYAN,G. T.; BROWN,R. R.; MORRIS, C. R.; ANDPRICE, J. M.In Vivo Elution of Tryptophan Metabolites and Other Aromatic Nitrogen Compounds from Cholesterol Pellets Implanted into Mouse Bladders. Cancer Res., 24:586-95, 1964.

11. BRYAN,G. T.; BROWN,R. R.; ANDPRICE, J. M. Studies on theEtiology of Bovine Bladder Cancer. Ann. N. Y. Acad. Sci.,108:924-37, 1963.

12. •.Incidence of Mouse Bladder Tumors Following Implantation of Paraffin Pellets Containing Certain TryptophanMetabolites. Cancer Res., 24:582-85, 1964.

13. CHAPMAN,W. H. A Method for Producing Bladder Tumors inMice, with a Technique Allowing Easy Interpretation of theDifference between Hyperplasia and True Tumor. J. Urol.,88:518-26, 1962.

14. CLAYSON,D. B.; JULL, J. W.; ANDBONSER, G. M. The Testingof Ortho Hydroxy-Amines and Related Compounds by Bladder Implantation and a Discussion of Their Structural Requirements for Carcinogenic Activity. Brit. J. Cancer, 12:222-30, 1958.

15. DOBRINER,K.; HOPMANN,K.; ANDRHOADS,C. P. The Metabolism of /3-Naphthylamine by Rats, Rabbits and Monkeys.Science, 93:600-601, 1941.

16. DUNNING, W. F.; CURTIS, M. R.; ANDMAUN, M. E. The Effectof Added Dietary Tryptophane on the Occurrence of 2-Acetyl-aminofluorene-Induced Liver and Bladder Cancer in Rats.Cancer Res., 10:454-59, 1950.

17. EHRHART, H.; GEORGII, A.; AND STANISLAWSKI,K. Untersuchungen überexperimentelle Leukämien. Überdie leukämo-gene Wirkung von 3-Hydroxyanthranilsäure bei RFH-Mäusen.Klin. Wochschr., 37:1053-59, 1959.

18. ESMARCH,O. Deposition of Methylcholanthrene in Some Organs of the Rat. Acta Pathol. Microbio!. Scandinav., 19:79-99,1942.

19. FISHER, R. A. Statistical Methods for Research Workers, 13théd.,pp. 96-97. New York: Hafner Pubi. Co., Inc., 1958.

20. HARTWELL, J. L. Survey of Compounds Which Have BeenTested for Carcinogenic Activity, 2d Ed., pp. 284-318. PublicHealth Service Publication «149.Washington: U. S. Gov'tPrinting Office, 1951.

21. HIEGER, I., ANDORB, S. F. D. On the Carcinogenic Activity ofPurified Cholesterol. Brit. J. Cancer, 8:274-90, 1954.

22. HORNING, E. S. Induction of Glandular Carcinomas of theProstate in the Mouse. Lancet, 2:829-30, 1946.

23. JULL, J. W. The Induction of Tumours of the Bladder Epithelium in Mice by the Direct Application of a Carcinogen. Brit.J. Cancer, 5:328-30, 1951.

24. LARSON,D. M.; GUTMANN,H. R.; ANDIRVING, C. C. Evaluation of the Carcinogenicity of Aminofluorenols by BladderImplantation. Proc. Am. Assoc. Cancer Res., 4:37, 1963.

25. MILLER, E. C.; MILLER, J. A.; ANDHARTMANN,H. A. N-Hy-droxy-2-acetylaminofluorene : A Metabolite of 2-Acetylamino-fluorene with Increased Carcinogenic Activity in the Rat.Cancer Res., 21:815-24, 1961.

26. MILLER, J. A.; WYATT,C. S.; MILLER, E. C.; ANDHARTMANN,H. A. The 7V-Hydroxylation of 4-Acetylaminobiphenyl by theRat and Dog and the Strong Carcinogenicity of A'-Hydroxy-4-acetylaminobiphenyl in the Rat. Cancer Res., 21:1465-73,1961.

27. PAMUKCU,A. M.; BROWN, R. R.; AND PRICE, J. M. Tryptophan Metabolites in the Urine of Turkish Cows with UrinaryBladder Cancer. Cancer Res., 19:321-25, 1959.

28. PRICE, J. M., ANDBROWN, R. R. Studies on the Etiology ofCarcinoma of the Urinary Bladder. Acta UnióIntern. ContraCancrum, 18:684-88, 1962.

29. PRICE, J. M.; WEAR, J. B.; BROWN,R. R.; SATTER,E. J.; ANDOLSON,C. Studies on Etiology of Carcinoma of Urinary Bladder. J. Urol., 83:376-82, 1960.

30. RUSCH,H. P.; KLINE, B. E.; ANDBAUMANN,C. A. The Nonad-ditive Effect of Ultraviolet Light and Other CarcinogenicProcedures. Cancer Res., 2:183-88, 1942.

31. TROLL, W.; BELMAN, S.; AND RINDE, E. N-Hydroxy AcetylAmino Compounds, Urinary Metabolites of Aromatic Aminesin Man. Proc. Am. Assoc. Cancer Res., 4:68, 1963.

32. TROLL, W., AND NELSON, N. N-Hydroxy-2-napthylamine, aUrinary Metabolite of 2-Naphthylamine in Man and Dog.Federation Proc., 20:41, 1961.

33. WEISBURGER, E. K., AND WEISBURGER, J. H. Chemistry,Carcinogenicity, and Metabolism of 2-Fluorenamine andRelated Compounds. Adv. Cancer Res., 5:331-431, 1958.

34. WEISBURGER,J. H.; WEISBURGER,E. K.; ANDMORRIS, H. P.Aminofluorenols as Urinary Metabolites of N-2-Fluorenyl-acetamide. J. Nati. Cancer Inst., 27:145-52, 1961.

35. WILEY, F. H. The Metabolism of /3-Naphthylamine. J. Biol.Chem., 124:627-30, 1938.

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1964;24:596-602. Cancer Res   George T. Bryan, R. R. Brown and J. M. Price  Suspended in CholesterolMetabolites and Other Aromatic Nitrogen Compounds Mouse Bladder Carcinogenicity of Certain Tryptophan

  Updated version

  http://cancerres.aacrjournals.org/content/24/4_Part_1/596

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/24/4_Part_1/596To request permission to re-use all or part of this article, use this link

on July 2, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from