Download Effect of L-Tryptophan Excess and Vitamin

[CANCER RESEARCH 47, 1244-1250, March 1, 1987]
Effect of L-Tryptophan Excess and Vitamin B6 Deficiency on Rat Urinary
Bladder Cancer Promotion1
Diane F. Birt,2 Alan D. Julius, Ryohei Hasegawa, Margaret St. John, and Samuel M. Cohen
The Eppley Institute for Research in Cancer [D. F. B., A. D. J., S. M. C.J and Department of Pathology and Microbiology, College of Medicine IR H M S J
S. M. C.J, University of Nebraska Medical Center, Omaha, Nebraska 68105-1065
2-napluIn lamine, benzidine, and 4-aminobiphenyl (6).
Further evidence for the carcinogenicity of tryptophan me
To further evaluate the role of tryptophan and vitamin H„
in bladder
carcinogenesis, male Fischer 344 rats were fed 0.2% ¡\'-|4-(5-nitru-2- tabolites was provided by studies using the pellet implantation
technique (6-8); however, these results remain difficult to in
furyl )-2-tliia/olyl|f(irmamidf (FANFT) in semipurified diet or were given
terpret. Reports in the late 1960s and early 1970s began to
semipurified diet alone for 4 weeks. One week later, rats from each group
raise considerable doubt as to the role of tryptophan in bladder
were assigned for the remainder of the experiment to one of four exper
carcinogenesis because findings when indole was coadminisimental diets, labeled as follows: group 1, control semipurified; group 2,
i.-tryptnplian excess (2'7.);group 3, vitamin Bt-deficient (1.0 mg/kg diet);
tered with AAF (9) were similar to those observed with trypto
or group 4, i -tryptophan excess, plus vitamin Bt-deficient diet. All
phan (1). However, indole is produced from tryptophan via an
surviving rats were killed at 80 weeks of the experiment. Throughout the
entirely different pathway than that of kynurenine and related
study, body weights were reduced in the groups fed FANFT and, at 70
amines. A recent study also demonstrated no significant differ
and 80 weeks, body weights were reduced in the groups given tryptophan
ences between the level of various tryptophan metabolites in
excess. The incidence of urinary bladder carcinoma was highest in the
the urine of bladder cancer patients versus that of controls (10).
group treated with FANFT, followed by diet with control tryptophan and
Interpretation of the latter study is difficult, because the patients
vitamin B*levels (40%). The disease incidence was reduced in the vitamin
were from Boston, where the association between bladder can
Bfc-defieientgroup (13%) and of an intermediate range in the groups fed
cer
and abnormal tryptophan metabolism was shown to be
a tryptophan excess with or without vitamin B«deficiency (28-29%).
weaker than in other geographical areas (e.g., Wisconsin) (11).
Tumors at other sites were greatest in number in KANKI -treated rats
fed vitamin Bt-deficient diet with excess tryptophan and were significantly
Tryptophan has been tested recently as a promoter for urinary
bladder carcinogenesis (12-14). Cohen et al. (12) demonstrated
fewer in FANFT-treated rats fed vitamin Bt-deficient diet alone. Animals
that the feeding of DL-tryptophan after FANFT initiation sig
given diet deficient in vitamin Be consistently had depressed levels of
alanine aminotransferase activity and plasma pyridoxyl phosphate.
nificantly increased the bladder tumor incidence. When this
FANFT pretreatment decreased alanine aminotransferase activities in
work was repeated with a lower dose of FANFT and L-tryptorats in some groups and the feeding of tryptophan had variable effects
phan as promoter, the bladder tumor incidence again increased,
on alanine aminotransferase and plasma pyridoxyl phosphate levels.
however, not to a statistically significant level (13). Tryptophan
Urinary tryptophan metabolites were influenced by all treatments, but
did
not induce bladder tumors in either study without prior
the results did not correlate with tumor yields. Urinary bladder ornithine
initiation. Similar results have been found in mice (15) and
decarboxylase activity was not altered in vitamin Bt-deficient female rats.
dogs (16, 17).
These results do not support the hypothesis that increased dietary i An involvement of vitamin B,, in urinary bladder cancer is
tryptophan promotes bladder carcinogenesis in rats, but other dietary
suggested, because this vitamin is required as a cofactor for
factors might modify the process following FANFT initiation.
several enzymes in the metabolic pathway from tryptophan to
niacin (Fig. 1). Thus, vitamin B6 deficiency causes increases in
INTRODUCTION
several metabolites that are suspects in urinary bladder carci
The relationship of tryptophan to urinary bladder carcino
nogenesis (18). The administration of vitamin B6 to bladder
genesis has remained a dilemma since the original observation
cancer patients has corrected the metabolic abnormalities in
in 1950 by Dunning et al. (1) of an increased bladder cancer
tryptophan metabolism in many of them (19). Furthermore,
incidence following AAF3 administration simultaneously with
bladder cancer patients with abnormal tryptophan metabolite
DL-tryptophan. Several tryptophan metabolites, such as kynuexcretion have shown a high recurrence rate of urinary bladder
renine and anthranilic acid and their corresponding o-aminotumors, compared with that in patients with normal tryptophan
phenols, are aromatic amines and are present at elevated levels
metabolite levels (20). The evidence of a role for vitamin B6 in
in the urine of many bladder cancer patients (2-5). These
bladder cancer was strengthened by suggestions that pyridoxine
compounds are chemically and metabolically similar to known
can prevent bladder cancer recurrence (21). Unfortunately, tryp
human bladder carcinogens of the aromatic amine class, e.g.,
tophan metabolite levels were not measured in this study. In
addition, an earlier report showed that vitamin B«restriction
Received 6/19/86; revised 11/13/86; accepted 11/14/86.
enhanced bladder tumor incidences in Fischer 344 rats treated
The costs of publication of this article were defrayed in part by the payment
with 2-AAF (22). Early research on the cocarcinogenicity of
of page charges. This article must therefore be hereby marked advertisement in
tryptophan used diets marginal in vitamin B6 (1, 23, 24).
