in vivo elution of tryptophan metabolites and other...

In Vivo Elution of Tryptophan Metabolites and Other AromaticNitrogen Compounds from Cholesterol Pellets Implanted

into Mouse Bladders*

GEORGET. BRYAN,f R. R. BROWN,i C. R. MORRIS ANDJ. M. PRICEÂ§(Division of Clinical Oncology, University of Wisconsin Medical School, Madison, Wisconsin)

SUMMARY

The rate of elution of certain aromatic amine and quinoline metabolites of trypto-phan and some aromatic hydroxyl amines, hydroxamic acids, and other nitrogen-containing aromatic compounds from cholesterol pellets implanted into mouse bladders has been studied. The time required for 50 per cent of the chemical to diffuseout of the pellet (TÂ¿)varied from 0.7 of a day for the most rapidly eluted compound to107 days for the most slowly eluted substance. The rate of elution was observed tofollow first-order kinetics, and a specific elution-rate constant (K) was calculated thatprovided a measure of the probable extent and duration of exposure of the mousebladder mucosa to the potential carcinogens. It was suggested that studies of thistype should be done in conjunction with attempts to induce bladder carcinomas bythe pellet implantation technic.

The development of the technic of surgical introduction,into the mouse bladder, of a pellet containing a suspectedcarcinogen (15) was presented in an attempt to directlyexpose the bladder mucosa to a test compound. Metabolism by the liver of the chemical under test would beminimized, and a high, local concentration of this substance would be available for interaction with the bladder(4, 5). The chemical would diffuse from the pellet intothe urine, and, since the pellet was being constantlywashed with fresh urine, diffusion would continue for aconsiderable period of time (15). It was suggested thatthe rate of diffusion of the chemical from the pellet mightbe of importance in determining the carcinogenic activityof the test compound on the bladder mucosa and that this

* 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 bythe Elsa 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., 3:307, 1962).

t Part of this investigation was carried out during the tenureof a Special Fellowship from the National Cancer Institute,United States Public Health Service.

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

Â§American Cancer Societyâ€”Charles S. Hayden FoundationProfessor of Surgery in Cancer Research.

Received for publication August 6, 1963.

factor would have to be considered in the choice of avehicle and in the evaluation of results (5).

Paraffin was found to be unsuitable as a vehicle for thetesting of tryptophan metabolites, because pellets ofuniform chemical composition could not be fashioned andbecause the test compounds were leached from the waxslowly by urine under the in vivo conditions of the car-cinogenicity experiments (8). It seemed essential, before initiating additional large-scale bladder carcino-genicity studies with a different vehicle, that methodsshould be developed (a) to make pellets of uniform chemical composition, (6) to measure the concentration of testchemical present in a sample of a group of pellets priorto implantation into mice, and (c) to measure the rate withwhich these chemicals disappeared from the pellets invivo. Cholesterol was selected as the vehicle to be studiedfirst because of the reported experience of other investigators with this substance (1, 11). This report describesthe methods developed to measure the in vivo elution ofcertain tryptophan metabolites and other aromaticnitrogen-containing compounds from cholesterol pelletsimplanted into mouse bladders.

MATERIALS AND METHODSPreparation of chemicals.â€”The preparation of 3-hy-

droxyanthranilic acid, 3-hydroxy-L-kynurenine, xanthu-renic acid, the 8-methyl ether of xanthurenic acid, 8-hy-droxyquinaldic acid, and 4,8-quinolinediol was described(8). Kynurenic acid was synthesized according to Riegelet al. (19) and purified as described by Besthorn (3).JV-Hydroxy-2-acetylaminofluorene was provided as a

586

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BRYANet al.â€”Elution of Tryptophan Metabolites 587

gift by Dr. J. A. Miller (17). 2-Nitronaphthalene wasobtained as a gift from K. H. Ferber.1 2-Amino-l-naphthol hydrochloride was prepared by Na2S204 reduction of 2-nitroso-l-naphthol in alkaline solution and re-crystallized from dilute HC1 (14). 2-Naphthylacetamidewas synthesized from 2-naphthylamine by acetylationwith acetic anhydride (13) (m.p., 135Â°-136Â°C.).22-Naph-

thylhydroxylamine was synthesized by the method ofBaudisch and FÃ¼rst (2) from 2-nitronaphthalene byreduction with H2S in alcoholic NH3 and purified byvacuum sublimation (m.p., 127Â°C.).

