[CANCER RESEARCH 43, 4454-4457, September 1983]

Promoting Effects of Sodium L-Ascorbate on Two-Stage Urinary BladderCarcinogenesis in Rats1

Shoji Fukushima,2 Katsumi Imaida, Takao Sakata, Takehiko Okamura, Masa-aki Shibata, and Nobuyuki Ito

First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan

ABSTRACT

The promoting effect of sodium L-ascorbate on two-stageurinary bladder carcinogenesis in F344 rats initiated with A/-butyl-A/-(4-hydroxybutyl)nitrosamine at levels of 0.01 and 0.05% in

drinking water was studied. Administration of 5.0% but not of1.0% sodium L-ascorbate in the diet significantly increased the

incidence and number of preneoplastic lesions, papillary ornodular hyperplasia, papilloma, and cancer of the urinary bladder.In groups given 5.0% sodium L-ascorbate, the urine was characterized by an apparent elevation of pH, a decrease of osmo-

lality, and an increase of MgNH4PO4 crystalline. Addition ofsodium L-ascorbate to the diet also resulted in increase in the

content of ascorbic acid and its metabolite, dehydroascorbicacid, in the urine. These results show that an extremely highdose of sodium L-ascorbate (5.0%) promotes urinary bladder

carcinogenesis under the present experimental conditions, whilea high dose (1.0%) does not.

INTRODUCTION

There have been many reports on the relationship betweenvitamins and the development of cancers (21, 28), and ascorbicacid (vitamin C) has attracted particular attention with regard tothe prevention of cancer (4). Ascorbic acid is known to blocknitrosamine formation by reaction with nitrites and amines (19),and it has also been shown to inhibit bacterial mutagenesis byA/-nitroso compounds (12). Therefore, it seems possible that its

addition to foods may prevent cancer in humans.The two-stage process of chemical carcinogenesis, initiation

and promotion, is now known to occur in many organs, includingthe urinary bladder (2, 5, 9, 13, 20). Previously, we studied in ascreening experiment the promoting activities of various chemicals in urinary bladder carcinogenesis in male rats (10). In thesepreliminary experiments, sodium L-ascorbate, the ionic form of

ascorbic acid, appeared to have promoting activity in urinarybladder carcinogenesis.

This paper reports studies on the promoting effect of sodiumL-ascorbate on urinary bladder carcinogenesis in rats.

MATERIALS AND METHODS

Animals. Male 6-week-old F344 rats (Charles River Japan, Inc., Ka-

nagawa, Japan) were used in the present experiments. The rats werehoused 5 per plastic cage (33.5 x 37.5 cm, 17.5 cm deep) with woodchips for bedding in an animal room with a 12-hr light-12-hr dark cycleat 22 ± 2° (S.D.) and 60% relative humidity. Body weights, food

1This work was supported by Grants-in-Aid for Cancer Research from the

Ministry of Education, Science and Culture of Japan, and the Ministry of Healthand Welfare of Japan.

2To whom requests for reprints should be addressed.

Received September 9, 1982; accepted May 25, 1983.

consumption, and water intake were measured once a week. Theamounts of food and water consumed for 2 consecutive days of a weekwere measured on a per cage basis.

Chemicals. BBN3 was obtained from Izumi Chemical Co., Yokohama,

Japan. Sodium L-ascorbate, obtained from Wako Pure Chemical Ind.,

Ltd., Osaka, Japan, was reagent and food additive grade and was 99.8%pure (Lot DPP3987) in Experiment 1 and 99.2% pure (Lot DPG1180) inExperiments 2 and 3. It was added to Oriental M powdered basal diet(Oriental Yeast Co., Tokyo, Japan) at levels of 1.0 and 5.0%. Analysis ofa food sample showed that the actual levels of sodium L-ascorbate

added at levels of 1.0 and 5.0% were 0.94 and 4.91% initially and 0.92and 4.81% after 2 weeks at room temperature (Japan Food ResearchLaboratories, Tokyo, Japan).

