Effect of Sodium PhÃ©nobarbitaland Sodium Saccharin in AIN-76A

[CANCER RESEARCH 46, 6160-6164, December 1986]

Effect of Sodium PhÃ©nobarbitaland Sodium Saccharin in AIN-76A Diet onCarcinogenesis Initiated with AL[4-(5-Nitro-2-furyl)-2-thiazolyI]formamideand jY,yV-Dibutylnitrosaminein Male F344 Rats1

Katsumi Imaida and Ching Yung Wang2

Department of Chemical Carcinogenesis, Michigan Cancer Foundation, Detroit, Michigan 48201

ABSTRACT

Promoting activities of sodium phÃ©nobarbital(PB) and sodium saccharin (SS), incorporated in a semisynthetic diet (AIN-76A), on 2-stageCarcinogenesis initiated with Â¿V-|4-(5-nitro-2-furyl)-2-thiazolyl)formam-ide (FANFT) or yV,/V-dibutylnitrosamine (DBN) in male F344 rats wereinvestigated. For the first 4 weeks of the experiment, weanling maleFischer rats were fed Wayne diet containing 0.2% FANFT or drinkingwater containing 0.005% DBN. The control rats were given the basaldiet and normal drinking water. Beginning at the fifth week, the rats weregiven the AIN-76A diet or this diet containing 0.05 or 0.15% PB or 5%SS. The experiment was terminated at the end of 100 weeks. PBsignificantly increased the incidence of transitional cell carcinoma of thebladder of the rats that had been treated with FANFT (P = 0.027). PBalso increased the incidence of bladder carcinoma of the rats that hadbeen treated with DBN, but the increase was not significant (P = 0.081).SS in the AIN-76A diet increased the incidence of bladder carcinoma inthe rats which had been treated with FANFT or DBN, but the increasewas not significant (P = 0.059 and 0.327, respectively). Both high andlow doses of PB, but not SS, significantly increased the incidence ofhepatocellular carcinoma in the rats that had been treated with DBN.None of the control rats that had been fed the basal diet or the basal dietcontaining low or high PB or 5% SS developed either bladder or livercarcinoma. These results demonstrate that PB promotes urinary bladderCarcinogenesis of rats initiated with FANFT but not with DBN. Incontrast to incorporation in commercial rat chows, SS incorporated inthe AIN-76A diet is very weak in promoting bladder Carcinogenesis. Onthe other hand, PB, but not SS, promotes hepatocarcinogenesis initiatedwith DBN. Neither PB nor SS promoted DBN-induced Carcinogenesis ofesophagus or forestomach.

INTRODUCTION

Since Berenblum and Shubik (2) demonstrated two-stageCarcinogenesis in mouse skin, it has been widely accepted thatchemical Carcinogenesis consists of at least two steps: initiationand promotion. Several compounds have been shown to bepromoters of bladder Carcinogenesis in the rat. These includesodium saccharin, DL-tryptophan (3,4), sodium L-ascorbate (5),butylated hydroxyanisole, butylated hydroxytoluene (6), andother antioxidants (7), derivatives of retinoic acids (8, 9), andurine (10). Other substances, such as allopurinol (11), pheno-thiazine and 2,5-di-0-acetyl-D-glucosaccharo-(l,4)(6,3)-dilac-tone ( 12), are cocarcinogens of FANFT3 in bladder carcinogen-

esis of the rat, but they have not yet been tested for promotingactivities. Allopurinol has been reported to be a weak promoterfor bladder Carcinogenesis initiated with BBN (13).

Received 7/29/85; revised 1/13/86, 6/2/86; accepted 9/3/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This report from the A. Taubman Facility for Environmental CarcinogenesisResearch was supported by N1H Grant CA23800 and an institutional grant fromthe Uniled Foundation of Detroit. A preliminary report was presented previously(1).

2To whom requests for reprints should be addressed.3The abbreviations used are: FANFT, A'-(4-(5-nitro-2-furyl)-2-thiazolyl]form-

amide; BBN, A'-butyl-A'-(4-hydroxybutyl)nitrosamine; DBN, AVV-dibutylnitrosa-minc; PB. sodium phÃ©nobarbital;HCC, hepatocellular carcinoma; SS, sodiumsaccharin.