Ar-Acetyl-2-naphthylhydroxylamine was prepared bytwo different methods. Method A: 159 mg. of 2-naph-thylhydroxylamine was dissolved in 10 ml. of ethyl acetateand 0.3 ml. of acetic anhydride and warmed to 30Â°~40Â°C.

for 1 hour. The solvents were then removed in vacuo.The residue was dissolved in 20 ml. of 95 per cent ethanoland 20 ml. of 5 per cent NaOH and hydrolyzed at 50Â°-60Â°C.for 10 minutes. The solution was cooled promptly

and extracted twice with benzene. The aqueous phasewas neutralized to pH 6 when it became turbid and deposited a brown oil which solidified on cooling overnight.This solid was recrystallized from benzene and petroleumether yielding 84 mg. of faintly pink crystals meltingat 84Â°-85Â°C.No depression of the melting point was

observed when a sample of this product was mixed witha sample prepared by method B. Method B: 6.92 gm.of 2-nitronaphthalene were dissolved in 150 ml. of ethylacetate and 30 ml. of acetic anhydride. Approximately0.6 gm. of 10 per cent palladium on charcoal was added,and hydrogÃ©nationwas carried out under a pressure of 50p.s.i. until two equivalents of hydrogen were taken up.The catalyst was filtered off, and the solvent was removedby vacuum distillation. The residue was dissolved in 75ml. of ethanol, 200 ml. of 5 per cent NaOH was added,and the mixture was heated at 55Â°-60Â°C.for 10 minutes.Crystals of 2-naphthylacetamide which deposited duringhydrolysis were removed, after cooling, by three extractions with benzene. The aqueous layer was neutralizedto pH 6 with 12 N HC1, and after traces of benzene wereremoved by bubbling with nitrogen the oily depositsolidified. The dry solid was dissolved in chloroform,decolorized with charcoal, and crystallized by addingpetroleum ether. The yield was 1.03 gm. of light tan-colored rosettes, m.p. 84Â°-86Â°C.This product wasfurther purified by vacuum sublimation. When re-crystallized from aqueous solutions, the product underwent a phase change at 68Â°C.followed by melting at84Â°-86Â°C.The product gave a strong hydroxamic acid

test with ferric chloride in dilute HC1 and gave no meltingpoint depression when mixed with the product of methodA.

8-Hydroxyquinoline-.V-oxide was prepared from 8-hy-droxyquinoline (Eastman Organic Chemicals) by re-fluxing with 30 per cent hydrogen peroxide in glacialacetic acid (18) and was recrystallized from dilute ethylalcohol (m.p., 142Â°-143.5Â°C.).2,8-Quinolinediol wassynthesized from 8-hydroxyquinoline-A''-oxide by re-

fluxing with acetic anhydride, filtering the crystals of

1National Aniline Co., Buffalo, N. Y.1All the melting points were uncorrected.

2-hydroxy-8-acetoxyquinoline, warming these with diluteNaOH, and recrystallizing the final product from diluteacetic acid (m.p., 280Â°-287Â°C.,dec.). 2,6-Quinolinediolwas prepared from 6-methoxyquinoline (Eastman OrganicChemicals) by treatment with hydrogen peroxide inglacial acetic acid, refluxing the crude crystals of 6-meth-oxyquinoline-./V-oxide with acetic anhydride, and refluxingthe impure 2-hydroxy-6-methoxyquinoline thus obtainedwith 57 per cent HI and red phosphorous. The finalproduct was recrystallized from dilute acetic acid (m.p.,315Â°-320Â°C.,dec.). Commercial cholesterol was re-

crystallized from ethyl alcohol just prior to use (m.p.,151Â°C.). Stearic acid (m.p., 69Â°-70Â°C.)was obtained

from commercial sources (Matheson). All the compoundswere carefully purified before being used as analyticalstandards or being compressed into pellets.