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Research Grants CA 33368 and CA 32513 and Laboratory
The present studies were designed to further test tryptophan
Cancer Research Center Grant CA 36727 from the National Cancer Institute.
as a promoter of FANFT-induced urinary bladder cancer, to
Presented in part at the annual meeting of the Federation of American Societies
explore the effects of vitamin B6 deficiency in this system, and
for Experimental Biology, April 1984, St. Louis, MO (58) and at the annual
meeting of the American Association for Cancer Research, May 1986, Los
to assess the association between urinary tryptophan metabo
Angeles, CA (59).
lites and urinary bladder carcinogenesis.
1To whom requests for reprints should be addressed, at The Eppley Institute
ABSTRACT
for Research in Cancer, University of Nebraska Medical Center, 42nd St. and
Dewey Ave., Omaha, NE 68105-1065.
3The abbreviations used are: AAF, W-2-fluoroenylacetamide; FANFT, N-[4(5-nitro-2-furyl)-2-thiazolyl)formamide;
ALAT, alanine aminotransferase; PLP,
pyridoxal phosphate; ODC, ornithine decarboxylase; TPA, 12-O-tetradecanoylphorbol-13-acetate.
MATERIALS
AND METHODS
Experimental Diets. The control seni ipurified diet (Table 1) was
modified from the diet recommended by the American Institute of
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L-TRYPTOPHAN AND VITAMIN B. IN URINARY BLADDER CANCER
>¡—¡|-CH2-CH2
L^
-CH2CHCOOH
^ VIT.B8
NH2
^COOH
NH2
ANTHRANILIC
ACID
THYPTOPHAN
SEROTONIN
O
COOH
Fig. 1. Metabolic scheme indicating the in
volvement of vitamin B* in tryptophan metab
olism.
XANTHURENIC ACID
8-METHYL ETHER
O
H. (fYOOH
COOH
.,
,COOH
^N^
COOH
3-METHOXYANTHRANILIC
OCH3
NICOTINIC
ACID
QUINOLINIC
ACID
Nutrition Ad Hoc Committee on Standards for Nutritional Studies
(25, 26). Modifications included changing carbohydrate sources from
sucrose and cornstarch to dextrin and glucose monohydrate (dextrose)
and including vitamin H,,as PLP at a level .of 10.0 mg/kg diet. The
control L-tryptophan level was that found in 20% casein (approximately
0.24%, w/w) and the elevated level was the control amount, plus 2%
(w/w) L-tryptophan (obtained from Sigma Chemical Co., St. Louis,
MO), added at the expense of dextrin. PLP was given at a control level
of 10.0 mg/kg diet and at a suboptimal level of 1.0 mg/kg diet.
Previously, the latter level had allowed normal growth for up to 4
months (27) but was not sufficient to maintain normal vitamin Be
status, as indicated by depressed glutamic-pyruvic transaminase activ
ity. FANFT was obtained from Saber Laboratories (Morton Grove, IL)
and was added at 0.2% to the control diet for feeding, as described
below. The diets were freshly prepared every 3 weeks and stored at 4'C
between feeding. Food and water were freely provided throughout the
study, fresh diet was given weekly, and food consumption records were
kept for all animals throughout the study.
Animals. Male Fischer 344 rats were obtained at 4 weeks of age from
the Kingston plant of Charles River Breeding Laboratories (Wilming
ton, MA). After 1 week of quarantine in the Eppley Institute animal
facilities, the animals were randomly assigned to treatment groups.
During the next 4 weeks, 160 rats were fed the control semipurified
diet containing 0.2% FANFT, and another 160 were given the control
diet only. All rats were fed control diet in the fifth week of the
experiment and, beginning in the sixth week, groups of 40 rats from
the FANFT-treated and non-FANFT-treated groups were assigned to
each of the four experimental diets for the remainder of the experiment:
group 1, control semipurified; group 2, L-tryptophan excess; group 3,
vitamin fit-deficient; or group 4, L-tryptophan excess plus vitamin H,.
deficient.