Experiment 1. Rats were randomly divided into 4 groups of 20 ratseach. Groups 1 and 2 were given drinking water containing 0.01% BBNfor 4 weeks, 7 days a week, and then powdered diet containing 5.0%(Group 1) or 1.0% (Group 2) sodium L-ascorbate for 32 weeks, 7 days a

week. Group 3 was given only BBN for 4 weeks. Group 4 was givendrinking water without BBN for 4 weeks and then powdered diet containing 5.0% sodium L-ascorbate. The total observation period was 36

weeks.Experiment 2. Rats were randomly divided into 3 groups of 30 rats

each. Groups 1 and 2 were given 0.05% BBN in their drinking water for4 weeks and then given powdered diet containing sodium L-ascorbate

at levels of 5.0 and 1.0%, respectively, for 32 weeks. Control Group 3was given only 0.05% BBN for 4 weeks. The total observation periodwas 36 weeks.

For urine examination in Experiments 1 and 2, samples of urine wereobtained from 5 rats in each group in both experiments 2 days beforesacrifice. Rats were individually housed in metabolic cages without foodor water for 4 hr in the early morning. The pH, osmolality, and otherparameters of the samples were measured, and 2 ml of the remainderof the samples were concentrated and examined microscopically forurinary sediment.

The liver and kidneys were removed, weighed, and fixed in 10%phosphate-buffered formalin solution. The urinary bladder was inflatedby intraluminal injection of 10% phosphate-buffered formalin solution andwas then cut into 8 strips. The urinary bladder was examined histologi-

cally. For quantitative analysis, urinary bladder lesions were counted bylight microscopy, the total length of the basement membrane wasmeasured with a color video image processor (VIP-21CH; Olympus-

Ikegami Tsushin Co., Tokyo, Japan), and numbers of lesions wereexpressed per 10 cm of basement membrane (20). The liver and kidneywere also examined histologically.

Experiment 3. Rats were randomly divided into 3 groups of 10 ratseach. Sodium L-ascorbate was administered ad libitum to rats at levels

of 0, 1.0, and 5.0% in Oriental M powdered basal diet. Rats wereindividually placed in metabolic cages with glass collection tubes surrounded by ice without food or water for 4 hr in early morning (5 a.m. to9 a.m.) before sacrifice at Day 7. The urine was stored in the freezer. Atthe time of sacrifice, blood samples were collected from the abdominalaorta for blood analysis. One day later, sodium L-ascorbate contents in

the blood and urine were measured by a calorimetrie method by JapanFood Research Laboratories.

'The abbreviations used are: BBN, N-butyl-N-<4-hydroxybutyl)nitrosamine; PN

hyperplasia, papillary or nodular hyperplasia.

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Ascorbate and Rat Bladder Cancer

RESULTSExperiment 1. Rats given sodium L-ascorbate had no toxic

symptoms such as diarrhea. Data on the body weight, urinarybladder weight, BBN intake, water consumption, and sodium L-

ascorbate intake are given in Table 1. The increase in averagebody weight in Groups 1, 2, and 3 given BBN was not differentfrom that of controls during BBN administration. The averagebody weight in rats of Group 4 treated with sodium L-ascorbate

without BBN was slightly less in Week 28 and was clearly lessthan those of the other groups when rats were weighed beforesacrifice at Week 36 after overnight starvation. The urinarybladder weight was significantly higher in Group 1 than in ControlGroup 3. There were no differences in the average water consumption or BBN intake of groups given BBN for 4 weeks, butgroups given 5.0% sodium L-ascorbate, with or without BBN,

drank more water than did controls from Week 5 to the end ofthe experiment.

In urinalysis in Week 36, an apparent elevation of pH wasobserved in groups given 5.0% sodium L-ascorbate. Treatmentwith 1.0% sodium L-ascorbate also resulted in slight elevation ofpH in the urine. Groups of rats treated with 5.0% sodium L-

ascorbate showed a decrease of osmolality and increase ofcrystals in the urinary sediment. This was not the case with 1.0%sodium L-ascorbate. Crystals were of MgNH4PO4 based on their

morphological appearance by light microscopy (22). Other parameters were within normal ranges.

