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Exocrine pancreatic secretion in the syrian golden hamster Mesocricetus auratus—III. Effects of carcinogen administration and development of pancreas cancer
Comp. Biochem. Physiol. Vol. 77C, No. 1, pp. 191-197, 1984 Printed in Great Britain
0306-4492/84 $3.00 +000 ~fj 1984 Pergamon Press Ltd
EXOCRINE PANCREATIC SECRETION IN THE SYRIAN GOLDEN HAMSTER M E S O C R I C E T U S AURA TUS--III. EFFECTS OF CARCINOGEN ADMINISTRATION A N D DEVELOPMENT OF PANCREAS CANCER A. SCOTT HELGESON, TERENCE LAWSON a n d PARVIZ POUR Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105, USA. Telephone (402) 559-4090
(Received 10 March 1983)
Abstract--1. The short- and long-term effects of the administration of the pancreas carcinogen Nnitrosobis(2-oxopropyl)amine (BOP) on pancreatic exocrine secretion were examined in Syrian hamsters with and without stimulation by secretin and pancreozymin. 2. Protein concentration, flow rate, pH and ion content, (Na ÷, K ÷, Ca 2÷, Mg 2+, HCO3, CI-, HPO ] and SO4 ) were measured. 3. An immediate effect of BOP is the stimulation of flow rate in females and of protein secretion in both sexes. 4. Multiple doses of BOP significantly altered the parameters mentioned in Section 2 only in the later stages of tumorigenesis. When these animals were stimulated with secretin or pancreozymin large decreases in flow rate and protein content of secretions were observed as early as 8 weeks after BOP treatment. 5. Insulin-like immunoreactivity and growth hormone-like immunoreactivity were detected in collected pancreatic secretions.
INTRODUCTION
MATERIALS AND
T h e exocrine pancreas of Syrian hamsters has been insufficiently e x a m i n e d in biological studies a n d those o f Helgeson et al. (1980a, b) a n d o f R e b e r et aL (1977) are the only reports to measure baseline parameters of h a m s t e r exocrine physiology. Studies of this " b a c k g r o u n d " type are necessary to u n d e r s t a n d h o w alterations in values o b t a i n e d m i g h t serve as indicators of pancreatic disease. The study performed here utilized Syrian golden h a m s t e r s with a developing pancreatic cancer induced by a d m i n i s t r a t i o n of N - n i t r o s o b i s ( 2 - o x o p r o p y l ) a m i n e (BOP). B O P is a p o t e n t inducer in Syrian hamsters of pancreatic t u m o r s that resemble the s p o n t a n e o u s type of cancer which occurs in m a n ( P o u r et al., 1975, 1979). The p a r a m e t e r s examined in previous studies (Helgeson et al., 1980a, b) were used again in c o n j u n c t i o n with r a d i o i m m u n o a s s a y for insulin a n d growth horm o n e s which, according to P o u r (unpublished observation), should be present in the exocrine secretions o f m a n y animals, including h u m a n s . Since there are extensive alterations o f pancreatic islet m o r p h o l o g y in h a m s t e r s with developing pancreatic carcinoma, it was hoped t h a t insulin levels m i g h t be affected if insulin was indeed being secreted by the islets into the exocrine fluid. G r o w t h h o r m o n e has a strong relationship to m a n y growth abnormalities a n d it was t h o u g h t t h a t this h o r m o n e also m i g h t be present. F u r t h e r m o r e it was h o p e d t h a t possible alterations in the a m o u n t of these h o r m o n e s b r o u g h t on by disease would manifest at a time when cancer or other dysfunctions were in treatable stages. 191
METHODS
Animals Outbred 8-week-old male and female Syrian golden hamsters from the Eppley Colony were used. They were housed in plastic cages (Macrolon) on granular cellulose bedding in groups of 5 by sex and maintained under standard conditions (temperature, 22 + 2", humidity, 55 + 5~o light-dark cycle 12 hr-12 hr; 10 air changes per hr). Wayne Lab-Blox (Allied Mills, Chicago, IL) and water were given ad libitum. Animal weights were recorded daily. Surgery and collection o f secretions Hamsters were anesthetized with sodium pentobarbital (75 mg/kg; i.p.). The common pancreatic duct was cannulated distally to collect pancreatic juice and ligated proximal to the gallbladder to eliminate bile from the secretions. After cannulation, hamsters were placed in restraining cages and allowed to recover for 1 hr before further treatment to reduce the influence of anesthesia on pancreatic function (Peterson and Grossman, 1978). Secretions were collected in tared vials in an ice bath and frozen at - 10°C until needed. The collection periods were: period (1) lla.m.-2p.m., period (2) 2p.m.-5p.m. and period (3) 5 p.m.-9 p.m. After 20 hr, hamsters were killed, the pancreas excised, fixed in 10~ (v/v) buffered formalin, and prepared for histology by conventional methods. Smears of pancreatic secretion were prepared during each collection period to evaluate their cellular content. The number of cells was estimated by + (1-3 cells), + + (3-10 cells) and + + + (over 10 cells) per 10 power field. In the long-term studies, collections were performed at 8-week intervals on different groups of hamsters. The first collection was at 7 days after the last BOP injection and termed 0 time. The collections occurred 8-32 weeks after 0 time.