Rats were housed in groups of 5 on corncob bedding (Bed-o-Cobs;
The Anderson Cob Co., Maumee, OH) and maintained under standard
conditions of temperature [21 ±2°C],humidity [40 ±5% (SE)], a 12h light/12-h dark cycle, and 10 air changes/hour. Body weights were
recorded weekly for the first 10 weeks of the experiment and then every
other week for the remainder. Rats were checked twice daily and
moribund animals were killed. Surviving rats were killed at 80 weeks
of the experiment. Complete necropsies were performed in all cases
and the tissues, except the urinary bladder, were fixed in 10% buffered
formalin. The urinary bladder was inflated with Bouin's fixative and
washed with 70% ethanol. Tissues were embedded in paraffin and
stained with hematoxylin and eosin. Data are presented only for animals
Tablet
ACID
Control experimental diet*
Ingredient
Vitamin-free casein
DL-Methionine
Glucose monohydrate
Dextrin
Fiber»
Corn oil
AIN vitamin nn\' (modified as described in text)
AIN mineral mix'
Choline bitartrate
20.0
0.3
20.0
45.0
5.0
5.0
1.0
3.5
0.2
Total
100.0
* Ingredients purchased from Teklad Test Diets, Inc., Madison, WI.
* Nonnutritive fiber.
cOne % in diet provides: in mg/kg, thiamine •
HCI, 6.0; riboflavin, 6.0; pyridoxine HCI (vitamin H,.)-7.0; nicotinic acid, 30.0; calcium pantothenate, 16.0;
folk acid, 2.0; biotin, 0.2; in Mg/kg, cyanocobalamin, 10.0; vitamin K, 50.0; in
lU/kg, vitamin A, 4000.0; vitamin D, 1000.0; vitamin E, 50.0.
'3.5% in diet provides the following elements (in mg/kg): calcium. 5200.0;
phosphorus, 4000.0; sodium, 1020.0; potassium, 3600.0; magnesium, 500.0;
manganese, 54.0; iron, 35.0; copper, 6.0; zinc, 30.0; iodine, 0.2; selenium, 0.1;
chromium, 2.0; chloride, 1560.0; sulfate, 1000.0.
that died after 63 weeks of the experiment and for tissues which were
not autolyzed.
Blood was collected from the orbital sinus at 0, 4, 5, 10, 26, 52, and
78 weeks of the study from 10 rats randomly selected from each of the
8 experimental groups. Approximately 1 ml of blood was made 2.0 x
10 ' M in EDTA and maintained in an ice bath. The erythrocytes were
used for ALAT measurements and the plasma was used for PLP
determination, as described below. Urine was collected at 0, 4, 5, 10,
26, and 52 weeks from 8 rats in each experimental group randomly
selected from those animals which had not been bled. Rats were placed
individually in Nalgene plastic metabolism cages for 16 h from 4:30
p.m. The collection vial was kept in dry ice to freeze the urine as it was
collected. In addition, urine was collected from all surviving rats for 16
h from week 78 through week 80 of the experiment. The urine was
used to measure tryptophan metabolites, as described below.
Urinary bladder ODC activity was assessed in female F344 rats
prefed the control and vitamin B6-deficient diets for 26 to 28 weeks.
Females were used because of the need for catheterization of the urinary
bladder to induce ODC. One half of the rats were treated intravesically
with 0.3 ml (10%) dimethyl sulfoxide; the other half received 2 nm
TPA in 10% dimethyl sulfoxide, as described previously (28). Rats were
killed 6 h after instillation and the urinary bladder was removed and
1245
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L-TRYPTOPHAN
AND VITAMIN
B6 IN URINARY BLADDER CANCER
homogenized in 50 n\i phosphate buffer containing 0.1 DIMsodium
EDTA and 0.1 mM PLP (pH 7.2); the supernatant was used to deter
mine ODC activity. Bladders from 5 rats were pooled for each assay;
assays were conducted in duplicate in non-TPA-treated rats and in
triplicate in TPA-treated animals.
Analytical Methods. Vitamin H,. status was assessed by measuring
plasma PLP by the tyrosine apodecarboxylase method (29). Plasma
was stored for up to 6 months at —
70°C,and protein was precipitated
the day before the assay. Apotyrosine decarboxylase obtained from
Cooper Biomédical,Inc. (Malvera, PA), was used for the assay and was
purified by extraction in 10 mM sodium citrate buffer and precipitated
with ammonium sulfate (29). DL-['4C]Ornithine was purchased from
Amersham Corporation (Arlington Heights, IL) and the trapped radio
activity was compared with values for PLP standards. Utilization of
vitamin li,, was evaluated by determining erythrocyte ALAT activity
and its stimulation by PLP (30, 31). Erythrocytes were washed with
0.9% saline after removal of plasma and diluted with 19 parts of
redistilled water to prepare the hemolysate, which was stored frozen
for no longer than 1 week and incubated with alanine by described
methods (30, 31). Pyruvate was determined colorimetrically after re
action with 2,4-dinitrophenylhydrazine (Sigma).
Urinary tryptophan metabolites were assayed the day following over
night urine collection. Samples were thawed and eluted through a Bond
Elute Cis disposable column (Analytichem International, Harbor City,
CA) in the following order: 1 ml sample, 1 ml 0.02 M K11..PO, buffer,
pH 3.7 (solvent A); 1 ml 60:40 methanokwater (solvent B); l ml
methanol. Urinary tryptophan metabolites were separated by reverse
phase high pressure liquid chromatography on a Partisi! PXS 5/25
ODS column with a 35-min linear gradient, 0-100% solvent A-solvent
SOD-,
ir
O
o
UJ
S
O
10
20
30
40
50
WEEK OF STUDY
60
70
80
Fig. 2. Effect of FANFT treatment and tryptophan excess on body weights of
male rats. Data were pooled across vitamin H,, intake, since significant effects
were not observed. Group designations are: O, without FANFT, control trypto
phan; D, without FANFT, excess tryptophan; •,with FANFT, control trypto
phan; •,with FANFT, excess tryptophan. Bars, SE. Significant effects of FANFT,
tryptophan, week, and all two-way interactions observed by analysis of variance
(P< 0.001).
