Carcinogenesis vol.17 no. 11 pp.2435-2439, 1996

Promoting effects of dimethylarsinic acid on N-butyl-iV-(4-hydroxybutyl)nitrosamine-induced urinary bladdercarcinogenesis in rats

Hideki Wanibuchi2, Shinji Yamamoto, Hua Chen1,Kaoru Yoshida1, Ginji Endo1, Takaaki Hori andShoji Fukushima

First Department of Pathology and 'Department of Preventive Medicineand Environmental Health, Osaka City University Medical School,1-4-54 Ashahi-machi, Abeno-ku, Osaka 545, Japan2To whom correspondence should be addressed

Arsenicals are epidemiologically significant chemicals inrelation to induction of urinary bladder cancer in man. Inthe present study, we investigated the dose-dependentpromotion potential of dimethylarsinic acid (DMA), amajor metabolite of inorganic arsenicals in mammals, forrat urinary bladder carcinogenesis. In experiment 1, 6-week-old male F344 rats were treated with 0.05% N-butyl-A^-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks and thengiven one of several concentrations of DMA in theirdrinking water (groups 1-6: 0, 2,10, 25, 50 and 100 p.p.m.)for 32 weeks. The development of preneoplastic lesions andtumors (papillomas and carcinomas) in the urinary bladderwas enhanced by treatment with DMA in a dose-dependentmanner. A significant increase in multiplicity of tumors(papillomas and carcinomas) was observed even at a lowconcentration of DMA (10 p.p.m.). On the other hand, nopreneoplastic lesions and tumors were observed in therats treated with DMA alone. In experiment 2, differentconcentrations of DMA (groups 1-4: 0, 10, 25 and 100p.p.m.) in drinking water were administered to the ratsfor 8 weeks without prior initiation by BBN. A significantincrease in the 5-bromo-2'-deoxyuridine labeling index andalteration of the surfaces of the urinary bladder epithelialcells, as revealed by scanning electron microscopy, providedevidence of a dose-dependent increase in cell proliferationdue to the DMA treatment These results suggest thatDMA has the potential to promote rat urinary bladdercarcinogenesis and one of the mechanisms involved is itsstimulation of cell proliferation in the urinary bladderepithelium.

Introduction

Epidemiological investigations have revealed that arsenicalsare carcinogenic to humans, especially the skin and lung (1,2).In an area of Taiwan endemic for blackfoot disease, becauseof the ingestion of high concentrations of arsenicals in thedrinking water, high mortality from internal cancers, especiallycancers of the urinary bladder, kidney, liver and lung, and skincancer has been reported (3-7). Tumor incidence was foundto be dependent on the concentration of arsenicals in the water.In experimental animals, arsenicals have not exerted complete

•Abbreviations: DMA, dimethylarsinic acid; BrdU, 5-bromo-2'-deoxyuridine;SEM, scanning electron microscopy; BBN, W-butyl-W-(4-hydroxybutyl)nitros-amine; AsIII, arsenite; MMA monomethylarsonic acid; TMAO, trimelhylarsineoxide; AsBe, arsenobetaine.

carcinogenecity (1,8). Recently, we revealed the promotionpotential of dimethylarsinic acid (DMA*), a major metaboliteof inorganic arsenical in most animals, in the urinary bladder,kidney, liver and thyroid gland using a multiorgan carcinogesisbioassay in rats (9).

Several chemicals have been identified that exert promotingactivity in two-stage rat urinary bladder carcinogenesis (10-14).The promoting and carcinogenic activities of non-genotoxicchemicals appears to be related to increased cell proliferation(15-18). In the present study, we established a relatively lowdose threshold for DMA promoting activity in rat urinarybladder carcinogenesis and correlated this with cell prolifera-tion in the urinary bladder epithelium, as assessed by the 5-bromo-2'-deoxyuridine (BrdU) labeling index method, andepithelial surface alterations observed by scanning electronmicroscopy (SEM) in the urinary bladder of rats treated withDMA alone.

Materials and methods

Animals

A total of 189 6-week-old male F344 rats (Charles River Japan Inc., Hino,Japan) were used. The rats were housed with wood chips for bedding in ananimal room with a 12 h light/dark cycle at 22 ± 2°C and 44 ± 5%relative humidity. Body weights were measured weekly and drinking waterconsumption was measured throughout each administration period. Diet andwater were available to the rats ad libitum.