A. SCOTT HELGESONet al.
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pH was measured on thawed samples. Total protein was measured by the method of Lowry et al. (1951). Potassium, sodium, calcium and magnesium concentrations were measured by atomic absorption (IL-257, Instrumentation Laboratories, Houston, TX), and chloride and bicarbonate by an Oxford titrator and supplied reagent sets (Oxford Laboratories, Foster City, CA). Phosphate was measured by the method of Weissman and Pileggi (1974) and sulfate by that of Berglund and Sorbo (1960).
Treatment with secretagogues Boots secretin (25 IU/kg) (Warren Teed Pharmaceuticals, Inc., Columbus, OH) and pancreozymin (20 IU/kg) (Sigma Chemical Company, St. Louis, MO) were injected intraperitoneally in six doses 30 min apart. BOP treatment To assess acute dosage effects, hamsters were injected once with 20mg BOP/kg s.c. Pancreatic secretions were collected from 1 hr after BOP treatment. Hamsters, in which long-term effects of BOP were being studied, were given 4 weekly s.c. injections of 10mg BOP/kg. Both treatments have produced pancreatic tumors. However, the latency period is shorter with the latter scheme (Takahashi et al., 1977; Pour et al., 1977). Radioimmunoassays Radioimmunoassays were performed upon aliquots of the collected secretions to determine amounts of insulin and growth hormone present using ~zSI-hGH chromacode and ~251-insulin chromacode radioimmunoassay kits (Bio RIA, Montreal, Quebec. Canada). RESULTS
(A ) Immediate effects Jollowing a single dose o f BOP There were no significant differences in the pH of secretions from BOP-treated hamsters. The flow rate in period (1) was 98.5/~1/100g body weight and remained steady throughout period (2). Protein concentration was highest (83.1 mg/ml) during period (1) and then steadily dropped to a value of 25 mg/ml. There were no significant differences in ion concentrations from previously reported control values (Helgeson et al., 1980a), except for phosphate and sulfate, which were 10-fold higher than in controls, e.g. averages of 1.4mg/liter and 0.3 rag/liter, respectively. (B) Long-term effect o f 4-weekly BOP treatments (BOP-4) There were no differences in pH, flow rate or protein concentration at weeks 0 or 8. By week 16 the flow rate in the BOP-treated hamsters during period (1) (0-3 hr) was 55 pl/hr/100 g body weight compared with
85 ,ul/hr/100 g body weight in the untreated controls. Period (2) BOP-4s had a flow rate 54~o of the period (2) flow rate in UCs (e.g. 56/ll/hr/100 g body weight vs 106.1/.tl/hr/100g body weight). Protein concentrations did not vary significantly between collection periods. There were no differences in pH during week 24. Protein concentrations were higher in U C hamsters than in BOP-4 animals during period (1), and continued to follow this pattern during period (2), although differences between U C and BOP-4 were smaller (i.e. U C 25.9+_ 12.8mg protein/ml BOP-4 12.6 _+ 9.5 mg). Week 32. There were no differences in pH. The flow rate was reduced considerably in BOP-4, e.g. U C values period (2). 64/.L1/hr/100 g body weight vs BOP4, 14/~tl/hr/100 g body weight. By this time, in most cases, BOP-4 had developed advanced pancreatic carcinomas (see histology section). Protein concentration was reduced substantially in all BOP-4s during all periods. Concentration of ions. During week 0 there were no differences in the ion concentrations between BOP-4 and U C hamsters nor were there differences during weeks 8, 16 or 32, except in the case of sulfate, which was not present in detectable amounts in any of the secretions collected during week 32.
(C) 4 Doses o f BOP and stimulation with secretin or pancreozymin Table 1, week O. The pH of pancreatic juice from BOP-pretreated hamsters stimulated with secretin (BOP-4-Sec) was lower than that from pancreozymin-stimulated animals (BOP-4-PZ), e.g. 8.75 avg. vs 9.75 avg. Control animals, which had not been pretreated with BOP (UC), were found to have similar pH differences between UC-Sec and U C - P Z hamsters. Differences between BOP-4-Sec or BOP-4PZ and UC-Sec and U C - P Z animals were minimal in regard to flow rate and protein concentration, with the following exceptions: (1) U C - P Z hamsters during period (3) had a flow rate 40% higher than did BOP-4-PZ hamsters, e.g. 92/~l/hr/100 g body weight vs 55/~l/hr/100g body weight. BOP-4-Secs had a protein concentration twice that of UC-Secs in period (3), 18.8 vs 8.2mg/ml. Week 8. There were again differences in pH between BOP-E-Sec and BOP-4-PZ hamsters, such as an average of 8.85 for the former and 9.85 for the latter. The flow rate was 505o lower on the average in BOP-4-Sec or BOP-4-PZ than in UC-Secs or UCPZs, e.g. avg. 185gl/hr/100g body weight vs 120/.d/hr/100 g body weight. During period (2) flow
Table 1. Physiologicaleffectsof l0 mg/kg doses of BOP l x weekly for 4 weeks upon exocrine biophysiologicalparameters* of pancreatic secretory protein, pH and flow rate after secretagogues (week 0) Flow rate Protein concentration pH 1/hr/100 g.b.w, mg/ml Control Experimental Control Experimental C o n t r o l Experimental Collection period 8.8+0.04 8.6_+0.11 181.9_+12.7 163.9-+26.4 22.9-+1.8 26.0-+0.69 (1) 0-3 hr Secretin 9.4+0.06 9.5 -+0.04 88.4_+ 13.5 89.9_+ 14.8 35.6_+0.9 27.6_+1.64 Pancreozymin 8.8_+0.03 8.7_+0.10 68.9_+31.5 45.8_+2.1 24.3_+0.1 27.9_+1.24 (2) 3 6hr Secretin 9.3_+0.06 9.6_+0.06 43.7_+8.8 50.8_+9.2 23.1_+0.9 22.6+3.99 Pancreozymin 8.9_+0.05 8.8+_0.08 86.6_+ 1 6 . 6 88.5_+0.3 10.6_+2.1 23.8-+2.50 (3) 6 10hr Secretin 9.5-+0.03 9.4-+0.16 92.6-+9.9 6 8 . 9 - + 1 7 . 4 24.0_+2.2 25.3_+0.40 Pancreozymin *Computed from a mean of at least four animals.