Preparation of pellets.â€”Cholesterol, or stearic acid,and each chemical which was selected to be tested forcarcinogenic activity were separately ground to a finepowder in an agate mortar. The test compound was thencarefully mixed with 4 times its weight of cholesterol orstearic acid by grinding thoroughly in a mortar. Themixture was compressed into spheroidal pellets, \fa inchin diameter and weighing 18-22 mg. with a deep, roundedcup die in a Stokes pellet press.3 The dies were dustedfrequently with fine magnesium stÃ©aratepowder to prevent capping of the pellet. Pellets of comparable sizewere also prepared from pure cholesterol or stearic acid.The reproducibility of the chemical composition of thepellets was greatly facilitated by preparing lots containingat least 50 pellets.

Analyses of the pellets.â€”Methods of assaying the pellets were developed to verify the concentration of thechemical under test in the cholesterol or stearic acidvehicle. The concentration of 3-hydroxy-L-kynureninewas measured by the method described by Brown (6).Pellets containing this compound were dissolved in 2 ml.of chloroform, and this was extracted with 5.0 ml. of 5N HC1. One ml. of this extract was diluted with 5 NHC1 to a final volume of 50 ml. in a volumetric flask andthoroughly mixed. A 2-ml. aliquot of this solution waspipetted into each of three colorimeter tubes, and 1 ml.of 5 N HC1 was added. After diazotization with NaNOs,as previously described (6), the optical density at 367m/i was measured in a modified Beckman DU spectro-photometer. A standard curve was run from a solution,prepared as described above, containing known quantitiesof cholesterol and 3-hydroxy-L-kynurenine in the sameratio as in the pellets.

Pellets containing each of the other fifteen test compounds, with the exception of 2-amino-l-naphthol hydro-chloride, were prepared for analysis by ultraviolet spectro-photometry in an identical manner. The pellets weretransferred to 25-ml.-capacity volumetric flasks, dissolvedin 1 ml. chloroform, a few drops of concentrated NH4Ã›Hwere added, and the contents of the flasks were diluted tovolume with 95 per cent ethanol. After complete mixingof the clear solution, an appropriate aliquot, usually 0.1ml., was diluted to 3.0 ml. with 95 per cent ethanol, andthe optical density of duplicate samples was measured in

1Eureka Model. F. J. Stokes Corp., Philadelphia, Penn.



588 Cancer Research Vol. 24, May 1964

a Beckman DU spectrophotometer. The blank used ineach analysis was prepared from a pellet of pure cholesterolor stearic acid of mass comparable to the test pellet,dissolved, and diluted in the same manner. A standardcurve for each compound was prepared from a solutioncontaining known quantities of cholesterol or stearic acidand one of the test chemicals in the same ratio as in thepellets. From the standard curve, the quantity of thecompound present in each pellet could be ascertained.No satisfactory method could be developed for the analysis of pellets containing 2-amino-l-naphthol hydro-chloride that had been implanted because of the apparentinstability of this chemical under these in vivo conditions.

In some instances the paper Chromatographie method ofMason and Berg (16) as modified by Brown and Price (7)was used for the qualitative identification of these compounds. The papers were inspected with ultravioletlight. Ultraviolet absorption spectra were frequentlyrecorded, before and after the implantation of pelletsinto mouse bladders, from solutions of pellets containingcholesterol alone or cholesterol plus test compounds in aneffort to detect either absorption of urinary aromaticconstituents by the cholesterol vehicle or to determinewhether the test chemicals were stable during the processof making the pellets as well as in vivo.

