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(CANCERRESEARCH39, 1207-1217,April
1979]0008-5472/79/0039-0000$02.O0
Promoting Effect of Saccharin and DL-Tryptophan in Urinary
BladderCarcinogenesis1
Samuel M. Cohen,2 Masayuki Aral,3 Jerome B. Jacobs, and Gilbert
H. Friedell
Department of Pathology, St. Vincent Hospital, and Department of
Pathology, the University of Massachusetts Medical School,
Worcester, Massachusetts01604
we have been particularly concerned with the identificationof
possible morphological markers of neoplastic change inbladder
epithelium, using light microscopy and both scanfling and
transmission electron microscopy. This required amodel in which the
sequence of biological and pathologicalevents was predictable. In a
previous study, we identifiedas ‘‘subthreshold'‘a dose of
FANFT, 0.2%, which when fedfor 6 weeks produced no bladder tumors
if the rats werethen fed control diet until 84 weeks, although the
samedose produced tumors if fed for 8 weeks followed by controldiet
(34). The present investigation was based on theconcept that this
subthreshold dose of FANFT, an ‘‘incompletecarcinogen―atthe
levelofadministration,could serveas an initiator, permitting us to
explore the phase of tumorpromotion in this model system.
This concept, the use of minimal amounts of knownchemical
carcinogens as initiating agents and the subsequent administration
of agents known to be noncarcinogenic when administered by
themselves, has been used byinvestigators for many years in their
attempts to identifyvarious stages in the carcinogenic process
(67), beginningwith investigations of 2-stage murine skin
carcinogenesis(8-10). A similar process has subsequently been
demonstrated in other tissues, notably the liver (47).
For the urinary bladder (29, 31 , 32), it has been shown byHicks
that instillation into the rat bladder of a subcarcinogenic dose of
MNU followed by p.o. administration ofsaccharin or cyclamate
resulted in a high incidence ofbladder cancer, while saccharin or
cyclamate alone induced a very low incidence of bladder cancer.
Cyclophosphamide, known to induce necrosis and then a
markedhyperplasia of the bladder epithelium in rats, did not
inducebladder tumors in the same experimental system after
asubcarcinogenic dose of MNU.
In the experiments described in this report, we attemptedto
determine whether at least 2 stages existed in
bladdercarcinogenesis, using for initiation an agent would couldbe
given in the diet rather than instilled into the bladderand as
promoting agents in this system, saccharin andtryptophan.
Saccharin, administered in the diet rather thanin the drinking
water, was used because of its demonstratedeffectiveness as a
promoting agent in the MNU model asdescribed above. Tryptophan was
chosen as another possible promoting agent because of the suggested
relationship between abnormal tryptophan metabolite levels
andbladder cancer in humans (14, 48, 68) and because of avariety of
studies in experimental animals indicating apossible relationship
of tryptophan metabolites with bladder cancer (14, 48).
Previous studies in our laboratory on the FANFT bladdercancer
model in Fischer rats indicated that an early marker
ABSTRACT
The existence of at least two stages in bladder carcinogenesis
was evaluated in male Fischer rats using
N-[4-(5-nitro-2-furyl)-2-thiazolyi]formamide (FANFT) fed for
si@weeks at a level of 0.2% of the diet as the initiator.
Sodiumsaccharin and DL-tryptophan were fed at levels of 5 and 2%of
the diet, respectively, as possible promoting chemicals,and they
were fed either immediately after FANFT administration or after six
weeks of FANFT plus six weeks of controldiet. All surviving rats
were killed at the end of two years.Both chemicals significantly
increased the incidence ofbladder tumors following FANFT feeding
compared to sixweeks of FANFT feeding followed by control diet, and
theresults were similar whether saccharin or tryptophan feeding was
started immediately after FANFT feeding was concluded or after a
six-week delay. Saccharin was considerably more potent as a
promoting agent than was tryptophan, inducing higher incidences of
bladder tumors andhaving a shorter latent period. Long-term
administration ofFANFT induced a 100% incidence of bladder cancer.
Sequential epithelial changes were observed by scanning
andtransmission electron microscopy as well as by light microscopy.
Pleomorphic microvilli were present on the superficial cells of all
tumors examined and on the surfacecells of hyperplastic bladder
epithelium after six weeks ofFANFT plus six weeks of saccharin, but
not after six weeksof FANFT and six weeks of control diet. Rats fed
onlysaccharin, tryptophan, or control diet did not have
bladdertumors or pleomorphic microvilli on bladder epithelium.These
data suggest that saccharin and tryptophan mightact as
tumor-promoting agents during bladder carcinogenesis.
INTRODUCTIONWe have focused our attention for 10 years on
developing
and defining an experimental model for studying the pathogenesis
of bladder cancer, particularly the early stages(20, 27, 63). The
Fischer rat, an inbred strain, has beenfound to be an appropriate
animal, and the nitrofuranFANFT4 has proved to be an effective
organ-specific carcinogen, inducing a 100% incidence of bladder
cancer after arelatively short time when given p.o. For the last
few years,
, Supported in part by USPHS Grant CA15495 from the National
Cancer
Institute through the National Bladder Cancer Project. A
preliminary reportof this work was presented (19).