Carcinogen (BOP) affects hamster pancreas
193
Table 2. Physiological effects of 10 mg/kg doses of BOP 1 x weekly for 4 weeks upon exocrine biophysiological parameters* of pancreatic secretory protein, pH and flow rate after secretagogues (week 16)
Collection period (1) 0 3 h r Secretin Pancreozymin (2) 3 6 h r Secretin Pancreoxymin (3) 6 10hr Secretin Pancreozymin
Control
pH Experimental
8.74_+0.04 9.89_+0.09 8.71 -+0.08 9.88+0.01 8.78_+0.07 9.9_+0.04
8.83_+0.06 9,72_+ 0.41 8,79_+0.02 9.66_+0.34 8.81 + 0 . 0 9 9.86_+0.03
Flow rate #l/hr/100 g.b.w, Control Experimental 335.3_+38.2 213.4_+ 1,6 119.7_+ 13.5 86.0_+13.7 8 6 . 6 + 16.6 91.9_+6.5
105.0_+ 15.6 23.2_+ 1.6 42.7_+ 11.2 35.4_+20.7 88.6_+0.3 54.8_+15.7
Protein concentration mg/ml Control Experimental 18.3_+3.5 38.5_+0.6 6.0_+0.3 17.5_+3.5 7.9_+0.2 18.8+0.4
21.9+2.6 33.8-+2.1 13.8-+3.3 20.6_+l.7 7.3_+ 1.2 8.2+0.8
*Computed from a mean of at least four animals.
rate in both BOP-4-Sec and BOP-4-PZ hamsters was 0.33 times as high as in UC-Sec or UC-PZ animals. During period (3) there was little difference in flow rate between BOP-4-PZ or BOP-4-Sec and UC-Sec or UC-PZ animals. During period (1), protein concentration in BOP-4-Sec was 25 mg/ml, twice that in UC-Sec animals, while that of BOP-4-PZ animals was 25.2 mg/ml, a lower protein concentration than UC-PZ animals had at 44.7 mg/ml. BOP-4-Secs had three times the protein concentration of UC-Secs during periods (2) and (3). NOP-4-PZ animals had a slightly lower protein concentration than did UC-PZs through the latter 2 periods. Table 2, week 16. There was no effect upon pH. Flow rate, however, was affected dramatically in the BOP-4-Secs rate that was 33°/0 of the rate in UC-Secs, while the flow rate in BOP-4-PZs was 10~o that of UC-PZs. In period (2), the flow rate in BOP-4-Secs was 40% that of UC-Secs, as was also the case with BOP-4-PZs vs UC-PZs. In period (3) there was no difference between UC-Secs and BOP-4-Secs, while BOP-4-PZs had a flow rate 60?/o of UC-PZs. Protein concentrations were similar in period (1) in all groups. In period (2) BOP-E-Secs had twice the protein concentration of UC-Secs. There were no differences in BOP-4-PZs and UC-PZs during this period. During period 3 BOP-4-Secs and UC-Secs had similar protein concentrations, while BOP-4-PZ animals had 50°Jo of the protein concentration of UC-PZs.
(D ) Ion concentration qf~er secretin or pancreozymin There were no effects of BOP treatment which caused deviations in the ion concentrations of BOP4-Secs or BOP-4-PZs from these same concentrations in UC-Secs or UC-PZs during weeks 0, 8 or 16. (E) Pancreas pathology of BOP-treated animals (histology) Week O. Hyperplasia was observed in BOP-4s. Week 8. Ductular adenomas, focal hyperplasia and ductular hyperplasia were seen in BOP-4s. Week l6. Ductular adenomas and ductular dysplasia were seen in all BOP-4 hamsters. Fifty per cent had hyperplasia of the large ducts and 50~o had either adenocarcinomas or microadenocarcinomas. None of the pancreases examined from either group showed conditions which would manifest as physical obstruction of pancreatic secretory flow (R. Runge, personal communication). Week 24. A few tumors were observed macroscopically in BOP-4 hamsters and blockage of ducts CBP 77,1(" M
was evident, as well as invasion and destruction of acinar tissue by ductular adenocarcinoma cells. Week 32. Degenerative changes progressed in BOP-4s with more destruction of acinar tissues, with obvious physical blockage of ducts and many more macroscopic tumors.