PB is a well-known promoter of hepatocarcinogenesis (14-17). It also promotes thyroid Carcinogenesis in the rat initiatedwith 7V-nitrosomethylurea (18) or with A'-bis(2-hydroxypro-

pyl)nitrosamine (19). However, PB is an antipromoter of mammary Carcinogenesis induced by 4-aminobiphenyl in the rat(20). Our preliminary results have shown that PB promotesbladder Carcinogenesis initiated with FANFT in the rat (21).Contrary to our observation, it has been reported that PB doesnot promote bladder Carcinogenesis initiated with BBN in therat (22).

The present study investigated in various organs of the ratthe effects of 2 dose levels of PB on Carcinogenesis initiatedwith FANFT or DBN. In addition to bladder and liver tumors,DBN produced esophagus and forestomach tumors in the rat(23). Thus, promotion by PB could also be examined in esophagus and forestomach. SS was used as a positive control for thepromotion of bladder Carcinogenesis.

MATERIALS AND METHODS

Chemicals and Diets. FANFT was obtained from Saber Laboratories,Inc., Morton Grove, IL; DBN was prepared from n-dibutylamine andpurified by vacuum distillation (24); SS and PB were purchased fromSigma Chemical Co., St. Louis, MO. PB, 0.05 or 0.15%, was added toa semisynthetic basal diet [AIN-76A diet (25), which was prepared andpelleted by Teklad Test Diets, Madison, WI], SS was incorporated intothe basal diet at a concentration of 5%, mixed, and pelleted in the samemanner. Ethoxyquin had been omitted from the AIN diet. FANFT wasadded to powdered Wayne Rodent Blox (Allied Mills, Chicago, IL) ata concentration of 0.2%, mixed, and pelleted. These diets were storedat â€”20Â°C,and were brought to room temperature immediately before

being given to the rats.Animals. Weanling male F344 rats (Charles River Breeding Labo

ratories, Wilmington, MA) were housed 6/polypropylene rat cage withwood chips for bedding in an animal room with a 12-h light 12-h darkcycle at 24 Â±2Â°Cand about 60% relative humidity. They were randomly

divided into 3 groups: 2 groups were fed the Wayne diet and one groupthe FANFT diet. One group that was fed the Wayne diet was also givendrinking water containing 0.005% DBN. After being treated for 4weeks, all the rats were given the Wayne diet and normal drinkingwater for an additional week. The rats in each group were then randomized into 4 groups to give a total of 12 groups, as shown in Fig. 1, andthey were then housed 3 or 4 rats/cage.

After being in the experiment for 5 weeks as mentioned above.Groups 1, 5, and 9 were given the AIN-76A diet throughout theremaining period of the experiment. Groups 2, 6, and 10 were giventhe basal diet containing 0.05% PB. Groups 3, 7, and 11 were giventhe basal diet containing 0.15% PB, and groups 4, 8, and 12 were giventhe basal diet containing 5% SS. Drinking water and the diets wereprovided ad libitum throughout the experiment. The consumption ofdiet and water was monitored during the treatment with carcinogens.Body weights were measured periodically during the entire experiment.The experiment was terminated at the end of 100 weeks. Rats that diedduring this experiment or were sacrificed at the end of experiment werenecropsied. Complete necropsies were performed, and body, liver,kidney, and spleen weights were determined. The urinary bladder wasinflated with 10% phosphate-buffered formalin solution, fixed, and cutinto 6 to 8 strips. Esophagus and stomach were also inflated with