Chemicals

A'-Butyl-A'-(4-hydroxybutyl)nitrosamine (BBN) was obtained from TokyoKasei Co., Tokyo, Japan. DMA with a purity of 99% was obtained fromWako Pure Chemical Industries Ltd, Osaka, Japan.

Experiment 1

One hundred and forty four male F344 rats were used. Rats were administereddrinking water with (groups 1-6, 20 rats in each group) or without (groups7-8, 12 rats in each group) 0.05% BBN for the first 4 weeks and then theywere given one of several concentrations of DMA in the drinking water(groups 1-6: 0, 2, 10, 25, 50 and 100 p.p.m.; groups 7-8: 0 and 100 p.p.m)for 32 weeks. They were fed normal pellet diet (CE2; Clea Japan Inc., Tokyo,Japan). At week 34, fresh urine was collected by forced urination and pHwas measured with a pH meter (Horiba model F-15; Kyoto, Japan). The urinesamples were then analyzed for sodium, potassium, chloride and calcium (byHitachi-710 electrolyte analyzer, Tokyo, Japan). Arsenic compounds, arsenite(AsIII), monomethylarsonic acid (MMA), DMA, trimethylarsine oxide(TMAO) and arsenobetaine (AsBe) in the urine samples were determined bya modification of the method of Inoue el al. (19,20). A combined system withan IC (model IC 7000; Yokogawa Analytical Systems Inc., Tokyo, Japan) forthe separation and an ICP-MS (model HP4500; Hewlett-Packard Co., DE) asan element selective detector was used to analyze the different arsenic species.For separation of the five arsenic compounds, the IC was operated under thefollowing conditions: mobile phase, 10 mM tartaric acid; flow rate, 1.0 ml/min;column temperature, 50°C; injection volume, 50 \i\. The elute from the ionchromatography column was introduced directly into the nebulizer of the ICP-MS and analyzed at a monitoring mass of 75.

At the beginning of week 37, the rats were killed under ether anesthesiaand their urinary bladders were inflated by intraluminal injection of 10%phosphate-buffered formalin solution. After fixation, the urinary bladders weredivided sagitally and weighed. Macroscopically, the numbers of tumors ineach rat were counted. For histological analysis the bladders were cut intoeight strips, routinely processed for embedding in paraffin, sectioned andstained with hematoxylin and eosin.

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Table I. Average final body and bladder weights, and water intake and total DMA of rats in experiment 1

Group

456l

Treatment

BBN DMA (p.p.m.)

+ 0+ 2+ 10+ 25+ 50+ 100

0100

No. of rats

Initial

202020w20201212

Final

2020201920201211

Final

391 :400 :405 :3 9 0 :393 :389 :410 :383 :

body wt (g)

t 13't 14t 22t 17t 17t 21t 22t 10

Urinary

0.12 ±0.12 ±0.13 ±0.18 ±0.22 ±0.28 ±0.12 ±0.13 ±

blad(

0.020.010.010.08b

0.16b

0.430.020.01

Water intake(g/rat/day)

21.722.723.025.426.527.922.526.0

Total intake ofDMA (mg/rat)

010.151.1

140.8294.5619.7

0578.3

"Mean ± SD.•"Significantly different from group 1 at P < 0.01 (Student's /-test).

Experiment 2

Forty five rats were used. They were administered different concentrations ofDMA (groups 1-4: 0, 10, 25 and 100 p.p.m.) for 8 weeks without anyinitiation pretreatment.

For SEM, the urinary bladders of five rats in each group were inflated with2% glutaraldehyde in 0.1 M phosphate buffer, divided into 5 mm squarepieces and then routinely processed as previously described (21).

In each group, the available rats (groups 1-3: 10, 5 and 5 animals; group4; all animals died because of the toxicity of DMA after 4 weeks) received asingle i.p. injection of BrdU (Sigma Chemical Co., St Louis, MO) at a doseof 100 mg/kg body wt 1 h before sacrifice. Then, the urinary bladders wereinflated with 10% phosphate-buffered formalin solution, excised and dividedinto eight strips and then embedded in paraffin for histological analysis.Epithelial cells incorporating BrdU were immunohistochemically demonstratedin the sections by the avidin—biotin-peroxidase complex procedure using anti-BrdU monoclonal antibody as detailed earlier (22). Numbers of labeled cellsper 1000 cells were counted under the light microscope and labeling indiceswere expressed as percentage values.