(F) Concentration o.1"GHLIR and ILIR in pancreatic secretions Table 3. This table gives values for the amounts of growth hormone, including immunoreactivity (GHLIR) and insulin-like immunoreactivity (ILIR), in secretin (UC-Sec)- or pancreozymin (UC-PZ)-treated control hamsters and those which had been given BOP prior to secretagogue stimulation (BOP-4-Sec) or (BOP-4-PZ). In period (1) (0-3 hr), pancreozymin stimulation of controls resulted in secretion of 7 times the amount of GHLIR that secretin stimulated during week 0. This was also true during period (2) (3-6 hr), at which time 190 microinternational units of GHLIR were secreted per ml of pancreatic fluid after secretin stimulation, while 1640.5 microinternational units of GHLIR were secreted per ml after pancreozymin stimulation. In period (3) (6-10 hr) the animals stimulated with pancreozymin had 10 times the amount of GHLIR in their pancreatic secretions as those stimulated with secretin. Amounts of ILIR in pancreatic juice were similar whether stimulation was by secretin or pancreozymin, except in hamsters during period (3), at which time the amount of ILIR per ml in UC-Secs was 7~o that in UC-PZs. In BOP-4-Secs and BOP-4-PZs almost identical differences can be seen during all collection periods when examining secretion of GHLIR and, unlike the case of ILIR in UC-Secs vs UC-PZs, pancreozymin consistently evoked from 2.4-2.6 times as much ILIR secretion as in animals pretreated with BOP. There was no significant difference in GHLIR secretion during week 0 between UC-Sec and BOP4-Sec animals in periods (1) and (2); however, a slight difference was found during period (3). UC-PZs had no GHLIR differences in weak 0, but BOP-4-PZ hamsters excreted 2.2 2.8 times as much (ILIR) as UC-PZs. Week 8. When values for UC-PZ and UC-Sec are compared at 8 weeks, the differences between GHLIR amounts resulting from stimulation by the two hormones were less extreme during the first 2 periods than during the same periods in week 0, although pancreozymin still caused more GHLIR release than did secretin in period (3). The differences in GH secretions between UC-Sec and UC-PZ hamsters
1800+- 1 0 . 0 1490 +- 14.1 1740 +- 64.9
2575.0 + 106.1 1640.5 ± 225.6 t410.5 ± 409.0
(1) (~3 hr (2) 3 6 h r (3) 6-10 hr
(1) (~3 hr (2) 3-6 hr (3) 6-10 hr
*Computed from a mean of at least four animals.
177.8±10.3 190.0 ± 28.3 87.5 -!-__58.0
Week 0
(1) (~3 hr (2) 3-6 hr (3) 6-10 hr
195.0 ± 77.8
(3) 6-10 hr
No BOP Control Secretin 25 iu per kg body wt 6 times in 3 hr BOP 10 mg/kg 1 x 4 weeks then Pancreozymin 20 iu per kg body wt 6 times in 3 hr No BOP Control Pancreozymin
200.0±28.3 262.5 _+67.2
Period (1) (~3 hr (2) 3 6 h r
Treatment
BOP 10 mg/kg 1 x 4 weeks then Secretin 25 iu per kg body wt
~iu G H L I R per ml
29.4 ± 3.4 29.3 + 0.6 27.0 ± 5.7
47.5 + 2.1
82.5__+2.1 53.0 + 2.8
28.5+-2.1 37.0 +- 1.4 1.9 ± 0.6
13.5+--2.1
32.0_+2.8 20.5 _+ 5.0
MU ILIR per ml
425.0 +- 106.1 605,0 + 35.4 685.0 ± 91.9
656.3 ± 254.5
608.3_+231.4 882.4 -+ 407.4
370.3+__37.8 227.0 ± 53.7 82.5 ± 3.5
320.9+-165.1
MU ILIR per ml
40 ± 2.8 33 ± 2.8 23.7 ± 0.5
35.8 +- 14.3
52.1 +- 1 6 . 7 46.4 +- 18.0
56±2.83 34.5 ± 2.12 35 +__6.49
30.00+-4.97
71.00+-56.02 38.88 ± 10.6
Week 8
391.9+-218,9 522.6 _+372.6
#iu G H L I R per ml
1175.0 ± 106.1 385.0 + 91,9 337.5 ± 102.5
136.7 ± 56.1
886.7+-61.1 267.7 -+ 39.0
1110.0+268.7 840.0 +- 28.3 585.0 + 63.6
1290.0 ± 1 1 5 . 3
1706.7_+90.2 270.0 +- 268.5
47.0 4-__5.7 88.0 + 5.7 44.5 ± 12,0
25.2 ± 12.3
110.0_+54.7 28.7 +- 2.3
16.0+-5.66 32.0 + 1.41 29.0 ± 0.00
57.7+-1.5
45.4+14.9 42.2 +- 5.4
Week 16 ,uiu G H L I R MU ILIR per ml per ml
Table 3. Concentrations* of growth hormone and insulin in pancreatic secretions collected at least 24 hr following I x 4 weeks of 10 mg BOP/kg hamster body weight
Z
P
;>
4~
Carcinogen (BOP) affects hamster pancreas became much greater, since pancreozymin caused release of 6.6 times the GHLIR as secretin during period (3). There were no significant differences between UC-Sec and UC-PZ hamsters as far as ILIR secretion was concerned. BOP-4-Sec and BOP-4-PZ hamsters exhibited no significant difference in GHLIR secretion or ILIR. When compared with UC animals, BOP-4 animals showed variance in GH secretion after secretin stimulation, although not in period (l). Periods (2) and (3), however, had from 2-8 times as much secreted GHLIR. Secretin caused no differences in insulin nor did pancreozymin stimulation result in a difference for either hormone in BOP-4, as opposed to UC hamsters. W e e k 16. In UC-Sec and UC-PZ hamsters, secretin for the first time caused a greater secretion of GHLIR than did pancreozymin. Pancreozymin caused ILIR secretion, as can be seen when UC-Sec and UC-PZ hamsters are compared during periods (2) and (3). Period (2) UC-Sec's value of 840 iu GHLIR per ml was 2.2 times the value of period (2) UC-PZ, which was 385 iu per ml. The difference was less during period (3). ILIR levels in secretions of UC-Sec hamo/ sters were ~~,,/o those of UC-PZs during