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EFFECT OF PB AND SS ON BLADDER CARCINOGENESIS

WEEKS

GROUP TREATMENT

1 CONTROL

2 CONTROL-0-05% PB [

4 5 100

*0.05% PB

3 CONTROL-0.15% PB

4 CONTROL-5% SS

5 FANFT

6 FANFT-O.05% PB

7 FANFT-0.15 % PB

g FANFT-5% SS

9 DBN

10 DBN-0.05% PB

1 1 DBN-0.15% PB

12 DBN-5% SS :Fig. 1. Experimental protocol for detecting promoting activities of PB and SS

after initiation with FANFT or DBN. In each group, 42 rats were used, exceptGroup 9 (43) and Group 10 (41), at beginning of this experiment. H. Wayne diet;m, 0.2% FANFT in Wayne diet; B, 0.005% DBN in drinking water and Waynediet; D, AIN-76A diet.

formalin, immediately opened longitudinally, and then fixed on flatpaper. Other organs were also fixed with formalin solution, sectioned,and stained with hematoxylin and eosin. The slides were examinedunder a light microscope. Student's / test, Fisher's exact 2x2 contingency table, and the x2 test using the 2x3 contingency table were used

for statistical analyses (26).Fifteen 24-week-old male F344 rats were used to determine the effect

of PB and SS on urine pH. They were randomly divided into 5 groupsand fed the AIN-76A diet, the AIN-76A diet containing 0.05 or 0.15%PB, or 5% SS, or the Wayne diet, respectively, for 3 days. Instead ofmeasuring the urine pH only in the morning (27), fresh urine sampleswere obtained by forced urination at 10 a.m., 12 a.m., and 3 p.m. ThepH was measured with pH paper (EM Laboratories, Inc., Elmsford,NY; pH range, 4.0 to 7.0 and 6.5 to 10.0).

RESULTS

Toxicky. Rats on the FANFT diet or DBN-containing waterreceived approximately 0.58 g FANFT or 0.021 g DBN. Theeffective number, average body weight, and survival of the ratsare summarized in Table 1. Treatment with FANFT or DBNdid not affect the final body weights of the rats. During thefeeding period and at the end of the experiment, the rats in thegroups fed 0.15% PB weighed slightly less than the rats in theirrespective control groups. However, the ratio of liver weight to

body weight was slightly increased by PB treatment. The bodyweights of the 5% SS-treated groups were significantly less thanthe control groups. Mean survival periods were not significantlydifferent between the individual groups.

Bladder Tumors. Bladder lesions were classified into 4 morphological types as described previously (28): simple hyperpla-sia, PN hyperplasia, papilloma, and carcinoma. The histolÃ³gica!evaluations of the urinary bladder are shown in Table 2. Ratsin the groups which had not been treated with FANFT or DBNdid not develop proliferative lesions in the bladder. The incidence of carcinoma in the groups treated with FANFT followedby basal diet, 0.05 and 0.15% PB (Groups 5 to 7) was 20, 38,and 49%, respectively. Comparing Group 5 with Groups 6 and7 using the 2x3 contingency table (26), it was observed that PBsignificantly increased the incidence of carcinoma (P = 0.027)as well as that of proliferative lesions, including hyperplasias,papilloma, and carcinoma (P = 0.027). Using Fisher's 2x2

contingency table, the carcinoma incidence was significantlyincreased by 0.15% PB (P = 0.007) but not by 0.05% PB (P =0.069), although the incidence was also increased by 0.05% PB.

For the groups in which carcinogenesis was initiated withDBN followed by basal diet, 0.05 and 0.15% PB (Groups 9 to11), the incidence of carcinoma was 12, 5, and 23%, respectively. Comparing Group 9 with Groups 10 and 11, PB treatment did not significantly increase the carcinoma incidence (P= 0.081), nor did it increase the incidence of proliferativelesions (P > 0.5). These results demonstrate that PB promotesbladder carcinogenesis initiated with FANFT but not withDBN.

SS, which is a known promoter of bladder carcinogenesis inthe rat, slightly increased the incidence of bladder tumors inthe rats in which carcinogenesis was initiated with FANFT orDBN (Groups 8 and 12), but the increases were not statisticallysignificant. It should be noted, however, that P for FANFT-SSversus FANFT is 0.059, which is close to the commonly accepted value for demonstration of significance.

