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CancerLetters, 41(1988) 179-189 Elsevier Scientific Publishers Ireland Ltd.
179
INHIBITION OF DIETARY FAT-PROMOTED DEVELOPMENT OF (PRE)NEOPLASTIC LESIONS IN EXOCRINE PANCREAS OF RATS AND HAMSTERS BY SUPPLEMENTAL VITAMINS A, C AND E
RUUD A. WOUTERSEN and ANNEMARIE van GARDEREN-HOETMER
Department of Biological Toxicology, TNO-CIVO Toxicology and Nutrition Institute, P.O. Box MO, 9700 AJZeist (The Netherlandsl
(Received 25 February 1988) (Revised version 11 April 1988) (Accepted 20 April 1988)
SUMMARY
The effects of vitamins A, C and E on the development of putative preneoplastic foci in exocrine pancreas were investigated in azaserine- treated rats and iV-nitrosobis(2-oxoproypylamine-treated hamsters. The ani- mals were fed a semipurified diet high in saturated fat (20% lard) either or not supplemented with vitamin A, vitamin C or vitamin E. A separate group maintained on a diet low in saturated fat (5%1 lard) was incorporated as extra controls. The animals were given their diets 12 days after the last treatment with carcinogen. At 4 months postinitiation, the pancreata were quantitatively examined for both the number and size of early, putative pre- neoplastic lesions and the presence of neoplastic lesions. Rats as well as hamsters maintained on 5%1 lard exhibited a significantly lower number of putative preneoplastic pancreatic lesions than animals fed a diet containing 20% lard. Growth of acidophilic but not of basophilic foci was inhibited in rats of the high vitamin A and C group, whereas vitamin E exerted an inhi- bitory effect on growth of basophilic but not of acidophilic foci. In hamsters maintained on a diet high in vitamins A or C, the number of early ductular lesions was significantly decreased, whereas the number of (mi- crokarcinomas was increased. Vitamin E did not have any modulating effect on development of ductal lesions in hamster pancreas.
Key words: Pancreatic carcinogenesis; Rat; Hamster; Inhibition; Vitamins A, C and E.
INTRODUCTION
Research and public attention have been increasingly focussed on the hypothesis that vitamins and micronutrients may decrease the incidence of
0304~3835188/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
180
some cancers of epithelial origin such as adenocarcinomas of the pancreas. Carcinomas of the pancreas have a bad prognosis because diagnosis is usually late and treatment ineffective. Thus, prevention seems to be the best approach to successfully reduce the death rate due to this aggressive neoplasm.
A few studies have been reported concerning the possible inhibitory effects of synthetic retinoids on growth of (prelneoplastic pancreatic lesions induced in rats by azaserine (leading to acinar adenocarcinomasl or in hamsters by N- nitrosobis(2-oxopropyllamine (BOP) (leading to ductal adenocarcinomasl. Depending on both the animal model employed and the sex of the animals, syn- thetic retinoids have been found to enhance, to inhibit or to have no effect on pancreatic carcinogenesis ]3,5,7,8,10,15]. The conflicting nature of these data may be ascribed to 6) a difference in the types of retinoids used, since it has been proposed that distribution of retinoids over tissues might be altered by varying the retinoid structure, or (ii) a difference in the basal diets used, since in most studies unpurified (natural ingredients) commercial laboratory diets were used, which may vary in amount of (unlsaturated fat. Since both saturated and unsaturated fat enhanced pancreatic carcinogenesis in rats [9,13,14] and hamsters [4,6] it is not unlikely that the inconsistent effects of retinoids on pan- creatic carcinogenesis can be ascribed to differences in the diets used.
Up to now, the effects of natural vitamins on experimental pancreatic carcinogenesis have not been reported. Therefore, we studied the effects of vitamins A, C and E on pancreatic carcinogenesis in rats and hamsters maintained on a semipurified diet high in saturated fat (20% lard), with number and size of putative (prelneoplastic foci as parameters.
MATERIALS AND METHODS
Hundred male weanling Syrian golden hamsters (Cpb-TNO-colony) and 100 male weanling SPF albino Wistar rats (Cpb/WU; Wistar random1 were obtained from the TN0 Central Institute for the Breeding of Laboratory Animals, Zeist, Netherlands. The animals were either kept on softwood bed- ding in macrolon cages or in stainless steel cages, fitted with wire-mesh floors and fronts. They were housed under standard laboratory conditions, 5 animals per cage. Both rats and hamsters were fed a diet low in saturated fat (5% lard) or a diet high in saturated fat (20% lard), either pure or supple- mented with vitamin A, C or E. The diets were stored at - 20°C until use. The percentage composition of the diet is summarized in Table 1.