all collection periods. Differences in GHLIR secretion for BOP-4Sec and BOP-4-PZ hamsters in periods (1) and (3) were apparently significantly different. BOP-4-Secs secreted 2 times as much GHLIR during period (1) and 5.4 times as much during period (3). There were no apparent differences in GHLIR secretion during period (2). ILIR secretion results were not as readily discernible; however BOP-4-Secs secreted more ILIR than BOP-4-PZs except in period (1). The variability during period (2) in BOP-4-PZ hamsters was too large for a valid statement regarding significance. GHLIR secretions were compared and BOP-4-Secs were found as capable of GHLIR secretion as UCSecs during week 16, although times of maximum secretion differed as UC-Sec GHLIR secretion peaked during period (1), stayed high during period (2), and dropped during period (3). No UC-Sec value was as high as the period (1) and (3) peaks for BOP-4-Secs. ILIR secretion in periods (1) (3) was about 2 times higher in BOP-4-Secs than in UC-Secs. BOP-4-PZ hamsters secreted consistently smaller GHLIR concentrations than did UC-PZ hamsters, while UC-PZ hamsters generally secreted more ILIR per ml of pancreatic fluid, except in period (1), at which time BOP-4-PZ hamsters secreted a greater ILIR concentration than UC-PZs. DISCUSSION
Immediate B O P effects
The effects of BOP administered at 20 mg/kg are similar to effects from the action of pancreozymin, e.g. a small increase in flow rate and a large increase in protein concentration. We considered that the increased protein concentration might be due to a toxic reaction from BOP and a sloughing off of dead cells, but if this was the case there should have been large amounts of cellular debris in the exocrine fluid, which we did not find. Although Reber et al. (1978) believed that carcinogen had a toxic effect on duct ceils, since his hamsters secreted less HCO~, indi-
195
cating that duct cells were damaged, our hamsters had no such decrease in secretory rate for HCO3. The latter finding indicated that the HCO~--secreting mechanisms of ductular cells were intact and hence the ductular cell toxicity of 20 mg/kg BOP was minimal. Further evidence for the intact nature of the duct cells is provided by the unaltered calcium secretion, which paralleled protein secretion, thereby indicating that duct and acinar cells were intact. Calcium is not only secreted as an ion, but also excreted with zymogens from acinar cells where it helps to regulate stimulus secretion coupling (Sarles, 1977). BOP may stimulate extrusion of zymogens (pro enzymes found in zymogen granules of acinar cells) as does pancreozymin (Mutt, 1980), serving as a stimulus-secretion activator of acinar cells by the mechanism described by Kanno and Yamamoto (1977) or Mutt (1980). Since BOP probably acts in a manner akin to this because it does have an effect similar to secretagogues, we think the compound has a specific affinity for some component of its target cells. This component may be a secretagogue receptor such as that for pancreozymin. Information supporting this process is given by Hahne et al. (1981), who have shown proglumide to have a strong affinity for the pancreozymin receptor on ductular and acinar cells (Christophe et al., 1980). Proglumide has a side chain which is a keto analog of the dipropylnitrosamine group of compounds (which includes BOP). Alternatively, BOP or its metabolites may bind to receptors on chromatin as do steroid hormones (Eisenfield et al., 1980). Long-term B O P effects
The physiological effects of pancreatic carcinoma induction by 4 weekly BOP doses were minimal until 24-32 weeks past the last injection of BOP. Between weeks 24 and 32, hamsters given BOP 4 times (BOP4) failed to secrete as much protein as untreated control hamsters (UC) of the same age. This was probably not due to physical duct blockage as there was no evidence of extensive physical blockage until 32 weeks after the final BOP treatment. The characteristics of the secretions of BOP-4 and UC animals were similar in flow rate, protein, pH and ion content. Long-term effects on response to secretagogues pancreozymin or secretin
At 8 weeks after the final BOP injection there was a pronounced difference in the secretory characteristics of untreated control hamsters stimulated during collection of pancreatic fluid with secretin or pancreozymin, and BOP-treated animals stimulated in an identical fashion. According to our previous results (Helgeson et al., 1980b), we would have expected to find the same degree of flow rate increase caused by secretin administration in "Normal" (see Helgeson et al., 1980) animals as in BOP-treated animals, since base-line values of animals not stimulated with secretagogues were virtually identical to those of BOP treated animals not given secretagogues. Since the secretory mechanism in these hamsters appears intact, the difference between the parameters in both UC hamsters and BOP-4 hamsters given secretin or
196