Statistical analysis

The significance of differences between mean values was analyzed usingStudent's /-test. The significance of differences in lesion incidence betweengroups was assessed by Fisher's exact probability test Two-tailed Cochran-Armitage analysis was used to evaluate the dose-response relationships forlesion incidences in groups 1-6.

Results

Experiment 1Two rats, one in group 4 (BBN followed by 25 p.p.m. DMA)and one in group 8 (100 p.p.m. DMA only), died on day 7after DMA treatment was started. Final average body weightsand urinary bladder weights are shown in Table I. No statistic-ally significant variation in final average body weights wasnoted in any group. Water consumption and total intake ofDMA increased in a dose-dependent manner (Table I).

Macroscopically, the urinary bladder had multiple very largetumors in groups 4—6, as compared with group 1 (a controlgroup treated with BBN only). Urinary bladder weights alsoincreased with dose (Table I). The rats in group 8 (treatedwith DMA only) did not show any tumors.

Histological findings in the urinary bladder epithelia aresummarized in Table II. There was a DMA dose-dependentincrease in the incidence and multiplicity of urinary bladdercarcinomas, papillomas and preneoplastic lesions, such aspapillary or nodular hyperplasia (23), in the groups initiatedwith BBN. Two-tailed Cochran-Armitage analysis revealeddose relationships of these lesion incidences (each P < 0.001).On the other hand, no preneoplastic lesions and tumors wereobserved in the rats treated with DMA alone.

Figure 1 shows data for the multiplicities of papillomas,carcinomas and total tumors (papilloma and carcinoma) of the

3 - 1Papilloma

n

Carcinoma Tumor(papilloma + carcinoma)

...ill..!0 ? 10 25 50 100 0 ? 10 25 50 100 0 2 10 25 50 100

DMA concentration (ppm)

Fig. 1. Numbers of bladder lesions (papilloma, carcinoma and tumor) perrat treated with BBN followed by DMA in experiment 1. Tumor meanspapilloma and/or carcinoma. Significantly different from group 1 (DMA0 p.p.m.) at *P < 0.05, **P < 0.01, ***P < 0.001 (Student's /-lest).

* *

. o i

"V 0 6CO)c

a

"2 0 2CD

I**

0 ppm 1 n nnm ?s npm

DMA concentration

Fig. 2. BrdU labeling indices for the urinary bladder epithelium of ratstreated with DMA for 8 weeks. "Significantly different from group 1(DMA 0 p.p.m.) at P < 0.01 (Student's /-test).

urinary bladder in the rats treated with the initiator and DMA.These lesions were significantly increased, in a dose-dependentmanner, down to the 10 p.p.m. dose.

Data for urinary pH and concentrations of urinaryelectrolytes such as sodium, potassium, chloride and calciumat week 34 are shown in Table HI. The urinary pH did notdiffer significantly between groups. The concentrations of

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Table II.

Group

12345678

Induction of urinary bladder

Treatment

BBN DMA (p.p.m.)

+ 0+ 2+ 10+ 25+ 50+ 100

0100

lesions in rats

No. of ratsexamined

2020201920201211

treated with BBN followed

PN1 hyperplasia

Incidence (%)

14 (70)13 (65)14 (70)18 (95)20 (100)c

20 (100)c

00

by DMA

NoVrat

1.05 d1.30 d1.55 d2.37 d2.95 d4.10 d_-

i 95b

t 1.30t 1.47i 1.17h

t 1.88h

i 3.02"

in experiment 1

Papilloma

Incidence (%)

3(15)2(10)7(35)

11 (58)d

13 (65)d

17 (85)e

_-

NoVrat

0.15 ± 0.370.10 ± 0.310.40 ± 0.601.05 ± 1.18h

1.50 ± 1.36h

1.70 ± 1.17h

_-

Carcinoma

Incidence (%)

1(5)2(10)3(15)7 (37)c

10 (50)d

12 (60)c

00

NoVrat

0.05 ± 0.220.10 ± 0.310.15 ± 0.370.42 ± 0.6 lf

0.80 ± 0.95*0.70 ± 0.66h

_-

*PN; papillary or nodular."Mean ± SD.CP < 0.05, dP < 0.01, CP < 0.001 (significantly different from group 1, Fisher's exact probability test).fP < 0.05, *P < 0.01, bP < 0.001 (significantly different from group 1, Student's r-test).

Table m.