2 To whom requests for reprints should be addressed, at
Department of
Pathology, St. Vincent Hospital, Worcester, Mass. 01604.3
Present address: First Department of Pathology, Nagoya City
University
Medical School, Nagoya 467, Japan.4 The abbreviations used are:
FANFT, N-[4-(5-nitro-2-furyb)-2-thiazo
Iyljformamide; MNU, N-methyl-.N-nitrosourea.Received October 5,
1978; accepted December 22, 1978.
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0 6 2 4@ 78
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S. M. Cohen et al.
weeks. Group 7 was the control group and was fed controldiet for
the entire length of the experiment. Rats in Group 8were fed FANFT
for 6 weeks followed by control diet untilthe end of the
experiment. Group 9 was fed FANFT for 6weeks followed by 6 weeks of
control diet, 30 weeks ofFANFT, and then control diet until they
died. Group 10 wasfed control diet for 6 weeks, followed by 36
weeks of FANFT(same total period of FANFT administrRtaon as in
Group 9),and then control diet until they died. Rats in Group
11received FANFT for the entire time they were alive in
theexperiment. All of the rats in Groups 9 to 11 died due tobladder
cancer by the end of Weeks 78, 80, and 77 of the
experiment, respectively.All rats were weighed at the beginning
of the experiment
and at the end of Weeks 2, 6, 7, 9, and 12 and
monthlythereafter. Food consumption was determined at the
sameintervals. When a rat was found dead or was killed, acomplete
autopsy was performed and the tissues, exceptthe bladder, were
fixed in 10% buffered formalin andembedded in paraffin; sections
were then stained with
hematoxylin and eosin. When indicated, sections werestained with
other stains. The urinary bladders were inflatedwith Bouin's
fixative, washed 3 to 5 times in 70% ethanol,and then processed as
above.
The number of rats in each group at the start of theexperiment
is listed in Table 1. An additional 4 rats weresacrificed from each
of Groups 7 and 8 at the end of thesixth week of the experiment and
from each of Groups 1, 3,4, 6, 7, 8, and 11 at the end of the 12th
week to examine forearly lesions in the bladder. These rats are not
tabulated aspart of these groups in Tables 1 to 3. The bladders
from 2 ofthe 4 rats from each group at each time interval
wereinflated with Bouin's fixative and processed for light
microscopy as described above. The bladders from the other2 rats
from each group were inflated transurethrally througha 25-gauge
needle with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4,
under a constant pressure of 50 mm ofmercury to a volume of 0.4 to
0.5 ml, depending on the sizeof the rat, and processed for light,
transmission, andscanning electron microscopic examination as
describedpreviously (20, 33). Two or more rats from each group
weresimilarly examined at the end of the experiment, and
theremainder were processed only for light microscopy as
04 described above.
RESULTS
The rats in all groups gained weight at a rate similar tothe
control group except for the rats fed DL-tryptophanwhich gained at
a rate approximately 10% less than thecontrols (Table 1). Rats in
Groups 9 to 11, those on longterm FANFT feeding, grew at rates
comparable to those ofthe controls until after 52 weeks at which
time several ratshad gross hematuria due to large bladder tumors,
and therats gradually lost weight. A similar process was
observedinratsinGroups 1 and 2 after76 weeks, but not
allratsinthese 2 groups showed such changes. Most of the rats
fedtryptophan alone or after FANFT (Groups 4 to 6),
saccharinwithout previous FANFT (Group 3), or FANFT for only 6weeks
(Group 8) survived until the end of the experiment(Table 1). All of
the rats in Groups 9 to 11, which received
of irreversibility of proliferative epithelial lesions was
thepresence of pleomorphic microvilli on the luminal surfacesof
epithelial cells as detected by scanning electron microscopy (20,
27, 33, 34). Similar findings have been reportedby others using
different carcinogens administered to otherstrains of rats (3, 30),
and comparable changes have beenreported in humans with bladder
tumors (28, 35). Wetherefore utilized scanning electron microscopy
and lightmicroscopy in these studies to correlate
morphologicalfindings with biological events.
MATERIALS AND METHODS
Male Fischer rats were purchased from Charles RiverBreeding
Laboratories, Inc. (Wilmington, Mass.), and were4 weeks of age at
the beginning of the experiment. Theywere housed 4/cage, were
maintained at 24°and 50%humidity on a 12-hr light-dark cycle, and
had food andwater available ad libitum. FANFT (Saber
Laboratories,Morton Grove, III.), sodium saccharin (Sigma Chemical
Go.,St. Louis, Mo.), and DL-tryptophan (Sigma) were mixed in
apowered diet (Charles River rat chow) at doses of 0.2, 5.0,and
2.0% by weight, respectively. A sample of the sodiumsaccharin was
evaluated for the presence of o- and ptoluenesulfonamide by M. C.
Bowman (National Center forToxicological Research, Jefferson,
Ark.). No o-toluenesulfonamide was detected (limits of detection,
0.03 ppm), andapproximately 0.03 ppm of what was considered to be
ptoluene-sulfonamide was detected.
The rats were divided into ii groups as illustrated inChart 1.