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pancreozymin must lie in an inability of the BOP-4Sec or PZ pancreases to respond to these two hormones. The actual mechanism responsible for these differences is open to conjecture, but one possible explanation is that the "neoplastic" pancreatic cells do not bind secretin and pancreozymin effectively. Cells can lose their ability to bind hormones after transformation (Delarco and Todaro, 1980). However, this explanation presupposes a large percentage of pancreatic cells are transformed, which is not usually the case, since the loci of cancer development are generally few (R. Runge, personal communication). Although possible explanation is that BOP binds to the receptors for pancreozymin [especially in light of Hahne's results (1981)] and secretin, and either destroys or down regulates them permanently. The findings of Warren (1982) suggest that alterations in the acinar cell population in rats results in a diminished response to the effect of secretagogues because the diminutive responses of acinar cells to secretagogues reflect an insensitivity of acinar cell carcinoma secretion to extracellular Ca 2+. In cases of pancreatic acinar cell carcinomas, Warren also postulates an inability of c - A M P to stimulate protein secretion by acinar carcinoma cells due to a deficiency of the c - A M P dependent protein kinase system. To postulate a similar occurrence in the case of ductular cell carcinoma is logical since acinar cells and ductular cells are hypothesized to have the same stem cell (Warren, 1982). It is significant that pancreatic secretions from Syrian hamsters contain both insulin-like and growth hormone-like immunoreactivity. If the insulin-like or the growth hormone-like immunoreactivity are insulin and growth hormone then they could affect the induction and development of pancreas cancer. Both hormones stimulate D N A synthesis by forcing the cell into the S phase, and when cells are in S phase they are usually vulnerable to transformation by external effectors, such as viruses or cancer-causing chemicals (Grisham et al., 1980). BOP is a mitogen for ductular cells (P. Pour, personal communication) and consequently the chance of finding cells in S phase could be a combination of mitogenic effects of BOP plus those of insulin and growth hormone. Insulin levels in the blood and pancreatic fluid increase after secretin administration (Mutt, 1980). Pancreozymin increases plasma and pancreatic fluid insulin levels (Mutt, 1980). An important clinical aspect of these findings is their possible use as an early detection system for pancreatic cancer. Table 3 reflects large differences at various times in hormone content between BOPtreated and non-BOP-treated animals. With a more extensive study of this system or of a similar one, a workable system could be developed to detect pancreatic cancer based solely on exocrine secretion of endocrine hormones and abnormalities of their levels in duodenal aspirates. The latter would be a less traumatic way to examine pancreatic fluid than cannulation of the c o m m o n duct. Altered levels of endocrine hormone should be readily detectable. Hopefully, if cancer would be found, it will be in a state whereby conventional treatment and therapy would be adequate to control or eliminate it.
SUMMARY
Pancreatic exocrine secretion was studied in Syrian hamsters immediately following administration of 20mg/kg N-nitrosobis (2-oxopropyl)amine (BOP) and in hamsters which had been given 4 weekly doses of l0 mg BOP/kg. The chronically treated hamsters consisted of 2 experimental groups. G r o u p (1) was given BOP or used as controls without BOP treatment. G r o u p (2) animals were treated in the same manner, with experimental and control hamsters subdivided into secretin-treated or pancreozymintreated groups. Flow rate, pH, protein concentration, total protein secretion, and ionic concentrations of Na 4 , K + , C a 2 + , M g 2 + , H C O ~ , C I ,So~ and PO~ were measured. Acute effects of BOP are simi.ar to those of pancreozymin. Chronically BOP-treated hamsters showed little alteration of any of the abovementioned parameters until 24~32 weeks after carcinogen treatment, at which time 85% had pancreatic adenocarcinomas. Ductular blockage did not occur until 24~32 weeks, although a decreased flow rate and protein concentration in secretions were measured before this time. BOP-treated hamsters, stimulated with secretin and pancreozymin, had a reduced response to these secretagogues as early as week 8, with the reduction becoming quite obvious during week 16. Flow rate in these animals was one-third to one-tenth that of controls, protein concentration in response to pancreozymin was one-half to one-fourth that in controls. Ionic concentrations were unaffected by any of these treatments. The decreased response to secretagogue stimulation appears to be a receptormediated phenomenon. Insulin-like and growth hormone-like immunoreactivities were detected in the collected exocrine pancreatic secretions. Acknowledgements--This study was supported by a grant