In vitro elution of chemicals from the pellets.â€”Themethod described by Allen et al. (1) was used with minormodifications to determine the in vitro elution of the8-methyl ether of xanthurenic acid. Five pellets containing this compound compressed with cholesterol wereincubated with gentle shaking in separate flasks with 0.5ml. of distilled water at 38Â°C. After 3, 6, 9, and 12 hours

the pellets were removed, transferred to flasks containing0.5 ml. of water, and the incubation was continued; 0.2-ml.aliquots of the aqueous fluid were diluted to a final volumeof 3.0 ml. with 95 per cent ethanol, and the optical densityat 336 mu was measured in a Beckman DU spectrophotometer. Following completion of the incubation at 24 hoursthe pellets were analyzed for residual 8-methyl ether ofxanthurenic acid as described above.

Animal selection and care.â€”Swissalbino female mice4were housed in individual screen-bottomed metal cages.The animals were 60-120 days old at the time of surgeryand were fed a diet of Rockland5 pellets and water adlibitum. The mice were anesthesized with pentobarbitalsodium (Nembutal sodium, Abbott Laboratories) andether. Pellets were inserted into the mouse bladders bythe technic of Jull (15) as modified by Allen et al. (1).

In vivo elution of chemicals from the pellets.â€”Thepelletsused in the elution study were selected at random fromthose used for the carcinogenicity studies (9). Thepellet was weighed prior to introduction into the bladderof the mouse, and the content of the test compound wascalculated. Following implantation of the pellets intothe bladders of several mice, the animals were killed atappropriate times, and the pellets were recovered. Thequantity of test compound remaining in the pellet wasdetermined, and from this and the calculated content oftest chemical present prior to insertion of the pellet into

4Rolfsmeyer Company, Madison, Wis.8A. E. Staley Manufacturing Company, Decatur, 111.

the mouse bladder an estimate of the amount of compoundthat had disappeared from the pellet during the timeinterval that it was in the bladder in vivo could be made.The per cent of compound remaining in a pellet after xdays in the bladder was converted to its common logarithm. When the analytical results, representing severalpellets remaining in the mouse bladders for varying periodsof time, were plotted graphically versus the number ofdays each pellet had been exposed to urine, a straight-line relationship was observed. The slope, 6, of theregression equation best representing this linear relationship was computed by the method of least squares, andtested for statistical significance by the F test (20). Aspecific elution-rate constant, K, could be calculated foreach compound by multiplying the slope, b, by (â€”)2.303.The time required for 50 per cent of a compound to disappear from a pellet, Tj, was computed from K (12) andwas used as another index of the elution time. Coordinates (x,y) lying on the regression line were computedfrom the regression equation, and a graphic representationof this line was drawn for each compound studied (Charts1-16).

RESULTSIn vivo elution of chemicals from a cholesterol vehicle.â€”

Analysis of several pellets that were not inserted intomouse bladders revealed a satisfactory uniformity inchemical composition (Table 1). The widest range ofvalues was obtained with pellets containing the 8-methylether of xanthurenic acid, the four pellets analyzed varyingfrom 78 to 108 per cent of the calculated content. Therange of the averages for the groups varied from 91 to104 per cent of the calculated content of test chemical,with an average of 95 per cent for all compounds. Ultraviolet absorption spectra obtained for several pellets ineach group revealed no alteration of configuration whencompared with those available for the analytically purechemicals. Paper chromatograms of extracts of thesepellets demonstrated only one spot corresponding to thecompound known to be present in the pellets.

Spectrophotometric and paper Chromatographie analyses of pellets originally prepared from pure cholesterolshowed no evidence of absorption of organic urinary constituents after remaining in mouse bladders for 1 year.Furthermore, when pellets containing the test chemicalshad been present in the bladder long enough for half ormore of the chemical to be eluted from them, the ultraviolet spectrum of the chemical obtained from the pelletswas identical to that observed for pellets that had notbeen implanted in vivo.

Paper chromatograms run on extracts of the pelletsthat had been inserted into the mouse bladders revealedonly one spot, corresponding to the chemical known to bepresent in the pellet, with the exceptions of pellets whichhad contained 3-hydroxyanthranilic acid or 2-naphthyl-hydroxylamine. With each of these two exceptions, twoadditional spots of apparently minor quantitative importance were observed.