B, with a 1.5 ml/min flow rate, by described methods (32). Fluorescence
of the tryptophan metabolites was detected on an SPF-125 spectrofluorometer (American Instrument Co., Silver Spring, MD), with excitation
of 285 inn and emission of 360 nm. Fluorescent metabolites were
quantitated by comparison with the following standards: tryptophan,
5-hydroxytryptophan, tryptamine, 5-hydroxytryptamine (serotonin),
kynurenic acid, anthranilic acid, indole, indole-3-acetic acid, indole-3acetamide, indole-3-lactic acid, 5-hydroxyindole-3-acetic acid. We at
tempted to determine the following nonfluorescent metabolites by UV
absorption at 254 nm, but interfering urinary components made their
measurement meaningless: kynurenine, W-formylkynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, nicotinic acid, and xanthurenic acid. ODC activity was determined as described previously
(33).
Statistical Analysis of Data. Data on the incidence of lesions were
compared using \ ' tests (34). Body weight, food consumption, and data
collected from blood and urine were evaluated by analysis of variance,
using the appropriate package of the Statistical Analyses System (35).
The experimental levels consisted of the week of measurement, with or
without FANFT treatment, dietary tryptophan, and dietary vitamin B«.
RESULTS
Body weights are shown in Fig. 2. Vitamin B6 intake did not
influence body weights. Treatment with FANFT depressed
body weights at 5 and 10 weeks of the experiment. By 30 weeks,
significant effects of FANFT were observed only in the tryptophan-fed groups; by 70 and 80 weeks, a tryptophan excess
depressed weights in all groups and FANFT treatment reduced
body weight in tryptophan-supplemented
rats. Food consump
tion was depressed during FANFT treatment (13.5 ±0.3 g/
day), in comparison with consumption at other times (15.6 ±
0.1 g/day). Dietary vitamin B6 and tryptophan did not influence
food consumption.
The incidence of urinary bladder cancer in the treatment
groups is shown in Table 2. There were few urinary bladder
papillomas and, although all of these lesions were observed in
the FANFT-treated group, their incidence did not vary between
the dietary groups. Urinary bladder carcinomas were observed
only in FANFT-treated rats. The highest incidence was found
in the control dietary group and a significantly reduced inci
dence was found in the rats fed a vitamin B6-deficient diet.
Urinary bladder cancer incidences were intermediate in the rats
fed a tryptophan excess, regardless of vitamin B6 treatment.
Tumors of the kidneys, forestomach, and other sites are
reported in Table 3. Effects of FANFT treatment were not
apparent with these lesions and dietary effects were observed
only when total tumor numbers were considered. The most
tumors were found in the FANFT-treated group fed vitamin
B6-deficient diet with excess tryptophan, while significant re
ductions occurred in the FANFT-treated group fed a vitamin
B6-deficient diet.
Other findings in the kidneys included pelvic transitional cell
hyperplasia, which was observed in 4% of the rats in the control
Table 2 Urinary bladder lesions in male rats fed excess tryptophan and vitamin Bt deficient diets'
HyperplasiaFANFT
vi
tamin
treatmentWith tryptophanControll¡,.Control
With
Excess
With
Control
With
Excess
Control
Without
Without
Excess
Without
Control
WithoutDietary ExcessDietary
* Treatments described in Table
no. of
rats4038
Control
Inadequate
Inadequate
Control
Control
Inadequate
InadequateEffective
1 and in "Materials
no.
(%of
incidence)8(20)
no. (% of
incidence)4(10)
no.
(%of
incidence)0(0)
7(18)
4(11)
3(8)
39
12(31)
6(15)
2(5)
36
10(28)
4(11)
2(6)
40
0(0)
0(0)
0(0)
40
0(0)
0(0)
0(0)
40
0(0)
0(0)
0(0)
37Simple
0(0)Nodular/papillary
0(0)TumorsPapillomas 0(0)Carcinomas
and Methods." Results of , - tests indicated by a < * (P < 0.025)
no.
(%of
incidence)16(40)(i)
11(29)
5 (13) (a)
10(28)
0(0)
0(0)
0(0)
0(0)Total
no. (% of
incidence)16
(40)
14 (37)
7(18)
12(33)
0(0)
0(0)
0(0)
0(0)
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L-TRYPTOPHAN
AND VITAMIN
B«IN URINARY BLADDER CANCER
Table 3 Tumors in mate rats fed excess tryptophan and vitamin Bf deficient diets'
tumorsPapilloma tumorsOther
kidneytumors
no.(%ofincidence)0(0)1(3)3(8)1(3)0(0)0(0)0(0)1(3)Forestomach
no.(%incidence)3(8)*0(0)0(0)t*
tumors ex
FANFT1treatmentWithWithWithWithWithoutWithoutWithoutWithoutDietary0tryptophanControlExcessControlExcessControlExcessControlExcessDietary"vitamin
no.of
no.of
tumors(%
of
cluding
urinarybladder796
incidence)1'
B«ControlControlInadequateInadequateControlControlInadequateInadequateEffective
rats4037393740404038Kidney
rats3535383133333434No.