from the National Cancer Institute National Pancreatic Cancer Project No. 1 R26 CA20198. Special thanks to Ed Bass for technical aid and to Thorkatla Donnelly and Rita MacCauley for surgical expertise, as well as to Mardelle Susman for editorial assistance. REFERENCES
Berglund F. and Sorbo B. (1960) Turbidimetric analysis of inorganic sulfate in serum, plasma and urine. Scand. J. clin. Lab. lnt'est. 12, 147 153. Christophe J., Svoboda M., Calderon-Atlas P., Lambert M., Vandermeers-Piret M. C., Vandermeers A., DeschodtLanchman M. and Robberecht P. (1980)Gastrointestinal hormone-receptor interactions in the pancreas. In Gastrointestinal Hormones (edited by Glass G. B. J.), pp. 451476. Raven Press, New York. Delarco J. and Todaro J. (1980) Sarcoma growth factor (SGF): Specific binding to epidermal growth factor (EGF) membrane receptors. J. Cell. Phys. 102, 267 277. Eisenfeld A. J. and Aten R. F. (1980) Estrogen receptor in the mammalian liver. In Advances in Steroid Biochemist O' and Pharmacology (edited by Briggs M. H. and Corbin A.), pp. 91-117. Academic Press, New York. Grisham J. W., Greenberg D. S., Kaufman D. and Smith G. J. (1980) Cycle-related toxicity and transformation in 10TI/2 cells treated with N-methyl-N'-nitro-N-nitroso guanidine. Proc. nam. Aead. Sci. U.S.A. 77, 4813 4817. Hahne W. F., Jensen R. T., Lemp G. F. and Gardner J. D. (1981) Proglumide and benzotript-members of a different
Carcinogen (BOP) affects hamster pancreas class of cholecystokinin receptor antagonists. Proc. natn. Acad. Sci. U.S.A. 98, 6304-6308. Helgeson A. S., Pour P., Lawson T. and Grandjean C. J. (1980a) Exocrine pancreatic secretion in the Syrian golden hamster Mesocricetus auratus--I, basic values. Comp. Biochem. Physiol. 66A, 473-477. Helgeson A. S.+ Pour P., Lawson T. and Grandjean C. J. (1980b) Exocrine pancreatic secretion in the Syrian golden hamster. Mesocricetus auratus--II. Effect of secretin and pancreozymin, Comp. Biochem. Physiol. 66A, 479~483. Kanno T., Saito A. and Saito Y. (1977) Stimulus--secretion coupling in pancreatic acinar cells: Influence of external sodium and calcium on responses to cholecystokinin-pancreacrozymin and ionophore A23287. J. Physiol. 270, 9-18. Kanno T. and Yamamoto M. (1977) Differentiation between the calcium dependent effects of cholecystokinin-pancreozymin and the bicarbonatedependent effects of secretin in exocrine secretion of the rat pancreas. J. Physiol. 264, 787 799. Lilja H. S., Curphey J. J., Yager J. D. Jr and Longnecker D. S. (1978) Persistence of DNA damage in rat pancreas following administration of three carcinogens and/or mutagens. Chem.-Biol. Interact. 22, 287 295. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with folin phenol reagent. J. biol. Chem. 193, 265-275. Mutt V. (1980) Cholecystokinin: isolation, structure, and functions. In Gastrointestinal Hormones (edited by Glass G. B. J.), pp. 169 221. Raven Press, New York. Peterson H. and Grossman M. I. (1977) Pancreatic exocrine secretion in anesthetized and conscious rats. Am. J. Physiol. 233, 530 536. Pour P. (1978) Islet cells as a component of pancreatic ductal neoplasms--I. Experimental study: ductular cells,
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including islet cell precursors as primary progenitor cells of tumors. Am. J. Pathol. 90, 295 316. Pour P., Althoff J., Krtiger F., Schmfihl D. and Mohr U. (1975) Induction of pancreatic neoplasms by 2,2'-dioxopropyl-N-propylnitrosamine. Cancer Lett. 1, 3-6. Pour P., Althoff J. and Takahashi M. (1977) Early lesions of pancreatic ductal carcinoma in the hamster model. Am. J. Pathol. 88, 291-308. Pour P., Salmasi S. Z. and Runge R. G. (1979) Ductular origin of pancreatic cancer and its multiplicity in man comparable to experimentally induced tumors. A preliminary study. Cancer Lett. 6, 89 97. Reber H. A., Johnson F. E., Montgomery C. and Carl W. R. (1977) Pancreatic secretion in hamsters with pancreatic cancer. Surgery 82, 34-41. Sarles H. (1977) The exocrine pancreas. Int. Rev. Physiol. Gastro. Physiol. H. 12, 173-221. Sarles H., Demol P., Bataille D., Guy O. and Rosselin G. (1978) Mise on evidence d'une hormone ileale inhibitrice de la secretion pancreatigue (ACP:anticholecystokininepeptide). C.r. Acad. Sci. Ser. O. 286, 367-369. Takahashi M., Pour P., Althoff J. and Donnelly T. (1977) Sequential alterations of the pancreas during carcinogenesis in Syrian hamsters by N-nitrosobis (2-oxopropyl)amine. Cancer Res. 37, 4602-4607. Veghelyi P. V. and Kemeny T. T. (1962) In The Exocrine Pancreas (edited by DeReuck A. V. S. and Cameron M. P.), pp. 329-373. Little Brown, Boston. Warren J. R. (1982) Secretagogue response in rat pancreatic acinar carcinoma. J. natn. Cancer Inst. 69, 969-974. Weissman N. and Pileggi V. J. (1974) Inorganic ions in clinical chemistry principles and techniques (edited by Henry R. J., Cannon D. C. and Winkleman I. W.), pp. 639-754. Harper and Row, Hagerstown, MD.