Saccharin and tryptophan were begun immediatelyafter 6 weeks of
FANFT (Group 1) or after a 6-week delayduring which time they
received control diet (Group 2).Group 3 received control diet for 6
weeks and were then feddiet containing saccharin. Saccharin was
discontinued inGroups 1 to 3 at the end of the 83rd week of the
experimentbecause some of the rats in Groups 1 and 2 had
developedsevere hematuria and had bladder tumors. They
receivedcontrol diet until the end of the experiment. Rats fed
DLtryptophan (Groups 4 to 6) continued to receive the chemical in
their diet until the end of the experiment at 104
GROUP
WEEK
— I
— 0.2%FANFT@ 20% DL-TRYPTOPHAN
5.0% SACCHARIN
CONTROL
Chart 1. Experimental design. Control refers to administration
of controldiet without added chemicals. The rats in Groups 9 to 11
had died by the endof Weeks 78, 80, and 77, respectively.
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Cumulative consumption of chemicals and growth and survival
ofratsGroupNo.
ofrats atstartEffective
no. ofratsWt
of rats (g)Av. cumulative dose
(g)Av.survival(wk)No.
alive atend of ex
perimentWk52Wk 85Wk1041
.FANFT —@saccharin20i 9388 ±22@'379 ±35366 ±53i ,5@)5i
494
±i 06(32)@2.FANFT
—@control —*sac 2018377 ±2i392 ±35416 ±411 .5@93
±169(50)charm4653.Control
—*saccharin2020370 ±20381 ±22405 ±30508102
±819(95)4,FANFT-+ tryptophan2019339 ±i8363 ±25376 ±281 .6―102
±518(95)5.FANFT
—*control -@tryp 2020347 ±26372 ±26376 ±29223 i ‘5@)1 02
±71 7(85)tophan2116.Control
—@tryptophan201932i ±24348 ±29360 ±27232102
±1017(89)7.Control4242376±24398.5412 ±3599
±1227(64)8.FANFT—icontrol2020393±22407±33429±321.5iOi
±6i6(80)9,FANFT-@ control —@1616378 ±367.466
±80FANFT—@control10.Control—+ FANFT —÷2019377 ±297.171
±60controlii.FANFT4240375
±33i4.660 ±60
Promotion in Bladder Carcino genesis
Table 1
a Mean ±S.D.1) For groups receiving 2 chemicals, the average
cumulative dose given first is the amount of FANFT consumed. The
average
cumulative dose represents the average amount of the chemical
consumed per rat for the length of the experiment for the rats
survivingthe entire period of feeding the chemical.
(@ Numbers in parentheses, percentage.
long-term administration of FANFT, died by the end ofWeeks 78,
80, and 77, respectively, due to bladder cancer.Several of the rats
prefed FANFT and then fed saccharin(Groups 1 and 2) also died due
to bladder cancer before theend of the experiment. The decreased
number of rats in thecontrol group (Group 7) at the end of the
experiment wasdue to sequential killing of rats during the
experiment forelectron microscopic evaluation; none had bladder
cancer.All of the rats fed saccharin (Groups 1 to 3) had
intermittentdiarrhea which ceased upon discontinuance of
saccharinin the diet.
The estimated average cumulative consumption of eachchemical is
shown in Table 1 as g/rat/total time of administration of the
chemical. It represents a maximum estimateinasmuch as no attempt
was made to account for spillage,and it is estimated for the rats
surviving the entire length ofthe feeding period. Generally, the
rats fed saccharin in theirdiet consumed approximately 20% more
food than did thecontrol group or rats fed the other chemicals. The
relativeconsumptions of the chemicals were approximately 2.5 to3.0,
0.8 to 1.2, and 0.07 to 0.12 g/kg for saccharin, tryptophan, and
FANFT, respectively, with the relative consumption gradually
decreasing as the rats grew.
Bladder lesions observed in each group are summarizedinTable 2.
Each ratistabulatedinthe column of the mostadvanced lesion found in
the bladder. Usually, rats withpapillomas or cancer also had areas
of hyperplasia in thebladder. Tumors which were benign by
histological criteriawere designated as papillomas. A diagnosis of
cancer wasbased on the loss of differentiation to the surface,
presenceof nuclear pleomorphism, and the presence of mitoses.
Thepresence or absence of invasion was determined microscopically,
and if questionable the lesion was classified asnoninvasive. The
extent of the bladder lesions in the variousgroups is shown in
Table 3. Bladders having a carcinomaand sarcoma were classified as
to extent of invasion of only
the epithelial component since the sarcoma presumablyoriginated
in the submucosa. The sarcomas were usuallyanaplastic, spindle cell
tumors histologically and usuallyinvaded through the muscle wall.
Statistical comparison ofincidences was performed using the exact
method for 2 x2 tables.
Rats fed only the control diet, only saccharin (Group 3),or only
tryptophan (Group 6) had no lesions of the urinarybladder except
for occasional instances of mild, simpleepithelial hyperplasia with
the mucosa 4 to 5 cell layersthick rather than the usual 3. The
group fed FANFT for 6weeks followed by control diet (Group 8)
included one ratwith a papilloma and 4 with carcinomas, one of
whichshowed microscopic invasion of the submucosa. Thesetumors were
small, and there was only one tumor in eachaffected bladder. In
contrast, the rats fed FANFT for longterms (Groups 9 to 11) all had
bladder carcinomas, severalhad invasive lesions (Tables 2 and 3),
and the carcinomaswere usually large and often multiple.