(3)I"
(3)l'(3)2*+!'
1,'y212/3.*
(a)16
(6)0(0)0(0)l'(3)0(0)Effective
+ Ie
(9)l'(3)0(0)0(0)0(0)Othertumors2/1'3«i,*
I,' 1/1,*
(A)93411
I/I"2/1/2,*!,*
I,0!'I/l.'l'2*1«4/l/3,*I,'lrTotal
" Treatments described in Table 2 and in "Materials and Methods." Results of x2 tests indicated in the body of the table by a < b (P < 0.025).
* Transitional cell carcinoma in the renal pelvis.
' Renal cell carcinoma.
'' Forestomach papilloma.
' Forestomach squamous cell carcinoma.
'skin squamous cell carcinoma testis mesothelioma.
* Testis mesothelioma.
* Skin epidermal cyst.
' Skin fibroma.
' Leukemia.
* Lipoma.
' Skin keratoacanthoma.
"' Skin papilloma.
" Skin sebaceous carcinoma.
" Salivary gland fibrosarcoma.
* Glioma.
* Spleen sarcoma.
' Adrenal pheochromocytoma.
and vitamin B6-deficient groups and in 13% of the rats given a
tryptophan excess (P < 0.005), irrespective of FANFT treat
ment. Calcification was seen in 6% of the rats and chronic
nephropathy in 100% of the rats. In addition, forestomach
hyperplasia occurred in 36% of the rats, irrespective of treat
ment.
Vitamin B6 status at weeks 4 and 5 of the experiment, before
the experimental diets were fed, indicated a depressed ALAT
activity in the FANFT-treated rats (1.8 ±0.1 mg pyruvate
formed/ml RBC/h), in comparison with control rats (2.3 ±0.1
mg pyruvate formed/ml RBC/h; P < 0.005). Stimulation of
this activity by in vitro incubation with PLP (8% ±2%) and
plasma PLP (130 ±8 ng/ml) was not altered by FANFT
treatment. Results of these measurements at 10, 26, 52, and 78
weeks of the experiment are given in Table 4. ALAT activity
was consistently depressed in rats fed diets inadequate in vita
min B6. FANFT pretreatment decreased ALAT at 10 weeks in
rats fed excess tryptophan with inadequate vitamin B6 and at
26 weeks in rats fed excess tryptophan with adequate vitamin
H,.or inadequate amounts of vitamin B6 alone. The feeding of
excess tryptophan increased ALAT activity in erythrocytes from
non-FANFT-treated rats given inadequate amounts of vitamin
B6 at 10 weeks and decreased values in FANFT-treated rats
given adequate vitamin B6 at 10 and 26 weeks. The stimulation
of ALAT by in vitro incubation with PLP was elevated in rats
fed the vitamin B6-inadequate diets and the difference was
statistically significant at 52 weeks. No significant effects of
tryptophan or FANFT treatment were observed. Plasma PLP
was not influenced by FANFT treatment but was consistently
lower in rats fed the vitamin B6-deficient diet. Plasma PLP was
higher in rats fed excess tryptophan, with the control vitamin
B6 level at 10 weeks, and with the deficient vitamin H,,diet at
26 weeks. However, at 78 weeks of the study, plasma PLP was
reduced in the group fed excess tryptophan in the vitamin B6deficient diet, in comparison with those fed vitamin B6-deficient
diet alone.
Table 4 Effect of vitamin Bt deficiency, tryptophan excess, and FANFT on
vitamin Bt status'
vita
vitaminB«.04(a).51
MeasurementErythrocytestudy1010101026262626525252521026521010262652527878FANFT
treatmentWithoutWithoutWithWithWithoutWithoutWithWithWithoutWitho
tryptophanControlExcessControlExcessControlExcessControlEx
B»2.01
min
ALAT*%
c)1.75(b,
(A)2.19
(ft).16
(c)1
(a)0.94
(A)1.97(c,rf)2.02
.66
(a).23
(A).12
(rf)2.02
ft)0.93(a,
(d)1.73
(a)1.
ft)0.21
14 (a,
(c)0.67
(ft)0.83
(a)0.11
(ft)0.66
(a)0.08
(ft)0.59
(a)0.30
(ft)ll(a)8
(a)20
of in vitro stimu
byPLP of ALAT
lation
(%YPlasma
(a)32
(a)1
(a)26
(a)757
(A)73
(ng/ml/Wkof
PLP
(ft)I53(c)170(c)155(c)249
13
(a)81
(a)82
(a)114
(A)75(o)75(a)96
(ft)226
(ft)169
(c)146
(A)64(0)
(c)Inadequate
" Results of r test comparisons within each measurement and week indicated
by the parenthetical letters. Values not sharing a common superscript are signif
icantly different a<b<c<d(p<
0.05).
'Activity expressed as mg pyruvate formed/ml RBC/h. N = 9-10 for each
value. SE = 0.12. Significant effects of week (P < 0.001), FANFT (P < 0.05),
vitamin Be (P < 0.001), and week by FANFT by tryptophan (P < 0.02), week by
vitamin »,.(/'•0.05), week by tryptophan by vitamin B«(/' < 0.02), and week
by tryptophan by vitamin H,. </' < 0.05) interaction observed by analysis of
variance.