The loci of the experimentally determined coordinates,the linear elution curves, computed by the method ofleast squares from these values, the elution-rate constant,




2.0

1.5

5 i.ooocW(L

0.5

2.0

uiO.

5 LOoKWO.

oo

0.5

XANTHURENIC ACID 8-METHYL ETHER

OH

OCH,

T 1/2Â»0.7 days

KÂ»0.95 doyÂ«'1

2.0 2.0

1.5 1.5

UJa.

.0WuKUJa.oo

1.0

0.5 0.5

0.3 0.6 0.9 1.2 1.5 1.8 2.1DAYS

2.03-HYDROXY-L-KYNURENINEd

C-CHgCHCOOH

T 1/2=5.2 days

K-0.13 days"1

4,8-OUINOLINEDIOL 2

OH

2.0

1.5

.0

T 1/2-4.2 daysK Â»0.17 days"1

12DAYS

16

1.0

0.5

IO 15DAYS

20 25 30 12 16DAYS

CHART 1.â€”Elution curve of the 8-methyl ether of xanthurenic acid.CHART2.â€”Elution curve of 4,8-quinolinediol.

CHART3.â€”Elution curve of 3-hydroxy-L-kynurenine.CHART4.â€”Elution curve of 3-hydroxyanthranilic acid.

0.5

20 24

2.0

3-HYDROXY

ANTHRANILIC ACID

OH

T 1/2 = 5.3 days

K=O.I3 days'1

24 28




2.0

8-HYDROXYQUINALDIC ACID

OH

T 1/2 Â«6.1 days

K*0.ll doyÂ«'1

2.0

1.5

1.0

0.5

8 12 16 20 24 28DAYS

2.0

1.5

blIL

1.0111O

CE111a.oo

0.5

KYNURENIC ACID

OH

T 1/2 = 6.3 days

KÂ»O.II days'1

2.0

1.5

1.0

0.5

8 12 16 20 24 28DAYS

2.0

N-ACETYL-2-NAPHTHYLHYDROXYLAMINE

0H

1.5

a.Z

1.0 â€¢

a:Ua.oo

0.5

IO 20

T l/2=9.ldays

K=0.076days-l

2.0

1.5

1.0

0.5

30 40DAYS

50 60 70

2.0

1.5

LUa.

1.0

a:bJa.

0.5

8-HYDROXYQUINOLINE-N-OXIDE

T 1/2 = 9.9 days

K = 0.07 days'1

8

18 24

DAYS

CHART5.â€”Elution curve of 8-hydroxyquinaldic acid.CHART 6.â€”Elution curve of kynurenic acid.

CHART7.â€”Elution curve of Ar-acetyl-2-naphthylhydroxylamine.CHART8.â€”Elution curve of S-hydroxyquinoline-A^-oxide.

2.0

1.5

1.0

0.5

30 36 42




2.0

1.5

UJo.

IU)o.

(9O

_l

1.0

0.5

2.0

UJa.

uiuerbla.oO

1.0

9

2-NAPHTHYLACETAMIDE

0

T 1/2 = 23 dayÂ»KÂ«0.03 days "'

2.0

1.5

1.0

0.5

10 20 30 40

â€¢DAYS

50 60 70

11

2,6-OUINOLINEDIOL

HO

T 1/2 = 36 days

K'0.019 days~l

'

2.0

1.5

.0

0.5

10 20 30 40

DAYS50 60 70

2.0

1.5h-ul

a.z

tâ€”zUIote.UJ

a.oO

1.0

0.5

10

20r

u)a.

zUlueruia.

I.O

0.5

2,8- QUINOLINEDIOL

OH

T 1/2 = 33 doyi

K â€¢0.021 days"1

2.0

1.5

I.O

0.5

IO 20 30 40 50 60 70

DAYS

12

XANTHURENIC ACID

OH

OH

T 1/2 = 47 days

K = 0.015 days"1

2.0

1.5

0.5

IO 20 30 40 50 60 70

DAYS

CHART9.â€”Elution curve of 2-naphthylacetamide.CHART 10.â€”Elution curve of 2,8-quinolinediol.CHART 11.â€”Elution curve of 2,6-quinolinediol.CHART 12.â€”Elution curve of xanthurenic acid.