Rats fed FANFT for 6 weeks and then fed saccharin,either
immediately after the FANFT feeding (Group 1) orafter a 6-week
delay (Group 2), developed high incidencesof bladder cancer (p
-
Lesionsof the urinarybladderandothertissuesUrinary
bladderTumorsof othertissuesNodu
Calcilarorfica
Sim papil tion inInterstiEffec pie hy laryblad tialcelltive
no.Nor per hyper- Papil Can der betumorsGroupof
ratsmalplasiaplasia bomacersionsof testisTissueHistology No.
1. 19 0 0 1 0 18 6 13
S. M. Cohen et al.
Table 2
FANFT -+ saccharm
2. FANFT -@ control.-.+ saccharin
18 0 1 4 0 13 1 9 Islet cellLungEpididymis
AdenomaAdenomaMesothelioma
121
3. Control —@saccharm
20 18 2 0 0 0 18 Islet cellJawPituitary
AdenomaLiposarcomaAdenoma
2
4. FANFT -+ tryptophan
19 0 5 3 1 10 0 13 AbdomenLungEpididymisPituitary
FibromaAdenomaMesotheliomaAdenoma
1221
5. FANFT —@control 20-4 tryptophan
13 Islet cellLungPituitaryBreastEar duct
AdenomaAdenomaAdenomaFibroadenomaFibroma
FibromaAdenomaAdenocarci
nomaMesothelioma
14 Lung AdenomaEpididymis Mesothelioma
6. Control —@tryptophan
7. Control
8. FANFT—control
19
0 3 3 4 10
16 3 0 0 0
11311
11
121111
2
12@'
42 37 4 0 0 22 IsletcellLungPituitaryS.C.ForelegSkin
AdenomaAdenomaAdenomaFibromaParagangliomaSquamous cell
carcinoma
20 5 9 1 1 4 0 17 AbdomenIslet cellThyroidEpididymis
0 0 0 0 16 2 2 Earduct Squamous cellcarcinoma
9. FANFT .-* control.-@ FANFT -*
control
10. Control —@FANFT—+ control
16 1
19 0 0 0 0 19 5 1
11. FANFT 40 0 0 0 0 40 7 3
a One of these was malignant with seeding of the pelvic
peritoneal lining.
cant (p < 0.04 and < 0.006, respectively, for Groups 4
and5). As in the rats fed FANFT and saccharin, none of the ratsfed
FANFT and tryptophan had normal bladders.
Unlike the small, solitary tumors observed in rats fedFANFT for
6 weeks followed by control diet, the tumors inrats fed FANFT
followed by either tryptophan or saccharinwere frequently multiple
and usually large, filling the bladder lumen and distending the
bladder. Areas of hyperplasiawere generally present in other parts
of the bladder mucosanot involved by tumor. The extent of invasion
of the bladderlesions in the various groups is shown in Table 3,
but
invasion did not always correlate with size of lesions.Several
of the larger tumors had areas of necrosis, and
occasionally calcification was present in these necroticareas
(Table 2). Calculi were not observed macroscopicallyin any of the
groups of rats, but occasional microscopiccalculi were observed
trapped betwen papillary fronds of atumor. Such calculi were
observed in 2 rats in each ofGroups 1 and 10 and in 3 rats in Group
11.
Scanning electron microscopy performed at Weeks 6, 12,and 104 of
the experiment showed normal bladder mucosalsurfaces in the rats in
the control group and in the groups
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Extent of lesions of theurinarybladderNo.
ofPapillo
CarcinomaNon-Inva
SubmuGroupratsmasTotalsiveStalkcosaMuscleSerosa1.FANFT—'saccharin1901811―42012.FANFT
-+ control —*sac 1801 31
0@2001bcharm4,FANFT—@tryptophan19110721005,FANFT
-@
control—@204108@'0101tryptophan8.FANFT-@control2014301009,FANFT
—@control-.160162@'6'620FANFT—@control10.Control—*
FANFT—*1901946450control11.FANFT400406@'1817
Promotion in Bladder Carcino genesis
Table 3
(1 Sarcoma also present in one rat bladder.
t@Metastases present.(@ Sarcoma also present in 2 rat
bladders.
d Metastases present in 2 rats.
fed only saccharin or tryptophan. Occasional small blebs(Fig. 1)
were noted on the bladder surface of one of the ratsfed saccharin
at Week 104 of the experiment, but we haveseen these in control
rats in previous experiments.One ofthe 2 ratsfed FANFT for6 weeks
and examined at
that time had small foci of hyperplastic epithelium with
theluminal surface having numerous short, uniform microvilliand
microridges, but a few pleomorphic microvilli were alsopresent
(Fig. 2). The other rat in this group at 6 weeks hadonly uniform
microvilli with mild hyperplasia. The rats fedFANFT 6 weeks
followed by control diet for 6 weeks andexamined at that time
showed normal mucosa by lightmicroscopy, and a nearly normal mucosa
was observed byscanning electron microscopy with the surface being
flat(Fig. 3). The epithelial cells were polygonal with microridges
on the luminal surface, but a few foci were observedwith some
variation in cell size. Rats fed FANFT for 12weeks and then
examined showed marked epithelial hyperplasia with nodular and
papillary formation by light microscopy with numerous pleomorphic
microvilli present on theluminalcellsurfaceswhen examined by
scanning electronmicroscopy, similar to the appearance previously
described(33, 34). The rats fed FANFT for 6 weeks followed
bysaccharin for 6 weeks and then examined had moderateepithelial
hyperplasia (Fig. 4) with marked variation in cellsize and shape.