The hamsters were injected subcutaneously, once weekly, with 20 mg BOP/kg body wt at 5, 6 and 7 weeks of age and the rats were injected intraperitoneally with 30 mg azaserinelkg body wt at 19 days of age. BOP (Ash Stevens, Inc., 5861 John C. Lodge Freeway, Detroit, MI) and azaserine (Calbiochem-Behring Corp., La Jolla, CA) were dissolved freshly in 0.9% NaCl solution.
During the initiation phase, the animals were fed the Institute’s stock diet. Twelve days after the (last) treatment with carcinogen, the animals
181
TABLE 1
COMPOSITION OF THE DIETS (O/b)
Ingredients 200/b Lard 5% Lard
Casein 15.6 13.3 Soya protein isolate 7.9 6.7 &Methionine 0.24 0.2 Wheat starch 13.6 38.55 Pregelatinized starch 23.5 20.0 Cellulose 11.8 10.0 Trace mineral salts CO220’ 5.3 4.5 KH,PO, 0.71 0.6 Vitamin ADEK preparationb 0.53 0.45 Vitamin B mixture’ 0.35 0.3 Choline chloride 0.47 0.4 Lard 20.0 5.0
Energy content of the diet (kJ/kgl 16,950 13,960
a Mineral mix provides (in mg/kg diet): KH,PO,, 15,910; CaCO,, 17,100; NaCl, 5850; MgSO,, 4500; FeSO, * 7H,O, 1350; ZnCl,, 45; &SO, * 5H,O, 20; MnSO, . H,O, 180; CoCI, * 6H,O, 4.5; KI, 36.
b The vitamin (A,D,E and Kl preparation provides (per kg diet): 3000 IU vitamin A acetate + 3000 JU vitamin A palmitate; 3750 IU vitamin D,; 4.5 mg vitamin E; 12 mg vitamin K,.
e The vitamin B mixture provides (in mg/kg diet): thiamine HCI, 30; riboflavin, 22.5; pyridoxine HCI, 9; niacin, 135; calcium pantothenate, 60; biotin, 0.9; folic acid, 3; vitamin B,,, 0.015; inositol (incorpo- rated in the vitamin B mixture), 150.
were fed the respective diets as follows: Group 1, high saturated fat diet (20% lard); Group 2, high fat diet supplemented with vitamin A (Dohyfral extra A-500, stabilized on a carrier, containing 5 x lo8 W/kg in a 50:50 ratio of retinyl acetate and retinyl palmitate, supplied by Philips Duphar, Veenen- daal, Netherlands) at a level calculated to provide 100,000 IU/kg diet; Group 3, high fat diet supplemented with vitamin C (ascorbic acid, obtained from E. Merck AG, Darmstadt, F.R.G.1 at a 10 g/kg dietary level; Group 4, high fat diet supplemented with 600 mg/kg vitamin E (in the form of 50% DL-a-toco- pherol acetate, obtained from E. Merck AG, Darmstadt, F.R.G.1; Group 5, low saturated fat diet (5% lard). Each test group consisted of 20 male rats or 20 male hamsters. The animals were allocated to the different groups by a computerized randomization procedure.
Food and drinking water were available ad libitum. Body weight and food intake were recorded weekly. The general condition and behaviour of the animals were checked daily. Terminal autopsy of rats was on days 131, 132 and 133 after azaserine treatment and of hamsters on days 137, 138 and 139 after the third injection with BOP. The animals were anaesthetized with ether, exsanguinated by cannulating the abdominal aorta and then examined for gross pathological changes. The entire pancreas and the liver from each animal were excised, weighed and, together with kidneys and lungs, fixed in 10% buffered formalin. The pancreata were completely processed for
182
microscopy by conventional methods, step sectioned at 5 pm (approx. 20 sec- tions per rat pancreas and approx. 10 per hamster pancreas), stained with haematoxylin and eosin (H and El and examined by light microscopy.