CN= 32-40 for each value. SE = 146-164. Significant effects of week (P <
0.05) and week by vitamin H,, !/' •0.05) interaction observed by analysis of
variance.
* Effects of FANFT treatment were not observed; therefore, data were pooled
across this factor.
' Effects of dietary tryptophan were not observed; therefore, data were pooled
across this factor.
fN= 18-20 for each value. SE = 12. Significant effects of week (/»<0.001),
vitamin B«
(P < 0.001), and week by tryptophan interaction (P < 0.005) observed
by analysis of variance.
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L-TRYPTOPHAN
AND VITAMIN
B, IN URINARY BLADDER CANCER
Table 5 Effect ofFANFT treatment, dietary vitamin Bt deficiency, and tryptophan excess on urinary tryptophan metabolites'
FANFTWkof
studyAnthranilic
Metabolite
1010262652527979K
acid*
Without
FANFTControl
vitamin
tryptophan
tryptophan
tryptophan
tryptohan
B.ControlDeficiencyControlDeficiencyControlDeficiencyControlDeficiencyPooledPooledControl
(mmol/ml)0048560(a)344
(mmol/ml)0072
(mmol/ml)0015
(mmol/ml)000(a)124
(c)0(a)0(a)8(fl)250
10265279Serotonin'
MIu rollii .nul'
(a)193
(a)010
10265279Dietary
(a)10ll(a)20
b)69(a,
e)125(a,
(A)119
(A)0(0)0(o)114
(A)233
(a)276
(a)022
(A)1315
(a)60
/»90
(a,
(a,*)282
(c)50
(a)38
(a)12
(A)18
(a)155(A,c)100
A)123
(a,
(A)123
(a)199
(a)147
(a)2511
(A)596
(A)486
(A)2824
(a)1011
(A)1219
(a,*)28
(a)9
(A)16
(A)Excess
(c)With
(a)Excess
(A)
* Results off test comparisons within a time interval and metabolite indicated by the parenthetical letters. Values not sharing a common parenthetical letter are
significantly different: a < A< c (P < 0.05).
* N - 8 for weeks 10, 26, and 52; SE = 49. N = 34-38 for week 79; SE = 23. Significant effects of week (/' < 0.001), vitamin B»
(P < 0.01) and week by FANFT
(/' < 0.03) week by tryptophan i/ ' < 0.01 ) and week by vitamin Bt (P < 0.001 ) interactions observed by analysis of variance.
' Pooled across vitamin H,,level since significant effects were not observed. V - 16 for weeks 10, 26, 52, and non-FANFT-treated groups at week 79; SE = 58. N
= 70-76 for FANFT-treated groups at week 79; SE = 27-29. Significant effects of week (/' < 0.001), tryptophan (P < 0.002), and week by tryptophan (/' < 0.02),
week by FANFT (P < 0.05) and week by FANFT by tryptophan (P < 0.01) interactions observed by analysis of variance.
J Pooled across vitamin B»level, since significant effects were not observed. Numbers as indicated in Footnote c. SE = 3 for weeks 10, 26, 52, and non-FANFT
groups at week 79, and SE = 1.0 for FANFT-treated groups at week 79. Significant effects of week (P< 0.05), FANFT (P< 0.01), tryptophan (P< 0.001), and week
by FANFT (P < 0.001) and week by tryptophan (/' < 0.05) interactions observed by analysis of variance.
Urinary 5-hydroxytryptophan and serotonin were elevated in
FANFT-treated rats at 4 and 5 weeks of the study (12 ±1
mmol/ml for each metabolite), in comparison with nonFANFT-treated controls (9 ±1 mmol/ml for each metabolite).
Tryptophan (0.3 mmol/ml) and kynurenic acid (12 mmol/ml)
were not influenced by FANFT treatment at this time. Urinary
tryptophan and indole metabolite values at 10 through 79 weeks
of the study are shown in Tables 5 and 6. The only metabolite
for which excretion was altered by vitamin H,, deficiency was
anthranilic acid, an increased excretion of which was observed
in general in vitamin B6-deficient rats at 26 and 52 weeks.
Significant effects occurred at 26 weeks in FANFT-treated rats
fed a tryptophan excess and at 52 weeks in all groups, except
in non-FANFT-treated rats given excess tryptophan. Pretreatment with FANFT increased anthranilic acid excretion at 79
weeks and excess tryptophan elevated values at 52 weeks in
vitamin B6-adequate rats not treated with FANFT and at 79
weeks in vitamin B6-deficient FANFT-treated rats. However, a
tryptophan excess decreased values in vitamin B6-deficient, nonFANFT-treated rats at 52 weeks (Table 5). Kynurenic acid
excretion was elevated in rats given excess tryptophan at 10
weeks, irrespective of FANFT treatment, and at 26 and 52
weeks in the FANFT-treated groups (Table 5). Serotonin ex
cretion was elevated by tryptophan feeding at 10, 52, and 79
weeks in both the FANFT-treated and non-FANFT-treated
groups, FANFT treatment reduced serotonin excretion in the
groups fed a tryptophan excess at 79 weeks (Table 5). The
treatment protocol did not influence tryptamine, indole, indole3-acetic acid, indole-3-lactic acid, indole-3-acetamide, or 5hydroxyindole-3-acetic acid excretion.