Cell surfaces were covered with numerousuniform microvilli, and
some cells were covered with bothnumerous and uniform pleomorphic
microvilli (Fig. 5). Ratsfed FANFT for 6 weeks followed by
tryptophan for 6 weeksand then examined were nearly normal by light
and scanning electronmicroscopy, but
greatervariationincellsize(Fig. 6), and more foci with variations
in size were presentthan in the rats fed FANFT for 6 weeks followed
by controldiet. Scanning electron microscopy of tumors from
thevarious groups was similar to that previously described
forurinarybladder tumors in rats(33,34) with the surfaceshaving
numerous pleomorphic microvilli (Fig. 7) as well asnumerous uniform
microvilli and ropy microridges. The onerat in Group 1 that did not
have a bladder carcinoma hadmarked nodular hyperplasia, and the
luminal surface hadnumerous pleomorphic microvilli (Fig. 8).
The urinary bladders examined by scanning electronmicroscopy
were also examined by transmission electronmicroscopy. Changes
observed by transmission electronmicroscopy were similar to those
described previously (33,34) and were consistent with the types of
lesions observedby light and scanning electron microscopy. There
wasgradual loss of asymmetrical membrane and fusiform vesides as
the lesions progressed. No asymmetrical membranewas present in
microvilli.
One of the bladder tumors from Group 2 was transplanteds.c. to
weanling male Fischer rats and has been successfully carried
through 5 transplant generations. The histology of the original
tumor showed both a transitional cellcarcinoma and a highly
anaplastic sarcoma invadingthrough the bladder wall. Only the
sarcoma grew in thetransplanted tumors, and examination by
transmissionelectron microscopy demonstrated plump tumor cells
withlarge round to oval nuclei containing clumped chromatinand
occasionally bizarre nucleoli. The cytoplasm containedlarge
mitochondria with tubular cristae, well-developedGolgi caveolae,
and abundant polyribosomes and endoplasmic reticulum filled with
flocculent material. Occasionalstereocilia were present. The cells
lacked desmosomes andtight junctions, but some gap junctions were
observed.Basement membrane-like material and collagen were
produced. Myofilaments were not present. These features
areconsistent with a very poorly differentiated fibrosarcoma.
The most common non-bladder tumor was the interstitialcell tumor
of the testis (Table 2), which frequently occurredbilaterally and
appeared more often as the rats increased inage. Thus, few
interstitial cell tumors were seen in the ratsfed FANFT for long
periods since none survived beyondWeek 80 of the experiment. No
correlation was observedbetween the occurrence of testicular tumors
and the induction of bladder tumors. Testicular tumors are very
commonly found spontaneously in older male control rats (22).
Other tumors observed in this experiment are summarized in Table
2 and also occurred with greater frequencywith increasing age of
the rats. Endocrine tumors werelisted as adenomas without
attempting to differentiate between hyperplastic nodules and
adenomas. In addition to
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S. M. Cohen et al.
the tumors listed in Table 2, most of which originated invarious
endocrine organs or soft tissues, the older ratsoccasionally showed
mild, focal bile duct hyperplasia in theliver and mild, focal
interstitial nephritis. All of these nonbladder lesions are common
findings in older rats (22). Inaddition, a single rat in each of
Groups 8 and 10 hadhyperplastic nodules in the liver which are
occasionallyseen in older rats spontaneously (22).
DISCUSSION
The 2-stage process of carcinogenesis, initiation andpromotion,
was initially proposed and demonstrated in themurine skin cancer
model (8, 9). Several properties ofinitiation and promotion have
been characterized experimentally in that system (10, 67).
Initiation by itself does notresult in cancer but requires the
action of a promotingagent to produce a cancer. Initiation is
irreversible; application of the promoting substance can be delayed
for longperiods of time, and the carcinogenic process will
stillappear. Application of the promoting substance
withoutinitiation theoretically will not induce tumors, but
long-termadministration of promoting substances has usually
resuited in low incidences oftumors (11, 12, 52, 65). Application
of the promoter for periods adequate for promotion,but administered
before the initiator, does not result intumors. Although
theoretically a pure initiator could exist,in practice
subcarcinogenic doses of complete carcinogenshave been used as
initiating agents. In addition, it appearsthat the mechanisms of
initiation and promotion differbiochemically (67). Most initiating
agents are also mutagenic in various short-term in vitro assays,
whereas promoting agents generally are not mutagenic or have only
weakmutagenic activity. Promoting agents usually have the ability
to induce hyperpiasia of the target organ without initiation, but
this does not progress to cancer unless the tissuehas been
initiated.