In rats quantitative determination of the number of foci per cm3 of pancreas and of their size was carried out as described previously [1’7]. In hamsters, major attention was directed to intermediate and tubular ductal complexes and (early) carcinomas. The size and number of these putative preneoplastic and early neoplastic lesions were evaluated as described previously [18]. In rats and hamsters the area of the focus transections was determined with a grid inside the ocular as described before [1’7,18]. Body and organ weight data were statistically evaluated by two-way analysis of variance with initial body weight as covariable. Food intake figures were subjected to Kruskal-Wallis analysis of variance and Mann-Whitney U-tests. Food efficiency figures were evaluated by analysis of variance and least sta- tistical difference (LSD) tests. In rats, the calculated volumetric data of foci were evaluated by one-way analysis of variance. In hamsters, the number of lesions was statistically evaluated by a generalized linear model (error is Poisson, link function is log).
RESULTS
Food and energy intake (Table 2) In rats, food intake was slightly increased in the group maintained on a
low fat diet, whereas food conversion efficiency was slightly lower in this group as compared to rats maintained on 20% lard. Mean energy intake did not strongly differ between groups. In hamsters, food intake was slightly lower in the groups maintained on a high fat diet supplemented with vitamin C or E, whereas food conversion efficiency was appreciably lower in the 5% lard group than in controls. Hamsters maintained on a 5% lard diet or on a 20% lard diet supplemented with vitamin C or E showed a decreased mean energy intake as compared to the control group.
TABLE 2
MEAN FOOD AND ENERGY INTAKE AND FOOD CONVERSION EFFICIENCY’
Postinitiation diet group
Rats
Food Energy FCE
Hamsters
Food Energy FCE
20% Lard (control) 105.4 1786.5 0.49 35.9 608.5 0.13 20% Lard + Vit.A 109.7 1859.4 0.50 34.7 588.2 0.12 20% Lard + Vit.C 103.4 1752.6 0.49 32.1 544.1 0.12 20% Lard + Vit.E 104.3 1767.9 0.51 31.6 535.6 0.10
5% Lard 122.0 1703.1 0.43 35.1 490.0 0.06
a Food intake in g per animal per week, energy intake in kJ per animal per week, and FCE = food conversion efficiency for the first 28 days on the respective diets (g weight gain per g food consumed).
TA
BL
E
3
BO
DY
AN
D
PA
NC
RE
AT
IC
WE
IGH
TS
Die
t R
ats
(n =
20
) H
amst
ersb
z =
20
)
Bod
y w
eigh
t P
ancr
eati
c w
eigh
t B
ody
wei
ght
Pan
crea
tic
wei
ght
at a
uto
psy
(g)
Abs
olu
te
(g)
Rel
ativ
e (g
/kg)
at
au
tops
y (g
) A
bsol
ute
(g
) R
elat
ive
(g/k
g)
20%
L
ard
(con
trol
) 44
0.8
k 9.
6 1.
264
f 0.
046
2.88
f
0.10
11
9.5
+
4.5
0.34
7 f
0.02
2 2.
94
f 0.
18
201
Lar
d +
V
it.A
46
3.1
f 12
.4
1.34
9 f
0.04
3 2.
93
f 0.
09
117.
4 f
2.9
0.34
5 f
0.01
8 2.
97 f
0.
18
20%
L
ard
+
Vit
.C
448.
4 f
11.0
1.
136
f 0.
036
2.55
f
0.08
11
1.3
+-
3.0
0.32
4 +
0.
014
2.94
f
0.13
200/
o L
ard
+
Vit
.E
452.
9 f
7.4
1.23
6 f
0.03
4 2.
75
+
0.09
10
8.5
f 2.
2 0.
342
f 0.
018
3.15
f
0.14
5%
Lar
d 44
2.9
f 9.
0 1.
244
f 0.
066
2.81
I?
: 0.1
5 97
.0
+
2.3*
* 0.
285
-c 0
.013
2.
96 f
0.
13
‘Val
ues
ar
e m
ean
s f
S.E
.M.
Sta
tist
ics:
tw
o-w
ay
anal
ysis
of
vari
ance
(t
wo-
tail
ed):
**
P <
0.
01.
TA
BL
E 4
EFF
EC
TS
OF
VIT
AM
INS
ON
PU
TA
TIV
E
PRE
NE
OPL
AST
IC
FOC
I IN
RA
TS
W
= 2
0) IN
DU
CE
D
BY
AZ
ASE
RIN
E’,
b
Die
t O
bser
ved
tran
sect
ion
data
of
foci
C
alcu
late
d vo
lum
etri
c da
ta o
f fo
ci
Tot
al
Tra
nsec
tion
n/em
3 w
ith m
ean
diam
eter
(p
m)
Tot
al
Mea
n A
rea
as %
n/
cm2
area
n/
cm3
diam
eter
of
pan
crea
s
(mm
2 x
100)
13
6 19
2.5
272.