Metabolites not influenced by vitamin B6 intake of FANFT
pretreatment are shown in Table 6. Tryptophan, tryptamine,
indoleacetic acid, and 5-hydroxyindoleacetic acid excretion was
elevated in the groups fed a tryptophan excess at each measure
ment (Table 6). 5-Hydroxytryptophan excretion was elevated
in the tryptophan-fed groups at 10 and 52 weeks.
Mean urinary tryptophan metabolite concentrations mea-
Table 6 Effect of tryptophan excess on urinary tryptophan and its metabolites'
of
tryptophan
tryptophan
MetaboliteTryptophan*5-Hydroxy-tryptophanTryptamine*'Indoleacetic
(mmol/ml)0.5
study1026527910265279265279265279265279Control
(mmol/ml)4.8
.icul'"5-Hydroxy-indole-acetkacid**Wk
(a)10.2
(a)2.5
(a)5.0
(a)10
(a)33.3
(A)37.9
(A)32.8
(A)21
(a)8
(a)11
(a)21
(A)0.2
(A)9
(a)20
(A)20
(A)8.9
(a)21.1
(a)23.3
(a)24
(a)129.8
(A)48.0
(A)88
(a)20
(a)24
(a)39
(c)59
(A)55
(A)62
(a)30
(a)132
(a)40
(A)139
(a)Excess
(A)
" Results of r test comparisons within each metabolite indicated by the paren
thetical letters. Values not sharing a common parenthetical letter are significantly
different: a < A < c (P < 0.05). N = 32 at weeks 10, 26, and 52; N = 86-92 at
week 79.
* SE = 5 for weeks 10, 26, and 52 and 3 for week 79. Significant effects of
week (P < 0.001), tryptophan (P < 0.001) and week by tryptophan interaction (P
< 0.02) observed by analysis of variance.
' SE = 3 for weeks 10, 26, and 52 and 2 for week 79. Significant effects of
week (/' < 0.001) and week by tryptophan interaction (P < 0.05) observed by
analysis of variance.
'These metabolites were not detected in the urine of rats at 10 weeks of the
study.
' SE = 15 at 26 and 52 weeks and 9 at week 79. Significant effects of week (/'
< 0.001), tryptophan (P < 0.001) and week by tryptophan interaction (P < 0.001)
observed by analysis of variance.
fSE = 8 at 26 and 52 weeks and 5 at week 79. Significant effects of week (/'
< 0.05) and tryptophan (P < 0.001) observed by analysis of variance.
' SE = 24 at 26 and 52 weeks and 14 at week 79. Significant effect of tryptophan
(/' < 0.001) observed by analysis of variance.
sured at 78 to 80 weeks in carcinoma-bearing and in noncarcinoma-bearing rats were found not to differ.
Urinary bladder ODC activity was not altered in vitamin B6-
1248
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L-TRYPTOPHAN AND VITAMIN B«IN URINARY BLADDER CANCER
deficient rats. Non-TPA-treated, vitamin B(,adequate, and vi
tamin B6-deficient rats had values of 0.08 ±0.06 and 0.08 ±
0.05 nm CO2 formed in 30 min per mg protein, respectively,
and TPA-treated vitamin B6-adequate and vitamin B,, deficient
rats had values of 1.9 ±0.3 and 1.8 ±0.3 nm CO2 formed in
30 min per mg protein, respectively.
DISCUSSION
The results of this study were unexpected. Vitamin B6 defi
ciency inhibited urinary bladder carcinogenesis when normal
tryptophan levels were fed. Thus the expected increase in tryptophan metabolite excretion in vitamin B6 deficiency did not
promote urinary bladder cancer. Furthermore, dietary trypto
phan excess did not increase urinary bladder cancer in rats fed
adequate vitamin B6. However, twice as many urinary bladder
carcinomas were observed in rats fed a tryptophan excess in the
presence of vitamin B6 deficiency, in comparison with vitamin
B6-deficient rats that had normal tryptophan levels, but the
difference was not statistically significant because the incidences
were low. In addition, more tumors at other sites (largely
benign, cutaneous tumors) were observed in rats fed tryptophan
excess in a vitamin B6-deficient diet than in rats fed inadequate
vitamin B6 alone. These results suggest that tryptophan may
promote tumors when vitamin B,, intake is marginal, but not
when the intake of vitamin B«,
in adequate.
The lack of agreement between the results of the present
study and earlier investigations, in which tryptophan was dem
onstrated to promote urinary bladder cancer (12, 14), may be
related to several factors. Research with DL-tryptophan has
generally suggested bladder tumor-promoting activity (12), but
experiments utilizing L-tryptophan have given either negative
results (14, 36) or results with marginal significance (13). In
addition, different diets have been fed. The control diet used in
this experiment contained 10 mg PLP/kg, a level somewhat
above that recommended for an AIN-76A diet and selected for
the control diet to assure adequacy under conditions of stress.
•
Secondly, earlier investigations used natural ingredient diets
(12-14), whereas the present study used a semipurified diet.
The latter was necessary because of the need for diets deficient
in vitamin B6, in addition to those having a tryptophan excess.