Liver Tumors. Liver lesions were classified into 3 types:hyperplastic foci, neoplastic nodule, and HCC (29). The resultsare summarized in Table 3. The incidence of HCC was 7, 27,and 43%, for DBN (Group 9), DBN-0.05% PB (Group 10),and DBN-0.15% PB (Group 11), respectively. Thus, ingestionof PB significantly increased the incidence of HCC (P < 0.05for 0.05% PB and P < 0.001 for 0.15% PB) in the rats withDBN-initiated carcinogenesis.

Table 1 Average body and liver weights and survital period of rats treated with 0.2% FANFT or 0.005% DBN followed by 0.05 or 0.15% sodium phÃ©nobarbitalor 5%sodium saccharin

TreatmentGroup

Initiation12345

0.2%FANFT60.2%FANFT70.2%FANFT80.2%FANFT90.005%DBN100.005%DBN1

1 0.005%DBN120.005% DBNPromotion0.05%

PB0.15%PB5%SS0.05%

PB0.15%PB5%SS0.05%

PB0.15%PB5%

SSInitialno.

ofrats424242424242424243414242Effectiveno.ofrats394242344040393943374040Body

wtInitial72.5

Â±9.7Â°75.8

Â±7.975.0Â±8.674.8Â±8.375.3Â±8.773.1

Â±8.973.1Â±7.372.4Â±7.767.0Â±4.671.7

Â±8.269.9Â±10.070.4Â±6.5Final465.7

Â±66.9442.9Â±52.1392.5

Â±46.4*375.5Â±47.6*493.3

Â±64.2440.2Â±45.4e397.4Â±57.9e404.6Â±56.9e462.0

Â±70.8437.9Â±48.7397.5Â±37.9'389.5Â±56.2'LiverWt15.3

Â±1.913.7Â±2.913.5

Â±4.112.2Â±3.116.2Â±3.510.4

Â±1.614.7Â±4.412.8Â±2.515.8

Â±3.616.5+2.914.9Â±3.211.9Â±3.0%

wttobodywt3.3

Â±0.53.1Â±0.63.6

Â±1.33.1Â±0.83.4

Â±0.53.5Â±0.24.2Â±1.1e3.2

Â±0.53.6Â±0.54.1

Â±0.8''4.0

Â±OY3.1Â±0.8Survival(wk)95.1

Â±8.786.190.394.693.086.790.792.393.384.890.28.06.29.78.27.58.09.68.39.27.694.8

9.8" Mean Â±SD.* Significantly different from Group 1. at P < 0.001.c Significantly different from Group 5. at P < 0.001.d Significantly different from Group 9. at P< 0.01.' Significantly different from Group 9, at P < 0.001.'Significantly different from Group 9, at P < 0.02.

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Table 2 HistolÃ³gica!findings of the urinary bladder in rats treated with FANFT or DBN followed by 0.05 or 0.15% sodium phÃ©nobarbitalor 5% sodium saccharin

TreatmentGroup

Initiation1

234

5 0.2% FANFT6 0.2% FANFT7 0.2%FANFT8

0.2%FANFT90.005%DBN100.005%DBN1

1 0.005%DBN12

0.005% DBNPromotion0.05%

PB0.1 5%PB5%SS0.05%

PB0.15%PB5%

SS0.05%

PB0.15%PB5%

SSEffective

no. ofrats39

4242344040393943374040Normal3942

4234155

(P = 0.009)*8 (P =0.078)*,(P

=0.027)'16(/Â»=0.464)*3029

(P =0.269)*27(P =0.505)*,(P

>0.5)'26(P = 0.409)'Simple

hyperplasia0

00016216120Incidence"Papillary

ornodular

hyperplasia00

0064440303Papilloma0

000

1010632224Carcinoma0

000

8(20)15 (38) (/>= 0.069)*19(49)(/> =0.007)*,(P=0.027)'15<38)(/>

=0.059)*5(12)2

(5) (P =0.283)*9<23)(/>=0.152)*,(/>=

0.081)'7(18),(/>= 0.327)*

" When papillary or nodular hyperplasia, papilloma. and carcinoma were present in the bladder, only carcinoma was indicated. Similarly, only papilloma orpapillary or nodular hyperplasia was indicated when lower grade lesions were present.