The histopathological lesions of the urinary bladder observedin rats are summarized in Table 2. As described previously (11),the lesions found in the urinary bladder epithelium were classifiedinto 4 types: simple hyperplasia, PN hyperplasia, papilloma, andcancer. Simple hyperplasia consisted of diffuse or focal thickening of the epithelium with 4 to 8 layers of transitional epithelialcells. In PN hyperplasia, the epithelium was 6 to 8 cells thick,and in most cases the changes were strictly localized. Cellularatypism and mitotic figures were not seen in the areas ofproliferating cells. Hyperplasia showed either exophytic growth,with a delicate fibrovascular core and protrusion into the lumenof the urinary bladder, or endophytic growth. These lesions area form of preneoplastic hyperplasia (11, 17, 18). Papilloma wasdefined as a benign epithelial tumor in which the transitionalepithelial cells were arranged in branched finger-like processeswith a delicate fibrovascular core or showed an endophyticgrowth pattern. Cellular irregularity was slight and few mitoticfigures were present in the epithelial proliferation of papillomas.Cancer was of the transitional cell type showing varying degreesof atypia. Mitotic figures were frequently seen. The incidencesof simple hyperplasia and PN hyperplasia were significantlyhigher in Group 1, given BBN followed by 5.0% sodium L-

Tabie1Average body weight, bladder weight, BBN intake (up to Week 4), water consumption (Weeks 5 to 36), and sodium L-ascorbate intake (Weeks 5 to 36) of rats in

Experiments 1 and 2

TreatmentBody wt (g)Urinary bladderwtSodium

L-ascor-GroupExperiment

11234Experiment

2123BBN

(%)0.010.010.010.050.050.05bate(%)5.01.05.05.01.0Initial107108109109127131133Final424430442418398420417Absolute(g)0.20

±0.04a'b0.17

±0.050.16±0.060.13±0.060.45

±0.27"0.1

6±0.040.16±0.04WaterRelative

(organ/ BBN intake (mg/consumptionbodywt%)0.05

±0.01°0.04

±0.010.04±0.010.03±0.010.14

±0.18*0.04

±0.010.04±0.01kg/day)11.811.511.551.049.048.5(ml/rat/day)25.921.421.726.926.822.421.6Sodium

L-ascor

bate intake(mg/kg/day)275552027892474472

a Mean ±S.D.6 Significantly different from Group 3 at p < 0.05.c Significantly different from Group 3 at p < 0.01." Significantly different from Group 3 at p < 0.001

Table 2Histological findings in the urinary bladder of rats treated with BBN followed by sodium L-ascorbate

TreatmentGroupExperiment

11234Experiment

2123BBN

(%)0.010.010.010.050.050.05SodiumL-as

corbate(%)5.01.05.05.01.0Simóle

hvoer-Effectiveno. of plasia inci-

ratsdence2020202025293020

(100)"c9(45)7(35)025(100)"20

(69.0)17(56.7)Papillary

or nodular hyperplasiaIncidence17(85)d10(50)8(40)025(100)°11

(37.9)8(26.7)No./10cmof

BM"4.9

±5.70'"1.1

±1.30.5±0.607.0

±3.0°0.6

±1.00.3±0.5PapillomaIncidence12(60)9(45)5(25)023

(92)"12(41.4)12(40)No./10cmof

BM1.1

±1.20.8±1.30.5±1.003.1

±1.6"0.4

±0.60.5±0.5CancerIncidence000024

(96)°01

(3.3)No./10cmof

BM00001.4

±0.7"00.0

±0.2

BM, basement membrane.6 Significantly different from Group 3 at p < 0.001.c Numbers in parentheses, percentage." Significantly different from Group 3 at p < 0.01.e Mean ±S.D.

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S. Fukushima et al.

ascorbate, than in Control Group 3. Moreover, quantitative analysis of the lesions showed that the number of lesions of PNhyperplasia per 10 cm of basement membrane was significantlyincreased in Group 1. Treatment with 1.0% sodium L-ascorbate

tended to increase the number of PN hyperplasia. However,there were no differences in the incidences or numbers ofpapillomas in Groups 1, 2, and 3. The mucosa of rats treatedwith 5.0% sodium L-ascorbate (Group 4) was characterized by

normal histology. Lymphocytic infiltration in the submucosa ofthe urinary bladder was observed in 1 of 20 rats in Group 4. Noinflammation, calculous deposit, or foreign body reaction wasobserved histologically. Other histopathologtoal changes werenot seen.