Although multistage carcinogenesis has been studiedprimarily in
the murine skin cancer model, other organsrecently have also been
demonstrated to undergo a similarprocess. Phorbol administered
systemically rather thantopically has had weak promoting activity
for skin carcinogenesis in mice (6), but it had greater promoting
activity forliver and lung tumors in mice following initiation
withdimethylnitrosamine(4)and forthe
breastinratsfollowinginitiation with 7,12-dimethylbenz(a)anthracene
(5). Initiationand promotion involving hepatic carcinogenesis has
alsobeen reported, with a variety of chemicals used as
initiatingand promoting agents (21, 47). For the urinary bladder,
the2-stage process has been demonstrated in rats with a
singleintravesical instillation of MNU followed by either
sodiumsaccharin or sodium cyclamate (29, 31, 32). The
singleinstillation of MNU did not induce tumors, but saccharin
orcyclamate alone given p.o. resulted in a very low incidenceof
bladder tumors after 2 years. Cyclophosphamide injectedi.p. did not
demonstrate promoting activity in the MNUexperimental model.
Six weeks of 0.2% FANFT was chosen in the presentexperiment as
the initiating agent because previous experiments in our
laboratories had demonstrated that, althoughrats fed 0.2% FANFT for
6 weeks developed mild bladder
epithelial hyperplasia, this had regressed when the ratswere
then fed the control diet so that the epithelium wasnormal at 52
(33) and 84 (34) weeks when examined by lightand electron
microscopy. The appearance of tumors in therats fed 0.2% FANFT for
6 weeks followed by control diet inthe present experiment was
unexpected in the light of theseprevious studies. To our knowledge,
neither the carcinogennor our laboratoryprocedures had changed
except thatthepresent experiment was continued to a total of 104
weeks.In previous experiments (27, 33, 34), examination of
theurinary bladders by scanning electron microscopy showedthe
mucosal surface to have uniform microvilli but notpleomorphic
microvilli after 6 weeks of 0.2% FANFT. In thepresent experiment,
however, pleomorphic microvilli werepresent in 1 of 2 bladders
examined at 6 weeks. This mightindicategreatersusceptibilityof
thisgroup of ratsto thecarcinogen than in previous experiments, but
male inbredFischer weanling rats from the same supplier were used
inboth instances and under the same conditions. An alternative
explanation is that, at 6 weeks, a small percentage ofthe rats fed
0.2% FANFT have pleomorphic microvilli, andby chance we observed
some in one rat in this experimentand not in previous experiments.
Since pleomorphic microvilli appear to be a marker of
irreversibility in the FANFTFischer rat model (27, 33, 34), finding
them in few rats after6 weeks of FANFT is consistent with the low
incidence ofbladder tumors observed when these rats are then
fedcontrol diet until a total of 2 years as in the
presentexperiment.
The results of this experiment and the studies of Hickswith MNU
are consistent with the 2-stage theory of carcinogenesis for the
urinary bladder. Features consistent withthe theory and similar to
the murine skin model are thesequential administration of 2
chemicals to induce cancer,the use of a low dose of a complete
carcinogen as theinitiating agent, and the lack of (or very low)
carcinogenicactivity of the promoting chemicals when
administeredwithout previous initiation. Previous experiments in
ratswith one-generation administration of saccharin or of cyclamate
plus saccharin have resulted in very low incidences ofbladdertumors
(15, 16, 29, 31, 32, 43, 46, 49), and negativeresults have been
observed in some species (23) includingmice (37), hamsters (2), and
a few other studies in rats (26,53, 56, 61, 62). In the present
experiment, the number ofrats in each group might well have been
insufficient todetect a low level of carcinogenic activity of
saccharin aspreviously reported. Moreover, rats in the present
experiment did not receive saccharin until after the sixth week
ofthe experiment when they were 10 weeks old. In
previousexperiments evaluating saccharin, feeding of the
chemicalwas begun at an earlier age. It should be noted,
however,that our studies were done to study the possible
promotingeffect of saccharin in this model system, not the
carcinogenic activity of saccharin. Two-generation studies,
theadministration of saccharin over 2 generations includingduring
pregnancy and lactation, resulted in higher mcidences of bladder
tumors than did one-generation studies(7).
Two possible explanations can be proposed regardingthe low
incidence of bladder tumors when only saccharin isadministered,
whereas high incidences occur when sac
1212 CANCERRESEARCHVOL. 39
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Promotion in Bladder Carcino genesis
charm follows either MNU or FANFT administration. Thelow
incidence resulting with saccharin alone may reflectthe background
incidence of initiated cells in the urinarybladder occurring due to
spontaneous changes, factors inthe diet, or endogenous factors. Low
levels of nitrosamines,for example, have been found in many grain
diets and canalso be formed endogenously from amines and nitrite
(55,59). An alternative explanation is that saccharin is a complete
carcinogen but has only weak initiating capabilities.This would be
similar to the results in skin carcinogenesiswhere long-term
administration of promoting agents resultsin a low incidence of
tumors (11, 12, 52, 65). Evaluation ofsaccharin as an initiating
chemical in the mouse skin modelshowed it to have weak but not
statistically significantactivity (54). If saccharin is a complete
carcinogen, thensaccharin following either MNU or FANFT may
represent asynergistic reaction resulting in a summation effect
(44, 60,64). At present, there are insufficient data available to
statewhich of these alternatives are correct. Reversing the
Sequence of administration might provide relevant new information,
but the long period of time required for the promoting phase for
the urinary bladder will make that experimentdifficult to design
properly for adequate evaluation.