5 38
5 ,5
45
(rm
)
Aci
doph
ilic
fo
ci
20%
Lar
d (c
ontr
ol)
41.8
1 f
5.01
6.
48 +
0.7
8 44
3 f
99
709
f 94
53
9 k
66
256
f 54
87
f
31
2034
-t
196
241
-t
10
3.25
+ 0
.79
20%
Lar
d +
Vit.
A
29.2
8 f
3.71
5.
24 k
0.
35
453
f 87
56
3 k
68
404
* 67
14
8 ”
34
39 f
12
16
06 f
17
9 22
3 f
7 1.
63 +
O-2
9*
20%
Lar
d +
Vit.
C
35.8
0 2
3.69
5.
32 k
0.
32
439
k 13
1 70
6 +
72
51
4 f
62
196
+: 3
5 39
f
8 18
93 f
21
2’
230
2 6
1.99
+ 0
.24*
20%
Lar
d +
Vit.
E
43.7
0 +
5.4
2 6.
27 -
C 0
.45
385
f 80
70
8 +
99
59
1 +
74
30
4 2
59
73 f
27
20
62 2
21
6 24
8 f
8 3.
00 f
0.
59
5% L
ard
40.2
5 f
5.49
5.
28 +
0.4
9 47
3 2
112
789
k 10
7 59
2 *
107
194
+
40
56 f
17
21
04 f
24
4 22
6 -t
9
2.35
f
0.43
68
96
136
192.
5 2
272.
5
Bas
ophi
lic
foci
20°/
oLar
d(co
ntro
lI
13.4
1 f
1.47
1.
57 2
0.
15
505
f 87
40
6 f
78
392
+ 6
5 14
5 +
31
45 f
9
1492
k
167
118
-e 6
0.
20 f
0.
02
20%
Lar
d +
Vit.
A
14.0
1 f
2.30
1.
26 2
0.
07
594
+
92
532
+‘
108
398
2 71
12
4 2
32
28 f
6
1675
f
245
105
f 2
0.18
f
0.03
20%
Lar
d +
Vit.
C
11.5
1 +
1.
52
1.37
+ 0
.18
418
+ 1
04
526
rc_
98
271
rc_ 5
1 10
2 f
28
38 f
16
13
55 2
14
2 10
7 -t
4
0.16
f
0.04
20O
k1 Lar
d +
Vit.
E
9.81
f
1.76
1.
82 f
0.
41
293
f 72
35
3 e
70
348
+- 8
0 88
-c
27
16 +
6
109’
7 k
174d
12
1 f
9 0.
13 +
0.0
2*
5% L
ard
8.41
+
1.07
1.
17 2
0.
08
333
f 93
37
5 +
83
26
1 -r
- 45
55 k
13
13
k
5 10
38 2
15
4*
107
r 3
0.10
f
0.01
+*
* V
alue
s ar
e m
eans
i
S.E
.M.
b D
ata
are
base
d on
H a
nd E
-sta
ined
pa
raff
in
sect
ions
. *
Num
ber
of f
oci
with
dia
met
er
larg
er
than
38
5 pm
was
sig
nifi
cant
ly
low
er
than
in
cont
rols
. d
Num
ber
of f
oci
with
dia
met
er
larg
er
than
19
2.5
pm w
as s
igni
fica
ntly
lo
wer
th
an
in c
ontr
ols.
St
atis
tics:
an
alys
is
of v
aria
nce
(one
-tai
led)
: *P
< 0
.05;
**P
<
0.01
.
185
TABLE 5
EFFECTS OF VITAMINS ON PUTATIVE PRENEOPLASTIC AND NEOPLASTIC LESIONS IN HAMSTERS INDUCED BY BOP’
Type of lesions No. of lesions observed observed
20% lard 20% lard 20% lard 20% lard 5% lard + vit.A + vit.C + vit.E
(191b (191 (201 (201 (191 Intermediate ductal complex 49 29* 23** 41 14*** Tubular ductal complex 14 17 10 9 5* Carcinoma in situ 1 1 0 3 1 Microcarcinoma 1 0 2 3 1 Carcinoma 0 2 1 1 0
a Data are based on H and E-stained paraffin sections. b The number of animals examined is given in parentheses. Statistics: generalized linear model tone-tailed); error is Poisson; link function is log; *P < 0.05; **p< 0.01; ***p< 0.001.