Experiment 2. Rats given sodium L-ascorbate at low and highdoses showed slight reduction in body weight (Table 1). Theintake of BBN in different groups was almost the same. Theaverage food consumption of groups given sodium L-ascorbate

was also similar to that of the control, but water consumption ofrats given 5.0% sodium L-ascorbate was more than that of

controls from Week 5 to Week 36 (Table 1).Urinalysis showed a shift to alkaline pH, a decrease of osmo-

lality, and an increase of MgNH4PO4 crystals in rats given 5.0%sodium L-ascorbate. Urine of rats in Group 1 also contained

occult blood and an increased number of RBC in the urinarysediment. Other urinary parameters examined were within normal ranges.

Macroscopically, rats in Group 1, given BBN followed by 5.0%sodium L-ascorbate, had more tumors of the urinary bladder

than did rats in Group 3 given BBN alone. The absolute andrelative weights of the urinary bladder were significantly increased in Group 1 (Table 1).

Histopathological lesions of the urinary bladder are shown inTable 2. The incidence of simple hyperplasia was significantlyhigher in Group 1 given 5.0% sodium L-ascorbate than in the

control group. The incidence and the number of PN hyperplasiaper 10 cm basement membrane were also significantly increasedin Group 1. The incidence and the number of papilloma andcancer per 10 cm basement membrane were also significantlyincreased by 5.0% sodium L-ascorbate. However, 1.0% sodiumL-ascorbate did not significantly increase the occurrence of any

bladder lesions in rats pretreated with BBN.Experiment 3. The sodium L-ascorbate contents in the blood

and urine of the various groups are presented in Table 3. Valuesof total ascorbic acid and dehydroascorbic acid in the blood ofrats treated with sodium L-ascorbate at levels of 1.0 or 5.0%

were significantly higher than those of the control. In the urine,total ascorbic acid increased significantly in rats treated withsodium L-ascorbate. These values clearly demonstrated a dose

dependency. Values of dehydroascorbic acid in the urine werealso significantly elevated when compared to the control.

DISCUSSION

It is clear that PN hyperplasia is a preneoplastic lesion of raturinary bladder (11,17,18). Our study was done to evaluate thepromoting activity of sodium L-ascorbate in urinary bladder car-cinogenesis in a relatively short-term experiment. Effects were

determined on the induction of preneoplastic lesions rather thanadvanced stages of urinary bladder carcinogenesis in Experiment1 after initiation with a low dose of BBN. In this experiment,administration of 5.0% but not of 1.0% sodium L-ascorbate

increased the occurrence of PN hyperplasia in the urinary bladderof rats pretreated with BBN. This is consistent with our preliminary findings (10). In Experiment 2, we examined the promotingactivity of sodium L-ascorbate on urinary bladder carcinogenesis

after initiation with a higher dose of BBN. In this experiment,addition of sodium L-ascorbate at a level of 5.0% to the diet

resulted in a marked increase not only in PN hyperplasia but alsoin papilloma and cancer. Thus, the promoting effect of an exceedingly high dose of sodium L-ascorbate was more clearly

demonstrated after stronger initiation.Moreover, the inductions of papilloma and cancer in the group

treated with 5.0% sodium L-ascorbate were significantly higher

in Experiment 2 than in Experiment 1. Thus, although both dosesof BBN caused definite initiation of urinary bladder carcinogenesis, the promoting activity of sodium L-ascorbate was clearer

after the stronger initiation in Experiment 2. These results areconsistent with results on the promoting effect of saccharin in 2-stage carcinogenesis in rat urinary bladder initiated by 2 differentdoses of BBN (16).