2.0

1.5

Uo.

1.0

ccUJo.oD

0.5

13

2-NAPHTHYLHYDROXYLAMINE

OH

TI/2 = 54 days

K â€¢0.013 days "'

2.0

1.5

1.0

0.5

10 20 30 40DAYS

50 60 70

2.0

1.5

LLl

a.

z 1.0

KUla.

oO

0.5

2.0

15

N-HYDROXY-2-ACETYLAMINOFLUORENE

OII

/CH3

N

T 1/2 Â«107days

K=0.0065 days

1.5

I.O

0.5

15 30 45 60 75 90 105

DAYS

2.0

1.5

1.0

enLUci

caO

0.5

2.0

1.5

lÃ¼CL

111oerLÃœa.

eo

as

2-NITRONAPHTHALENE

NO,

T 1/2 = 97 days

K=0.007I days ~'

2.0

1.5

1.0

0.5

20 40 60 80

DAYS100 IZO 140

16

XANTHURENIC ACID8-METHYL ETHER

4-- A T 1/2= 6.2 days

Â»- â€¢ T 1/2= 6.2 days

K=0.ll days"1

KÂ»0.lldays'1

2.0

1.5

1.0

0.5

12 16DAYS

20 24 28

CHART 13.â€”Elution curve of 2-naphthylhydroxylamine.CHART 14.â€”Elution curve of 2-nitronaphthalene.

CHART 15.â€”Elution curve of JV-hydroxy-2-acetylaminofluorene.CHART 16.â€”Elution curves of the 8-methyl ether of xanthurenic acid from a stearic acid vehicle. Experiment 1, A A;

experiment 2, O O.




TABLE 1CONTENT,PRIOR TO IMPLANTATION,OF RANDOMLYSELECTED

CHOLESTEROLPELLETS CONTAININGTEST COMPOUNDSANDRELATIVERETENTIONOFTHECOMPOUNDSIN THECHOLESTEROLPELLETSin Vivo

10

COMPOUND8-Methyl

ether of xanthurenicacid4,8-Quinolinediol3-Hydroxy-L-kynurenine3-Hydroxyanthranilicacid8-Hydroxyquinaldic

acidKynurenicacidAr-Acetyl-2-naphthylhy-droxylamine8-Hydroxyquinoline-JV-oxide2-Naphthylacetamide2

,8-Quinolinediol2,6-QuinolinediolXanthurenic

acid2-Naphthylhydroxylamine2-NitronaphthaleneN-Hydroxy^-acetylami-nofluoreneWAVE

LENGTH(HIM)*336270â€”339253331286242283258270340320304290PELLETS

ANALYZEDNo.455454445545455Per

centofcalculated

contentfoundRange78-10885-9887-10386-98103-10684-10688-9789-9296-10390-9692-10294-9692-10395-9894-97Aver

age9395969410494939199939995999695-bt

(days-Â«)0.41ÃŽ0.072Â§0.058Â§0.057Â§0.049Â§0.048ÃŽ0.033Â§0.031Â§0.013Â§0.0090Â§0.0084Â§0.0064Â§0.0056Â§0.0031Â§0.0028Â§

* Wavelength where compound being analyzed exhibited maximal ultraviolet absorption, with minimal interference from cholesterol.

t The ratio of the log of the per cent of the compound left inthe pellet divided by the number of days.

Ã•Significant, 0.05 > P > 0.01\â€ž.â€¢iÃ x T>^ n Â«iÂ¿Significancecalculated for theÂ§Highly significant, P < 0.01Ã•, , , . . ,'slope, 6 (obtained by the

method of least squares) by theF test.