* P value by Fisher's exact 2x2 contingency table with comparison to the respective control.c x2 tests for Groups 5, 6, and 7 or for Groups 9, 10, and 11 using a 2x3 contingency table.

Table 3 Histological findings of the liver and esophagus in rats treated with FANFT or DBN followed by 0.05 and 0.15% sodium phÃ©nobarbitalor 5% sodiumsaccharin

Treatment Liver Esophagus

Group123456789101112Initiation0.2%

FANFT0.2%FANFT0.2%FANFT0.2%FANFT0.005%DBN0.005%DBN0.005%DBN0.005%

DBNPromotion0.05%

PB0.15%PB5%

SS0.05%

PB0.15%PB5%SS0.05%

PB0.15%PB5%

SSEffective

no. ofrats394242344040393943374040CarcinomaNormal3726213230222033274020Foci1

(2.6)Â°9*(21.4)II*

(26.2)2

(5.9)6(15.0)6(15.0)8(20.5)4(10.3)5(11.6)2(5.4)07

(17.5)Nodule1

(2.6)7r(16.7)10*

(23.8)04

(10.0)11e(27.5)10

(25.6)2(5.1)g

(18.6)2\"(56.8)23*(57.5)12

(30.0)HCC000001

(2.5)1(2.6)03

(7.0)10e(27.0)11"(42.7)1

(2.5)Normal374242344040393953511Hyperplasia000000005(11.6)5(13.5)11(27.5)5(12.6)Papilloma000000007(16.3)11

(29.7)2(5.0)2

(5.0)In

situ000000006(14.0)6(16.2)4(10.0)4(10.0)Squamouscell0000000020

(46.5)12(32.4)18(45.0)18(45.0)

" Numbers in parentheses, percentage of effective number.* Statistically different from each control group, at P < 0.01.' Statistically different from each control group, at P < 0.05.d Statistically different from each control group, at P < 0.001.

Treatment with FANFT alone (Group 5) did not produceHCC, nor did it increase the incidence of liver nodules whencompared with the control group (Group 1). Furthermore, PBdid not significantly change the incidence of liver nodules inrats that had been treated with FANFT. PB alone slightlyincreased the incidence of liver nodules but did not produceHCC. SS did not increase the incidence of liver tumors in therats initiated with DBN or FANFT.

Tumors of Esophagus and Forestomach. Lesions of the esoph-

Other Organs. Tumors observed in other organs were unrelated to the treatment. The major tumors observed were asfollows: malignant lymphoma; pituitary adenoma and carcinoma; adrenal pheochromocytoma; lung adenoma; and testic-ular interstitial cell tumor.

Urinary pH. The average urinary pH of rats treated with theAIN-76A diet, the AIN-76A diet containing 0.05 or 0.15% PB,5% SS, or the Wayne diet, respectively, were: 5.7, 5.7, 5.7, 5.5,and 6.5 (10 a.m.); 5.8, 6.0, 6.2, 5.7, and 6.7 (12 a.m.); and 5.7,

agus were classified as hyperplasia, papilloma, carcinoma in 5.6, 5.7, 5.5, and 7.0 (3 p.m.). The urinary pH of rats treatedsitu, and SCC (30). The results are shown in Table 3. Esopha-

geal tumors were observed in the rats of Groups 9 to 12, whichhad been treated with DBN. However, neither PB nor SSsignificantly changed the incidence of esophageal tumors. Therats in other groups did not develop tumors of this organ.

DBN also induced papilloma and SCC in the forestomach(Table 4). Again, neither PB nor SS significantly changed theincidence of forestomach tumors.