Reports on urinary bladder cancer promotion by saccharin
have cited results similar to those in the present investigation
of tryptophan. Urinary bladder cancer has been promoted by
saccharin in studies which used natural ingredient diets, but
promotion with saccharin did not occur when the compound
was fed in the AIN-76A semipurified diet.4 It is also interesting
that the urinary bladder tumor yield was greater in the control
FANFT-treated group (group 1) than was previously observed
in rats treated with the same dose of FANFT in a natural
ingredient diet (13). This increased effect of FANFT in AIN76A diet was observed previously (37).
In a recent study, tryptophan failed to increase urinary blad
der carcinogenesis induced by Ar-butyl-Ar-(4-hydroxybutyl)-nitrosa m ine in male F344 rats when given 10 weeks of treatment
with 5% sodium saccharin, which promoted cancer (38). Thus,
tryptophan may not promote when conditions are such that
other promoters are applied or if the dose of carcinogen is too
large, inducing a significant tumor incidence, even without
subsequent promoter administration. Such a relationship was
previously observed with sodium saccharin promotion follow
ing administration of N-methyl-A^-nitrosourea (39).
A possible consideration in diet composition is the effect of
4 S. Y. Wang, personal communication.
diet on urine, since urinary constituents have been demon
strated to be important in urinary bladder carcinogenesis. Urine
has been shown to be essential for tumor formation with im
planted pellets (40) and to be a promoting substance for urinary
bladder cancer (41, 42). In addition, when an in vitro assay was
used to induce ODC in bladder cells, there were multiple factors
showing ODC activity in the urine, and the tryptophan metab
olite anthranilic acid increased both ODC activity and the
mitotic rate (43).
Vitamin B,, status was depressed in rats fed the vitamin B,,
deficient diets, as determined by the measurements of erythrocyte ALAT, plasma PLP, and urinary anthranilic acid. Body
weight depression, however, was not observed in vitamin B6deficient rats. The inhibition of urinary bladder cancer in vita
min B6-deficient rats may have been due to an inhibition of
urinary bladder ODC activity in these rats. ODC requires PLP
as a cofactor and as the rate-limiting enzyme in polyamine
synthesis, it is believed to be important for promoting carcino
genesis (44). Indeed, treatment with difluoromethylornithine, a
competitive inhibitor of ODC, halted urinary bladder carcino
genesis by Af-methyl-Ar-nitrosourea in heterotopically trans
planted rat urinary bladder (45). However, our results indicated
that urinary bladder ODC in female rats was not influenced by
vitamin B6 deficiency. Unfortunately, we could not measure
this factor in male rats; however, males were used for the
carcinogenesis study.
The possibility that tryptophan promotes tumors only when
vitamin B,, is inadequate is a plausible one, since early studies
by Dunning et al. used diets marginal in vitamin B,, ( 1. 23, 24).
Also, vitamin B,,intake varies considerably among humans and
certain pathological conditions, as well as drug and food con
sumption habits, reduce vitamin B6 status. More vitamin B6 is
required during pregnancy and when oral contraceptives are
used (46, 47). Some genetic diseases cause changes in vitamin
B6-dependent enzymes due to a decreased binding of pyridoxal
5'-phosphate to the apoenzyme (48). Isoniazid ingestion (49)
and abuse of marijuana (50) and alcohol (51) also influence
vitamin B6 metabolism. More disturbing is the report that 31%
of adolescents (52) and 28% of hospitalized elderly patients
(53) in the United States have low vitamin B6 levels, examples
that are probably related to poor dietary utilization. Pyridoxal
phosphate binds to lysine during the processing or storage of
foods and the utilization of this bound compound is poor (54).
The requirement for vitamin B,, also increases with rising die
tary protein levels (55). The current trend toward high protein
diets could stress vitamin B,.status, leading to a deficiency, and
could result in an elevated risk of cancer development if this
condition potentiates the toxicity of tryptophan. This is partic
ularly important, since tryptophan has been used as an antidepressant at levels of 6-10 g/day (56) and as an aid in inducing
sleep (57).
Another possible factor in the early studies (1, 23, 24) was
confounding by urinary bladder infections which may have been
involved directly in urinary bladder carcinogenesis or which
may have stressed vitamin B, and increased requirements.
The present results do not support the hypothesis that excre
tion of urinary tryptophan metabolites is associated with pro
motion of urinary bladder cancer by tryptophan. For example,
excessive tryptophan, when fed to vitamin B6-deficient rats,
caused the greatest elevations of several tryptophan metabolites,
while urinary bladder tumor yield was intermediate between
two diet groups that had lower overall excretion rates of urinary
tryptophan metabolites. In addition, there was no association
between the excretion of any one metabolite and the presence
of urinary bladder cancer in individual rats. However, other
12492017. © 1987 American Association for Cancer Research.
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L-TRYPTOPHAN AND VITAMIN B«IN URINARY BLADDER CANCER
tryptophan metabolites which were not measured in the present
experiment may have been associated with urinary bladder
carcinogenesis. Some of these metabolites, particularly 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and xanthurenic
acid, were associated with urinary bladder cancer in earlier
studies (11, 19).
29.
30.
31.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
Effect of l-Tryptophan Excess and Vitamin B6 Deficiency on Rat
Urinary Bladder Cancer Promotion
Diane F. Birt, Alan D. Julius, Ryohei Hasegawa, et al.
Cancer Res 1987;47:1244-1250.
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