K. and the 50 per cent elution time, Tj , are shown inCharts 1-15. The slope, o, of the regression equationand the level of statistical significance of b are indicatedin Table 1. The slope, 6, of the elution curve for the8-methyl ether of xanthurenic acid and for kynurenicacid was significant, with a P value lying between 0.05and 0.01. The slopes of the elution curves of the remaining compounds were determined to be highly significant, since P was less than 0.01 in all cases. The elution-rate constant, K, varied from a value of 0.95 days"1 forthe 8-methyl ether of xanthurenic acid to a value of0.0065 days"1 for A^-hydroxy-2-acetylaminofluorene. Itwas not possible to measure the rate of elution of 2-amino-1-naphthol hydrochloride from the cholesterol pellets,because these pellets turned a dark brown or black colorwithin a few hours after implantation into the urinarybladder. This discoloration was observed to persist inpellets remaining in the bladder for as long as 1 year.Pellets containing this compound gradually darkened overa period of several months if stored at room temperature;however, their original light grey color was retained for

oUJ

LÃœÃœ

Â£CLlJ<L

17

0 4 8 12 16 20 24 28

HOURSCHART17.â€”Invitro elution curve of the 8-methyl ether of

xanthurenic acid.

months if the pellets were stored in a refrigerator in thedark.

In vivo elution of the 8-methyl ether of xanthurenic acidfrom a stearic acid vehicle.â€”Thelinear elution curves, theelution-rate constant, K, and the 50 per cent elution tune,Tj , for the 8-methyl ether of xanthurenic acid suspendedin stearic acid and studied in two separate experimentsare shown in Chart 16. The P value of the slope of bothcurves was less than 0.01 and was highly significant.The calculated elution-rate constant, K, and the 50 percent elution time, Tj , for both curves were in good agreement, but were considerably different from the valuescalculated for the elution of this compound from a cholesterol vehicle (Chart 1).

In vitro elution of the 8-methyl ether of xanthurenic acidfrom a cholesterol vehicle.â€”Theelution curve obtained forthe 8-methyl ether of xanthurenic acid from cholesterolpellets incubated with water in vitro is demonstrated inChart 17. Since, following the surgical implantation ofpellets into their bladders, mice excrete 0.5-3.0 ml. ofurine per 24 hours,6 a volume of 2.5 ml. of water wasselected for the in vitro eluting fluid. At the end of the24-hour period of incubation an average of 98 per cent(90-103 per cent) of the 8-methyl ether of xanthurenicacid was recovered from the five pellets and their elutingfluids. About 8 per cent of this compound was elutedfrom the pellets in 24 hours and the rate of elution invitro did not follow first-order kinetics.

DISCUSSIONThe data in Table 1 demonstrate that pellets of uniform

chemical composition, in which the analytical values ofthe test compound closely resembles the calculated values,can be manufactured if careful mixing of the test chemicaland its vehicle is achieved prior to compression in a pelletpress. This contrasts with the wide variations of chemicalcomposition observed for many of these same compoundssuspended in paraffin (8).

The characteristics of a suitable vehicle for bladderimplantation experiments were discussed by other investigators (1, 5, 10, 11, 15). It was agreed that the

*G. T. Bryan and C. R. Morris, unpublished data.




compound under test should diffuse into the urine at anoptimum rate for tumor induction, but this rate was notdetermined. Allen el al. (1) observed that 3-hydroxy-anthranilic acid was carcinogenic when tested in pelletsin which cholesterol was the vehicle but not if paraffinwax was the vehicle. The diffusion of this compound fromthe two types of pellets into 1 ml. of water kept at 38Â°C.was studied. About one-half of the 3-hydroxyanthranilicacid diffused out of cholesterol pellets in 15 days whereasonly 6 per cent of the chemical was eluted from paraffinwax in 10 days (1). Previous experience (8) suggestedthat many of the tryptophan metabolites studied in thisseries of compounds diffused very slowly from paraffinwax in vivo and that failure to detect a significant incidence of bladder tumors with any one of these chemicalsmay have been, in part at least, related to this slow elution.The rate of elution of those compounds studied here underin vivo conditions, though variable (Charts 1-15), wasmuch more rapid than expected and was not at all uniformly prolonged.