Body weight and pancreas weight The figures in Table 3 demonstrate that addition of 100,000 IU/kg vitamin
A, 10 g/kg vitamin C or 600 mg/kg vitamin E to a semipurified diet high in saturated fat did not influence final body or pancreatic weight of the hams- ters and rats. Hamsters maintained on a low fat diet showed a significantly lower body weight at autopsy than hamsters fed a 20% lard diet.
Microscopy The results of the quantitative analyses of rat pancreatic H and E-stained
paraffin sections are presented in Table 4. Statistical analyses of the calcu- lated volumetric data of rats revealed a distinct inhibitory effect of vitamin A as well as vitamin C on growth of the acidophilic foci resulting in a signifi- cant decrease in 61 the area of pancreas occupied by focus tissue and (ii) the number of foci with a diameter over 385 pm. Vitamin E did not have any effect on number or growth of the acidophilic foci, but, in contrast to vitam- ins A and C, inhibited growth of the basophilic foci as seen by a decrease in 6) the area of pancreas occupied by basophilic focus tissue, and (ii) the num- ber of foci with a diameter over 192.5 pm. The findings with vitamin E were comparable with those seen in the group maintained on a low fat diet: a significant effect on growth of basophilic foci but no effect on the acidophilic foci. Neither vitamin A nor vitamin C inhibited or promoted growth of baso- philic foci.
In hamsters (Table 51, an inhibitory effect on development of putative preneoplastic ductular lesions (see Ref. 18 for the characterization of these lesions) was seen with vitamin A and C, but not with vitamin E. Moreover, vitamins A and C only caused a decrease in the total number of intermediate ductal complexes present in hamster pancreata. The incidence of (mi-
186
crolcarcinomas was increased rather than decreased by vitamins A and C. A significant decreased number of (large) ductal complexes of both categories (intermediate and tubular1 was seen in hamsters fed a diet low in saturated fat as compared to animals of the 20% lard group.
DISCUSSION
The results of the present short-term (Cmonthl study with azaserine- treated rats (model for acinar adenocarcinomal as well as BOP-treated hams- ters (model for ductular adenoearcinomal demonstrate an inhibitory effect of vitamins A and C on (il growth of acidophilic foci in rats and (ii) incidence of early lesions (intermediate ductal complexes) in hamsters.
Our findings with natural vitamin A are in accordance with those reported by other workers studying the effects of synthetic retinoids on pancreatic carcinogenesis. Roebuck et al. [15] found a decrease in number and size of acidophilic but not of basophilic foci in pancreata of azaserine- treated rats given a diet high in synthetic retinoids. Furthermore, the results of our study demonstrated an inhibitory effect of vitamin A on the incidence of early ductal pancreatic lesions, whereas the incidence of carcino- mas was higher in hamsters maintained on a high fat diet supplemented with vitamin A than in animals fed a high fat diet only. Birt et al. [3] found a reduction in pancreatic adenoma incidence by feeding retinoids to female hamsters, whereas in male hamsters an increase in the incidence of adeno- mas was observed. They found a consistently elevated incidence of pan- creatic carcinoma in males in another experiment and concluded that retinoids enhance pancreatic tumour yields in males to a greater extent than in females [5]. Moreover, a significant inhibitory effect of retinoids on the incidence of ductal pancreatic carcinomas in hamsters has never been reported. Our findings and those reported in literature suggest that, in hamsters, vitamin A only has an inhibitory effect on growth of early lesions and seems not to be effective enough to reduce tumour yield. There are at least 2 explanations for this phenomenon, which was also found with vitamin C: (il the early lesions involved are in reality not preneoplastic and (ii) these vitamins alter, somehow, the cell response making them to circumvent the preneoplastic stage.
The difference observed between the effects of vitamin A on pancreatic carcinogenesis in hamster and those in rat strongly supports our previous recommendation to use both animal models simultaneously in a comparative way for studying the effects of modulating factors on pancreatic carcinogenesis. Because of the close morphological similarities between BOP- induced tumours and pancreatic cancer occurring in man [ll], the failure of vitamin A to lower the incidence of (microlcarcinomas in hamsters might be more relevant than the inhibitory effect of vitamin A on growth of acido- philic acinar lesions observed in rats. Interestingly, in a parallel short-term (I-month) study in hamsters supplemented with pearotene (results presented
187
in a separate paper) we found a significant decrease in number of tubular ductal complexes and no increase in the incidence of more advanced lesions as compared to controls. On the basis of the present results with vitamin A and those reported with retinoids as well as the knowledge that (&carotene is less toxic than vitamin A, we have started a long-term study with rats and hamsters supplemented with p-carotene instead of vitamin A to find out whether p-carotene has a protective effect on the development of pancreatic cancer.