Thyroid Tumors. Thyroid tumors were classified as follicularadenoma or adenocarcinoma, papillary adenoma or adenocar-cinoma, or C-cell adenoma or adenocarcinoma (18). The incidence of thyroid tumors is given in Table 4. It is noted thatneither FANFT, DBN, nor the sequential treatment with thesecarcinogens and PB or SS significantly increased the incidenceof thyroid tumors over the control groups.

with the AIN-76A diet was lower than that of rats treated withthe Wayne diet, regardless of whether the urine was collectedin the morning or in the afternoon.

DISCUSSION

The present study confirms our preliminary results that PBis a promoter of rat urinary bladder carcinogenesis initiatedwith FANFT (21). This study also demonstrates that PB,although it promotes hepatocarcinogenesis, does not promoteDBN-induced carcinogenesis in bladder, forestomach, or esophagus. In comparison with previously reported results (3, 4, 18,21, 22), our data show that SS is a better promoter of bladdercarcinogenesis when it is incorporated in commercial diets thanin the AIN-76A diet.

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Table 4 Incidence of tumors of thyroid and forestomach in rats treated with FANFTor DBN followed by 0.05 or 0.15% sodium phÃ©nobarbitalor 5% sodium saccharinNeither PB nor SS treatment significantly affected the incidence of thyroid or forestomach tumors.

TreatmentFfTpftivtGroup123456789101112Initiation0.2%

FANFT0.2%FANFT0.2%FANFT0.2%FANFT0.005%DBN0.005%DBN0.005%DBN0.005%

DBNPromotion0.05%

PB0.15%PB5%SS0.05%

PB0.15%PB5%SS0.05%

PB0.15%PB5%SSno.

ofrats394242344040393943374040Normal282330302827273426242534C-cellAdenoma919113108631412146Carcinoma000110020000ThyroidForestomachPapillaryAdenoma100000000000Adeno-carcinoma000001200000FollicularAdenoma001001102010Adeno-carcinoma100013301100Normal394242344040393935313928Papilloma000000006416Squamouscellcarcinoma000000002206

It is known that commercial rat chows contain componentssuch as antioxidants that may affect the carcinogenesis process(5-7). Therefore the AIN-76A diet was used as the basal dietduring the promotion phase of the experiment. Ideally, it wouldbe preferable to use chemically defined diets for both theinitiation and promotion phases so that factors that may affectinitiation by FANFT and DBN could be avoided. However, incontrast to commercial rat chows (3-7, 18, 22, 28), there areno initiation data available for either FANFT, DBN, or BBNadministered in conjunction with semisynthetic diets. Furthermore, in order to confirm our previous study (21), the sameprotocol was used in the present experiment. Since the Waynediet was used only during the initiation stage and 1 week beforethe beginning of the promotion stage for all the groups, it isexpected that the use of the Wayne diet should not compromisethe results of the promoting effects of PB or SS.

We have previously shown that the administration of 0.05%PB following the treatment of 0.2% FANFT for 6 weekssignificantly increases the incidence of bladder carcinoma andthe combined incidence of carcinoma and papilloma as well asthe combined incidence of hyperplasia, papilloma, and carcinoma (21). In that experiment, the incidence of bladder carcinoma in the group fed FANFT alone was 43% which wasconsidered to be too high for a promotion experiment. Therefore, the feeding period of FANFT was shortened to 4 weeksand consequently the incidence of bladder carcinoma was decreased to 20%. As it turned out, ingestion of PB increased theincidence of carcinoma of the bladder (P = 0.027; 2x3 contingency table), and the increase was significant by 0.15% PB butnot by 0.05% PB. Ingestion of PB also significantly increasedthe combined incidences of proliferative lesions, including hy-perplasias (P = 0.027; by 2x3 contingency table). The increasewas significant by 0.05% PB but not by 0.15% PB (using the2x2 contingency table). Because only 2 doses of PB were used,this dose-related promotion by PB could not be demonstratedby the method of probit analysis.