The elution of the 8-methyl ether of xanthurenic acidand 3-hydroxyanthranilic acid (1) was studied in vivoand in vitro from cholesterol. It was found that the 8-methyl ether of xanthurenic acid diffuses much moreslowly from a cholesterol vehicle under in vitro conditions(Chart 17) than under in vivo conditions (Chart 1).Allen et al. (1) suggested from in vitro measurements thatthe diffusion of 3-hydroxyanthranilic acid from cholesterolwas proportional to the square of the amount of thiscompound present at any one time. The data obtainedin the present experiment for the fifteen compoundsstudied, including 3-hydroxyanthranilic acid, indicatethat the rate of elution from cholesterol in vivo is insteaddirectly proportional to the concentration of the testsubstance present in the pellet. It is possible that diffusion of a test chemical from a pellet occurs in vitro (in awater solution) in a manner different from diffusion invivo (in a mouse bladder). Thus, in the future, it wouldseem advisable to study elution of compounds from pellets under the identical in vivo conditions of the carcino-genicity experiments.

The demonstration that the elution of a test compoundfrom a pellet follows first-order kinetics provides a methodof measurement of the probable extent and duration ofexposure of the bladder mucosa to a potential carcinogenand suggests that the rate of elution may be increased ordecreased by increasing or decreasing, respectively, theconcentration of the test chemical in the vehicle. Therate of elution of a compound may also be altered by theuse of a different vehicleâ€”e.g., stearic acid (Chart 16)in place of cholesterol (Chart 1) with the 8-methyl etherof xanthurenic acid. Compounds that might be expectedto have comparable rates of elution because of structuralsimilarities did, in fact, show this relationship. Thus for3-hydroxy-L-kynurenine (Chart 3) and 3-hydroxyanthranilic acid (Chart 4), K was 0.13 days"1; for 8-hydrox-

yquinaldic acid (Chart 5) and kynurenic acid (Chart 6),K was 0.11 days"1; and for 2,8-quinolinediol (Chart 10)and 2,6-quinolinediol (Chart 11), K was 0.021 days"1and 0.019 days"1, respectively. Minor alterations of

substituent groups on ring structures produced major

changes in the elution half-life (Ti) and the specificelution-rate constant (K)â€”e.g., the 8-methyl ether ofxanthurenic acid (Chart 1), xanthurenic acid (Chart 12)and kynurenic acid (Chart 6).

The evidence presented indicates that no significantreaction occurred between the test compound and thevehicle. Furthermore, most of the chemicals tested werestable under the in vivo conditions of the assay for car-cinogenicity. The extraneous spots observed on paperchromatograms of extracts of pellets containing 3-hydroxyanthranilic acid or 2-naphthylhydroxylamine werejudged to be of little quantitative significance. Theseobservations suggest that any significant incidence oftumors observed with one or more of these compounds inthe bladder carcinogenicity studies may be induced bythe chemicals under test. 2-Amino-l-naphthol hydro-chloride was the only compound to show obvious evidenceof decomposition in vivo in the cholesterol pellets. Sincethese pellets were produced by compression without theuse of heat and since other compounds were recoveredquantitatively from pellets following compression, thedecomposition of 2-amino-l-naphthol hydrochloride in thepellet cannot be attributed to pyrolysis. It would be ofinterest to ascertain whether 2-amino-l-naphthol hydro-chloride is as unstable when suspended in heated paraffinwax.

ACKNOWLE DGMENTS

We are grateful to Mrs. Betty Decker, Mrs. Paula White, andMiss Norma Yess for assistance with the mouse surgery and withsome of the analytical methods.

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1964;24:586-595. Cancer Res George T. Bryan, R. R. Brown, C. R. Morris, et al. Mouse BladdersNitrogen Compounds from Cholesterol Pellets Implanted into

Elution of Tryptophan Metabolites and Other AromaticIn Vivo

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