Group

12345678

Urinary pH and electrolytes in rats

Treatment

BBN DMA (p.p.m.)

+ 0+ 2+ 10+ 25+ 50+ 100

0100

treated with

No. ofsamples

1010101010101010

BBN followed by DMA

Urinary pH

6.36 ± 0.23'6.41 ± 0.246.57 ± 0.25b

6.68 ± 0.21c

6.58 ± 0.236.58 ± 0.206.05 ±0.186.65 ± 0.20

at week 34 in

Na(mEq/l)

94 d105 d103 d85 d67 d80 d97 d

t 33t 58t 53t 56: 27b

: 33t 47

57 ± 34

Experiment 1

K(mEq/l)

303 ± 36340 + 58309 + 87224 + 112209 + 50s

176 ± 35°302 + 61146 ± 43

Cl (mEq/1)

170 ± 35264 ± 38C

169 ± 65135 ± 44108 ± 35C

116 ± 37°189 ± 6180 ± 43

Ca (mg/dl)

13.0 ± 4.011.1 ± 1.29.7 ± 3.77.4 ± 2.8f

6.6 ± 1.8°7.1 ± O.r

11.5 ± 1.86.7 ± 1.8

"Mean ± SD.bP < 0.05, CP < 0.01 (significantly different from group 1, Student's r-test).

Table IV.

Treatment

BBN

++

Urinary concentration

DMA (p.p.m.)

050

of arsenic compounds in rats treated

No.

1010

of samples DMA

0.2510.87

(mg/1)

±0.08± 2.03b

with BBN

TMAO

0.03 ±5.71 ±

followed

(mg/1)

0.013.22b

by DMA

AsBe

0.38 :0.30 :

at week

(mg/1)

t 0.06t 0.13

34 in experiment 1

AsIII (mg/1)

01.28 ± 0.5lb

Metabc(mg/1)

0.02 ±1.20 ±

)lite A

0.020.60b

"Mean ± SD.bSignificantly different from group 1 at P < 0.001 (Student's f-test).

Table V. Presence of bladder epithelium surface alterations in rats following treatment with DMA for 8 weeks (experiment 2)

Group DMA (p.p.m.) No. of rats examined SEM findings

Ropy or leafy microridges Short uniform microvilli Pleomorphic micTOvilli

1234

01025

100

5555

•Group (mean of group): - , no change; ±, slight; +, moderate; + + , marked,dumber of rats in which the morphologic feature was observed.

Na, K and Ca ions were decreased in the rats treated withDMA in a dose-dependent manner, with statistical significancebeing reached in the 50 and 100 p.p.m. DMA groups. Thewater intake and urinary volume were increased in a dose-dependent manner.

Table IV shows the urinary concentrations of arseniccompounds in the rats of group 1 (treated with BBN alone)and group 5 (treated with BBN followed by 50 p.p.m.DMA). DMA was the main excreted arsenic product. TMAO,AsBe and AsIII were also observed. An unidentified arsenic

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compound (metabolite A) was eluted immediately after theexpected peak for MMA, although MMA did not appear.Experiment 2SEM findings for the urinary bladder epithelial surfaces of therats treated with DMA are shown in Table V. Ropey or leafymicroridges and short, uniform microvilli, which appear inhyperplasia of the urothelium (16,24), were observed in adose-dependent manner. Pleomorphic microvilli appeared inthe group treated with the highest dose of DMA. The urinarybladder surfaces of control rats had a normal appearance andno necrotic lesions were observed in any groups.

BrdU labeling indices of the urinary bladder epithelium inthe rats treated with DMA (10 or 25 p.p.m.) were significantlyincreased over the untreated control rat values (Figure 2).

Discussion

In the present study, DMA showed dose-dependent promotingactivity for rat urinary bladder carcinogenesis initiated withBBN. This result is in accordance with our previous studyusing a rat multiorgan carcinogenesis model (9), where DMAexhibited promoting potential at 50 p.p.m. in the drinkingwater. In the present study, the promoting activity was observedin a dose-dependent manner from a very low dose of DMA(10 p.p.m.), although at the lowest dose (2 p.p.m.) no differencein tumor incidence or multiplicity was evident as comparedwith the control group. These data are in agreement with anepidemiological report on bladder tumor mortality in an areaof Taiwan endemic for blackfoot disease, where the arsenicconcentration in drinking water was found to be extremelyhigh (3-7).