The 6-week delay between the end of FANFT administration and the
beginning of either saccharin or tryptophanfeeding, although
relatively short, results in return of thebladder mucosa to an
essentially normal appearance bylight and electron microscopy (20,
33, 34). Also, FANFT israpidly eliminated from the rat via the
urine and feces (58),and little if any would be expected to be
present after 6weeks on control diet. Since both saccharin and
tryptophaninduced bladder tumors even after the 6-week delay
afterFANFT administration, the initiated changes were irreversible
at least for that period of time as was the stage ofinitiation in
the mouse skin model.
Other properties of saccharin are also consistent with itsbeing
at least a promoting agent. In general, saccharinlacks mutagenic
activity in short-term in vitro assays (18,24, 36, 40, 57, 66). The
weak mutagenic activity occasionallyfound might be due to
impurities present in the test preparations (24, 57). These
impurities may be acting as initiatorsin vivo with the saccharin
acting as the promoter. However,it seems highly unlikely that these
impurities can accountfor all of the activity attributed to
saccharin since they arepresent at very low levels. For the
impurities to account forthe carcinogenic activity of saccharin,
they would have tobe some of the most potent carcinogens ever
discovered.Our material was examined foro- and
p-toluenesuifonamidelevels, which were very low (
-
S. M. Cohen et al.
impurities in carcinogens and noncarcinogens by high-pressure
liquidchromatography and the Salmonella/microsome test. Cancer Res.
, 38:431-438, 1978.
25. Dunning, W. F., Curtis, M. R., and Maun, M. E. The effect of
addeddietary tryptophan on the occurrence of
2-acetybammnofluoreneinducedliver and bladder cancer in rats.
Cancer Res., 10: 454-459, 1950.
26. Fitzhugh, 0. G. , Nelson, A. A., and Frawley, J. P.
Comparison of chronictoxicities ofsynthetic sweetening agents. J.
Am. Pharm. Assoc. Sci. Ed.,40: 583-596, 1951.
27. Friedell, G. H., Jacobs, J. B., Nagy, G. K., and Cohen, S.
M. Thepathogenesis of bladder cancer. Am. J. Pathol., 89: 431-442,
1977.
28. Fulker, M. J., Cooper. E. H., and Tanaka, T. Proliferation
and ultrastructure of papillary transitional cell carcinoma of the
human bladder.Cancer,27:71-82,1975.
29. Hicks, R. M., and Chowaniec, J. The importance of synergy
betweenweak carcinogens in the induction of bladder cancer in
experimentalanimals and humans. Cancer Res., 37: 2943-2949,
1977.
30. Hicks, R. M. , and Wakefield, J. St. J. Membrane changes
duringurothelial hyperplasia and neoplasia. Cancer Res., 36:
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31. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J.
Co-carcinogenicaction of saccharin in the chemical induction of
bladder cancer. Nature,243:347-349,1973.
32. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J.
Evaluation of a newmodel to detect bladder carcinogens or
co-carcinogens; results obtamed with saccharin, cyclamate and
cycbophosphamide. Chem.-Biol.Interact., 11: 225-233. 1975.
33. Jacobs, J. B., Arai, M., Cohen, S. M., and Friedell, G. H.
Early lesions inexperimental bladder cancer: scanning electron
microscopy of cellsurface markers. Cancer Res., 30: 2512-2517,
1976.
34. Jacobs, J. B. , Arai, M., Cohen, S. M. , and Friedell, G. H.
A long-termstudy of reversible and progressive urinary bladder
cancer lesions in ratsfed
N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide. Cancer Res. , 37:
2817-2821, 1977.
35. Jacobs, J. B., Cohen, S. M., Arai, M. , and Friedell, G. H.
SEM on bladdercells. Acta Cytol., 21: 3—4,1977.
36. Kramers, P. G. N. The mutagenicity of saccharin. Mutat.
Res., 32: 81-92,1975.
37. Kroes, R., Peters, P.W.J., Berkvens,J.M.,Verschuuren, HG.,
DeVires,T., and Van Esch, G. J. Long-term toxicity and reproduction
study(including a teratogenicity study) with cyclamate, saccharin
and cycbohexylamine. Toxicology, 8: 285-300, 1977.
38. Lutz, W. K. , and Schlatter, C. Saccharin does not bind to
DNA of liver orbladder in the rat. Chem.-Biol. Interact. , 19:
253—257,1977.
39. Matsushima, M. The role of the promoter L-tryptophan on
tumorigenesisin the urinary bladder. 2. Urinary bladder
carcinogenicity of FANFT(initiating factor) and L-tryptophan
(promoting factor) in mice. Jpn. J.Urol., 68: 731-736, 1977.
40. McCann , J . Results of a battery of short-term tests on
highly purifiedsaccharin. In: Cancer Testing Technology and
Saccharin, appendix 2,pp. 91-108. Washington, D. C.: Office of
Technology Assessment,Congress of the United States, 1977.