Ascorbic acid has been shown to have protective effectsagainst nitrosation, the reaction of nitrite with secondary aminein vitro and in vivo (19,27). It also inhibited mutagenesis inducedby A/-methyl-A/'-nitro-A/-nitrosoguanidine and dimethylnitrosa-

mine in the Ames test (12). Furthermore, it was found to inhibitthe conversion of A/-hydroxy-A/-acetyl-2-aminofluorene to 2 more

potent carcinogens in vitro (6). Ascorbate also prevented induction of urinary bladder carcinogenesis by implantation of a tryp-tophan metabolite, 3-hydroxyanthranilic acid, into mouse bladder

(23) and inhibited the initiation of skin tumors by application of7,12-dimethylbenz(a)anthracene (24). Thus, ascorbic acid is rec

ognized to be important for cancer prevention. Consistent withthis, ascorbate treatment significantly prolonged the life of patients with cancer (3). However, unexpectedly, in the presentstudy, an extremely large dose of sodium L-ascorbate promoted

bladder carcinogenesis in rats. Moreover, ascorbate was recentlyfound to promote induction of sarcoma by 3-methylcholanthrene

in guinea pigs (1). It is known that ascorbic acid is synthesizedin rat liver and excreted by the kidney, and it is present in largeamounts with urea in the urine. In the present study, addition ofsodium L-ascorbate to the diet resulted in an increase in the

TablesAscorbate content in the blood and urine of rats treated with sodium L-ascorbateduring i week

BloodDose

¡ndiet(%)0

1.05.0Total

ascorbic acid(mg/100ml)0.74

±0.07a1.06 ±0.18"1.61 ±0.80"Dehydroascorbic

acid(mg/100ml)0.43

±0.050.61 ±0.0860.80 ±0.23°UrineTotal

ascorbic acid(mg/100ml)2.23

± 0.1229.30 ± 5.82"86.82 ±34.23CDehydroascorbic

acid(mg/100ml)1.83

±0.217.92 ±0.88

12.82 ±2.53°

' Mean ±S.D.* Significantlydifferent from the control at p < 0.001.: Significantlydifferent from the control at p < 0.01.

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Ascorbate and Rat Bladder Cancer

content of ascorbic acid and its metabolities in the urine, andthis excess of ascorbic acid in the urine may have contributedto induction of preneoplastic lesions, papilloma, and cancer inthe urinary bladder. Investigations are required on why extremelyhigh doses of sodium t-ascorbate promote urinary bladder car-

cinogenesis in rats.In the present study, apparent elevation of pH, low osmolality,

and increase of MgNH4PO4 crystal contents were established inurine after sodium L-ascorbate (5.0%) treatment. Administration

of 5.0% sodium saccharin p.o., which promotes urinary bladdercarcinogenesis, induces MgNH4PO4crystalluria(8,10). However,crystallurias have been induced by chemicals which do notpossess promoting activity (10). Therefore, any correlation between crystalluria and promotion of urinary bladder carcinogenesis requires further study. It also has been reported that sodiumo-phenylphenate and o-phenylphenol are carcinogenic for the rat

urinary bladder (7,14). Moreover, the carcinogenicity of sodiumo-phenylphenate seems to be more potent than that of o-phen

ylphenol. This evidence supports the view that the alkalinity ofurine relates to carcinogenic potency in the urinary bladder. Inthe present study, 1.0% sodium L-ascorbate did not induce

crystalluria and only slightly increased the pH of the urine, whilenot showing promoting activity for the urinary bladder. Alterationsin the composition of the urine could be responsible for tumordevelopment. Thus, promoting activity of sodium L-ascorbate

may be related not to its primary effect but rather to secondaryevents caused by the observed changes in composition of theurine. We need further investigation to resolve this point. It is ofobvious importance to establish whether ascorbic acid may havepromoting activity or not.

Sodium L-ascorbate is an antioxidant used as a food additive.

Wattenberg (25, 26) demonstrated that antioxidants have inhibitory effects on chemical carcinogenesis. The induction of cancers in various organs by carcinogens was inhibited by concomitant administration of antioxidants in the diet. However, recently,we found that these antioxidants, such as butylated hydroxy-

anisole and butylated hydroxytoluene, possess promoting activity in 2-stage urinary bladder carcinogenesis in rats (15). Themechanism of promotion by sodium L-ascorbate was suggested

to depend on its antioxidant properties. Therefore, many otherantioxidants may possess promoting activities. Moreover, sodium L-ascorbate might act as a promoting agent merely because

it is an anión, similar to saccharin, and not because it is anantioxidant.

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1983;43:4454-4457. Cancer Res   Shoji Fukushima, Katsumi Imaida, Takao Sakata, et al.   Bladder Carcinogenesis in RatsPromoting Effects of Sodium l-Ascorbate on Two-Stage Urinary

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