The effects of vitamin C on growth of early lesions in rats and hamsters were essentially similar to those found with vitamin A: inhibition of the growth of acidophilic but not of the basophilic foci in the rat model and of the intermediate ductal complexes in the hamster model. In hamsters, vitamin C enhanced rather than inhibited the number of (microlcarcinomas. The inhibitory effect on the early ductal lesions in hamsters might be related to the significant decrease in energy intake in these animals as compared to the high fat controls. Our observations in hamsters maintained on a high vitamin E diet, however, do not support this explanation since in this group a significant decrease in energy intake did not cause any effect on pancreatic carcinogenesis.
Reports on the efficacy of ascorbic acid in preventing tumour formation in various animal models are contradictory and we know of no reported data on the efficacy of this vitamin in a pancreatic tumour model. The mechanism of a preventive effect of vitamin C is unknown. It is suggested that anti-oxidant compounds such as ascorbic acid exert a scavenging effect on active molecu- lar species of carcinogens. Since, however, in the present study the animals were given the ascorbic acid-supplemented diet after carcinogen treatment, it seems justifiable to assume that the modulating effects found can be ascribed to an effect on vitamin C during the promoting phase of the carcino- genesis process.
Vitamin E is another anti-oxidant thought to have anti-carcinogenic properties. In our study, vitamin E was found to modulate the growth of basophilic but not of acidophilic acinar foci in rat pancreas, whereas no effect was seen on growth of the early or more advanced lesions in hamsters. The inhibitory effect on the growth of the basophilie foci in rats is interesting since this type of putative preneoplastic lesion is thought to be unrelated to pancreatic carcinogenesis [12,15,16]. Recently, however, we found that both ethanol and fat exert an enhancing effect on growth of both acidophilic and basophilic foci [17], whereas camostate (a synthetic trypsine inhibitor) given to rats for 4 months caused a highly significant decrease in number and growth of this type of putative preneoplastic foci (unpublished data). The present study also demonstrated that a diet with 20% lard enhances growth of basophilic, but not of acidophilic foci as compared to 5% lard. These results indicate that it is of paramount importance to know which phenotype (acidophilic and/or basophilicl is linked to a high progression probability and hence may be relevant to the formation of pancreatic cancer. Studies of Bax
188
et al. [1,2] indicated that enzyme-histochemical markers such as ATPase and yGT can be of great help to answer this important question.
From the results of the present short-term study it is concluded that: (il vitamins A, C and E have potential protective effects on the development of azaserine-induced acinar pancreatic cancer in rats; (ii) vitamins A and C inhibit development of BOP-induced early ductal lesions but enhance rather than inhibit development of (microlcarcinomas in hamster pancreas; (iii) vitamin E does not have a potential protective effect on pancreatic carcino- genesis in hamsters.
ACKNOWLEDGEMENTS
We wish to thank Mr P.M. Scherrenberg, MS A.A. van Tuyl and MS M. Stoepker for technical assistance, Dr L. van Beek for his advice on the nutri- tional aspects of the study, and Ir J.T.N.M. Thissen and Ir E. Schoen for advice on statistical analysis. We are grateful to Dr V.J. Feron and Profes- sor R.J.J. Hermus for evaluation of the draft. This work was supported by a grant from the “Praeventiefonds”, The Hague, Netherlands.
REFERENCES
1
2
3
4
5
6
10
11
Bax, J., Feringa, A.W., van Garderen-Hoetmer, A., Woutersen, R.A. and Scherer, E. (1986) Adenosine triphosphatase, a new marker for the differentiation of putative precancerous foci induced in rat pancreas by azaserine. Carcinogenesis, 7,457 - 462. Bax, J., Schippers-Gillissen, C., van Garderen-Hoetmer, A., Woutersen, R.A. and Scherer, E. (1988) Expression of adenosine triphosphatase and gamma glutamyl transferase during pro- gression of putative precancerous lesions induced in rat pancreas by azaserine. Carcinogene- sis, in press.
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