41. Miyakawa, M. , and Yoshida, 0. DNA synthesis of the urinary
bladderepithelium in rats with long-term feeding of
DL-tryptophan-added andpyridoxmne-deficient diet. Gann, 64:
411-413, 1973.
42. Mondal, S., Brankow, D. W. , and Heldelberger, C.
Enhancement ofoncogenesis in C3H/1OT'/2 mouse embryo cell cultures
by saccharin.Science,201: 1141-1142, 1978.
43. Munro, I. C. . Moodie, C. A. , Krewski, D., and Grice, H. C.
A carcinogenicity study of commercial saccharin in the rat.
Toxicob. Appb. Pharmacol., 32: 513-526, 1975.
44. Nakahara, W. Critique of carcinogenic mechanism. Prog. Exp.
TumorRes., 2: 158-202, 1975.
45. Okajima, E., Hiramatsu, T., Motomiya, Y., lriya, K., Ijuin,
M., and Ito, N.Effect of DL-tryptophan on tumorigenesis in the
urinary bladder and liverof rats treated with
N-nitrosodibutylamine. Gann, 62: 163-169, 1971.
46. Oser, B. L., Carson, S., Cox, G. E., Vogin, E. E., and
Steinberg, S. S.Chronic toxicity study of cycbamate:saccharin
(10:1) in rats. Toxicology,4:315—330,1975.
47. Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisieleski,
W. E. Effects ofvarying the exposure to phenobarbital on its
enhancement of 2-acetylaminofluorene-induced hepatic tumorigenesis
in the rat. Cancer Res.,33:2701-2705,1973.
48. Price, J. M. Etiology of bladder cancer. In: E. Maltry, Jr.
(ed), Benignand Malignant Tumors of the Urinary Bladder, pp.
189—261. Flushing, N.Y. : Medical Examination Publishing Co. ,
Inc. , 1971.
49. Price, J. M., Biava, C. G., Oser, B. L., Vogin, E. E.,
Steinfeld, J., andLey, H. L. Bladder tumors in rats fed
cycbohexylamine or high doses of amixture of cyclamate and
saccharin. Science, 167: 1@31-1132,1970.
50. Radomski, J. L., Glass, E. M., and Deichmann, W. B.
Transitional cellhyperplasia in the bladders of dogs fed
DL-tryptophan. Cancer Res., 31:1690-1694, 1971.
51. Radomski, J. L., Radomski, T. , and MacDonald, W. E.
Cocarcinogenicinteraction between D,L-tryptOphan and
4-aminobiphenyl or 2-naph
CANCER RESEARCH VOL. 391214
level could be eliminated, e.g. , by administering vitamin
B6,recurrencesshould be reduced or prevented.Such a resulthas
recently been reported by Byar and the Veterans Administration
Cooperative Urological Research Group (17), although urinary
tryptophan metabolite levels were not measured. The importance of
tryptophan metabolites in theetiology of bladder cancer and the
usefulness of vitamin B6in the treatment of bladder cancer,
particularly low-gradesuperficial papillary tumors, need further
evaluation.
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Promotion in Bladder Carcino genesis
APRIL 1979
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S. M. Cohen et al.
Fig. 1. Luminal surface of the bladder of a rat fed saccharin
(Group 3) and sacrificed at the end of the experiment (2 years).
The typical microridgeappearance seen in normal rat bladder is
present along with an occasional bleb. x 6,000.
Fig. 2. Pleomorphic microvilli present on the bladder luminal
surface of a rat fed FANFT for 6 weeks and then killed. X
10,000.Fig. 3. The luminal surface of the bladder of a rat fed
FANFT for 6 weeks followed by control diet for 6 weeks and then
killed. The bladder mucosa has
returned to near normal with a flat surface composed of large
polygonal cells with microridges. Occasional foci show some
variation in cell size. x 500.Fig. 4. Hyperplastic bladder
epithelium from a rat fed FANFT for 6 weeks followed by saccharin
for 6 weeks and then killed. x 200.
-@‘@:.@@
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1216 CANCER RESEARCH VOL. 39
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Promotion in Bladder Carcino genesis
__@___#@
Fig. 5. Higher magnification of the bladder shown in Fig. 4
showing pleomorphic microvilli on the surface in addition to the
short, uniform microvilli.x 4,000.
Fig. 6. The bladder surface of a rat fed FANFT for 6 weeks
followed by tryptophan for 6 weeks is near normal, similar to that
shown in Fig. 3, but foci withvariations in cell size are more
numerous. x 200.
Fig. 7. Transitional cell carcinoma in a rat fed FANFT for 6
weeks and then fed saccharin. The rat was killed at the end of the
experiment. x 200.Fig. 8. Numerous pleomorphic microvilli present
on the bladder surface of a rat fed FANFT for 6 weeks followed by
saccharin and killed at the end of the
experiment. This was the only rat in Group 1 that did not have a
bladder carcinoma. x 8,600.
1217
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-
1979;39:1207-1217. Cancer Res Samuel M. Cohen, Masayuki Aral,
Jerome B. Jacobs, et al. Bladder CarcinogenesisPromoting Effect of
Saccharin and dl-Tryptophan in Urinary
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