Considering the mechanisms of promoting activity of DMA,the BrdU labeling index, a reflection of the level of cellproliferation, of the epithelial cells of the urinary bladder wassignificantly increased after intake of DMA for 8 weeks. Theinduced increase in cell proliferation in the urothelium wasalso obvious from the SEM findings. In previous studies,surface alterations of the urothelium were discussed in termsof the promoting activities of chemicals in the rat urinarybladder (16,17,21,23-26). In this study, ropey or leafy micro-ridges, short uniform microvilli and pleomorphic microvilliwere observed and the incidences and degree of developmentwere DMA dose dependent. This result showed a discrepancyfrom that of a reduced increase in BrdU index, owing topossible toxicity, for the high DMA dose group. Further studywill be needed to confirm this. These data for BrdU index andSEM findings indicate that an increase in cell proliferationmay be one of the mechanisms by which DMA promotes raturinary bladder carcinogenesis.

Concerning the toxicity of DMA, in experiment 2 the ratsgiven DMA at 100 p.p.m. demonstrated a 50% mortalityrate. We selected living rats for SEM study because earlyproliferative changes of epithelial cells of the urinary bladdercan be easily detected using this approach. The toxicity ofDMA to rats is very characteristic. We recently found thatseven of 10 rats starting 200 p.p.m. DMA treatment at 6 weekof age were dead within 4 weeks (unpublished data). Incontrast, only one and none of 10 rats aged 8 weeks and 10weeks respectively at the commencement suffered mortality.These data reveal that chronic DMA administration is primarilytoxic for young rats. In experiment 1, rats received DMA fromwhen they were 10 weeks old and only two (in groups 4 and8) were dead at 4 weeks after the start. The main symptom of

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DMA toxicity is weight loss and, histologically, necrosis andfatty change around the central veins in the liver. Althoughwe could not detect epithelial cell necrosis in the bladder, theBrdU index as a marker of proliferative activity was decreasedby the high dose of DMA.

It has been reported that increasing cell proliferation of theurothelium in rat bladder carcinogenesis correlates with urinepH and urinary Na concentration (11,27-30). Thus, underconditions of equally increased urinary Na ion concentration,the value for urinary pH determined the promoting activity(18). On the other hand, with equal elevation of urinary pH,higher sodium concentrations in the urine are associated withtumor promotion (18). In this study, urinary pH did notdiffer among the DMA-treated groups and the urinary Naconcentration was decreased in a dose-dependent manner. Thelatter was presumbly due to increased water intake and urinaryexcretion. Clearly, the promoting action of DMA in rat urinarybladder carcinogenesis is not correlated with urine pH and Naconcentration and in that sense it differs from most otherreported promoters.

DMA is one of the major excreted metabolites of ingestedarsenicals in humans (31-35). Our previous study using amultiorgan carcinogenesis model (9) and the present studyrevealed that during chronic ingestion of DMA, 20% of theingested DMA is excreted as unchanged DMA in the urine.The urinary concentration of DMA was thus increased inaccordance with the ingested DMA. From these data, weconsider that DMA itself may act as a potent promotor in theurinary bladder. As other methylated and demethylated arsenicmetabolites were also observed, however, further experimentsmay be required to assess their influence.

It is not clear whether DMA should be regarded as acarcinogen or a promotor, although no tumors were found inrats of the DMA only group in the present study. Since it wasreported that DMA induces chromosomal alterations, DMA isprobably a potent clastogenic agent (36,37) capable of inducingDNA damage, such as DNA single-strand breaks (38,39) andDNA-protein cross-links (40). Clastogenic effects might alsobe important for bladder carcinogenesis. On the other hand, itwas recently reported that sodium arsenite increased rat hepaticornithine decarboxylase activity and hepatic heme oxygenaseactivity, but caused no DNA damage, indicating that it mightbe a promoter rather than an initiator of carcinogenesis (41).Since our present data revealed potent promoting activity ofDMA in two-stage rat urinary bladder carcinogenesis, we arenow conducting an on-going two year carcinogenicity studyof DMA to confirm whether DMA is a carcinogen or only apromoter for the urinary bladder.

AcknowledgementThis study was supported by a Grant-in-Aid for Scientific Research on PriorityAreas from The Ministry of Education, Science, Sports and Culture, Japanand a Grant-in-aid for Cancer Research from the Ministry on Health andWelfare, Japan.

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Received on March 19, 1996; revised on July 8, 1996; accepted on July 26, 1996

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