On the other hand, PB did not affect the incidence in rats ofurinary bladder carcinoma initiated with DBN (Table 2). Thismay be due to the low incidence of bladder carcinoma inducedby DBN alone. This is consistent with the observation thatingestion of 0.05% PB significantly promoted bladder carcinogenesis in the rats which had been fed 0.2% FANFT for 6 (21)but not for 4 weeks (Table 2). In addition, it has been reportedthat 2% BHA significantly increased in rats the incidence ofcarcinoma or papilloma of the urinary bladder initiated with0.05% BBN but not with 0.01% BBN (6). Alternatively, the

promotion by PB may be limited to specific initiating agents.This is consistent with the results of short-term experiments byNakanishi et al. (22) who reported that ingestion of 0.05% PBfollowing the initiation with BBN or 2-acetylaminofluorene inthe rat did not increase the incidence of hyperplasia of thebladder.

The present study demonstrates that PB is more potent thanSS in promoting bladder tumors in the rat (Table 2). PB alsoappears to be more potent than most other known promotersof rat bladder tumors. The effective concentrations of otherpromoters, such as tryptophan, butylated hydroxyanisole, bu-tylated hydroxytoluene, sodium ascorbate, and other antioxidants, are between 2 and 5% in the diet. The effective concentration of PB used in our studies (e.g., 0.15%) is far less thanthat required for these compounds.

Bladder carcinogenesis and bladder epithelial hyperplasiahave been promoted by the sodium salts of saccharin andascorbic acid but not by their free acids (7, 27, 31 ). Since theaddition of these sodium salts substantially increases the sodium content of the diet, this suggests that sodium ions maycontribute to the promoting activity of these compounds. However, the promoting activity of PB cannot be due to sodium ion,since the AIN-76A diet contains approximately 0.11% sodiumand PB, at a concentration of 0.15%, only contributes anadditional 0.014% sodium to the diet.

The results of urinary pH measurements showed that theurine of rats treated with the AIN-76A diet had a lower pHthan urine from animals fed Wayne diet. This observationsupports the hypothesis that the promotion of bladder tumorsby SS (31) and by sodium bicarbonate (27) may be linked to ahigh urinary pH. The higher urinary pH caused by ingesting5% SS in commercial rat chows as compared with 5% SS inthe AIN-76A diet may be the reason why bladder carcinogenesisinitiated with 0.2% FANFT in the rat is promoted by theingestion of SS only if it is in a commercial chow diet (4) butnot in the semisynthetic diet (Table 2). In any case, the presentdata demonstrate that bladder carcinoma promotion by PB isindependent of urinary pH.

PB treatment significantly increased the incidence of hyper-plastic foci and neoplastic nodules, but not that of HCC, in theliver of rats without prior treatment with DBN or FANFT(Groups 2 and 3). The incidence in rats of HCC and neoplasticnodules initiated with DBN was significantly increased by PB,and the promotion was related to the dose of PB. A dose-relatedpromotion in the rat of hepatocarcinogenesis initiated with 2-acetylaminofluorene has previously been reported (17, 32).

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The present study demonstrates that neither PB nor SSaffects DBN-induced carcinogenesis in forestomach or esophagus. Although reported to be a promoter of thyroid tumorsinitiated with /V-nitrosomethylurea (18, 33) or jY-bis(2-hydrox-ypropyl)nitrosamine (34), PB did not promote spontaneousadenocarcinoma of the thyroid gland (Table 4). PB also doesnot promote skin (35), lung (16), or kidney (36) tumors. Interestingly, 0.05% PB displays an antipromoting activity in 4-aminobiphenyl-induced mammary carcinogenesis in the rat

(20).

ACKNOWLEDGMENTS

We thank K. Zukowski and R. Overton for their assistance with theanimal experiments and the preparation of histolÃ³gica!specimens. Wealso thank Dr. J. Frederick for his assistance with statistical analysisand Dr. Charles M. King for a critical review of our manuscript.

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1986;46:6160-6164. Cancer Res Katsumi Imaida and Ching Yung Wang -Dibutylnitrosamine in Male F344 Rats

N,N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide and NAIN-76A Diet on Carcinogenesis Initiated with

Effect of Sodium Phenobarbital and Sodium Saccharin in

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Effect of Sodium PhÃ©nobarbitaland Sodium Saccharin in AIN-76A