0306-4492/84 $3.00 +000 ~fj 1984 Pergamon Press Ltd
EXOCRINE PANCREATIC SECRETION IN THE SYRIAN GOLDEN HAMSTER M E S O C R I C E T U S AURA TUS--III. EFFECTS OF CARCINOGEN ADMINISTRATION A N D DEVELOPMENT OF PANCREAS CANCER A. SCOTT HELGESON, TERENCE LAWSON a n d PARVIZ POUR Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105, USA. Telephone (402) 559-4090
(Received 10 March 1983)
Abstract--1. The short- and long-term effects of the administration of the pancreas carcinogen Nnitrosobis(2-oxopropyl)amine (BOP) on pancreatic exocrine secretion were examined in Syrian hamsters with and without stimulation by secretin and pancreozymin. 2. Protein concentration, flow rate, pH and ion content, (Na ÷, K ÷, Ca 2÷, Mg 2+, HCO3, CI-, HPO ] and SO4 ) were measured. 3. An immediate effect of BOP is the stimulation of flow rate in females and of protein secretion in both sexes. 4. Multiple doses of BOP significantly altered the parameters mentioned in Section 2 only in the later stages of tumorigenesis. When these animals were stimulated with secretin or pancreozymin large decreases in flow rate and protein content of secretions were observed as early as 8 weeks after BOP treatment. 5. Insulin-like immunoreactivity and growth hormone-like immunoreactivity were detected in collected pancreatic secretions.
INTRODUCTION
MATERIALS AND
T h e exocrine pancreas of Syrian hamsters has been insufficiently e x a m i n e d in biological studies a n d those o f Helgeson et al. (1980a, b) a n d o f R e b e r et aL (1977) are the only reports to measure baseline parameters of h a m s t e r exocrine physiology. Studies of this " b a c k g r o u n d " type are necessary to u n d e r s t a n d h o w alterations in values o b t a i n e d m i g h t serve as indicators of pancreatic disease. The study performed here utilized Syrian golden h a m s t e r s with a developing pancreatic cancer induced by a d m i n i s t r a t i o n of N - n i t r o s o b i s ( 2 - o x o p r o p y l ) a m i n e (BOP). B O P is a p o t e n t inducer in Syrian hamsters of pancreatic t u m o r s that resemble the s p o n t a n e o u s type of cancer which occurs in m a n ( P o u r et al., 1975, 1979). The p a r a m e t e r s examined in previous studies (Helgeson et al., 1980a, b) were used again in c o n j u n c t i o n with r a d i o i m m u n o a s s a y for insulin a n d growth horm o n e s which, according to P o u r (unpublished observation), should be present in the exocrine secretions o f m a n y animals, including h u m a n s . Since there are extensive alterations o f pancreatic islet m o r p h o l o g y in h a m s t e r s with developing pancreatic carcinoma, it was hoped t h a t insulin levels m i g h t be affected if insulin was indeed being secreted by the islets into the exocrine fluid. G r o w t h h o r m o n e has a strong relationship to m a n y growth abnormalities a n d it was t h o u g h t t h a t this h o r m o n e also m i g h t be present. F u r t h e r m o r e it was h o p e d t h a t possible alterations in the a m o u n t of these h o r m o n e s b r o u g h t on by disease would manifest at a time when cancer or other dysfunctions were in treatable stages. 191
METHODS
Animals Outbred 8-week-old male and female Syrian golden hamsters from the Eppley Colony were used. They were housed in plastic cages (Macrolon) on granular cellulose bedding in groups of 5 by sex and maintained under standard conditions (temperature, 22 + 2", humidity, 55 + 5~o light-dark cycle 12 hr-12 hr; 10 air changes per hr). Wayne Lab-Blox (Allied Mills, Chicago, IL) and water were given ad libitum. Animal weights were recorded daily. Surgery and collection o f secretions Hamsters were anesthetized with sodium pentobarbital (75 mg/kg; i.p.). The common pancreatic duct was cannulated distally to collect pancreatic juice and ligated proximal to the gallbladder to eliminate bile from the secretions. After cannulation, hamsters were placed in restraining cages and allowed to recover for 1 hr before further treatment to reduce the influence of anesthesia on pancreatic function (Peterson and Grossman, 1978). Secretions were collected in tared vials in an ice bath and frozen at - 10°C until needed. The collection periods were: period (1) lla.m.-2p.m., period (2) 2p.m.-5p.m. and period (3) 5 p.m.-9 p.m. After 20 hr, hamsters were killed, the pancreas excised, fixed in 10~ (v/v) buffered formalin, and prepared for histology by conventional methods. Smears of pancreatic secretion were prepared during each collection period to evaluate their cellular content. The number of cells was estimated by + (1-3 cells), + + (3-10 cells) and + + + (over 10 cells) per 10 power field. In the long-term studies, collections were performed at 8-week intervals on different groups of hamsters. The first collection was at 7 days after the last BOP injection and termed 0 time. The collections occurred 8-32 weeks after 0 time.
A. SCOTT HELGESONet al.
192
pH was measured on thawed samples. Total protein was measured by the method of Lowry et al. (1951). Potassium, sodium, calcium and magnesium concentrations were measured by atomic absorption (IL-257, Instrumentation Laboratories, Houston, TX), and chloride and bicarbonate by an Oxford titrator and supplied reagent sets (Oxford Laboratories, Foster City, CA). Phosphate was measured by the method of Weissman and Pileggi (1974) and sulfate by that of Berglund and Sorbo (1960).
Treatment with secretagogues Boots secretin (25 IU/kg) (Warren Teed Pharmaceuticals, Inc., Columbus, OH) and pancreozymin (20 IU/kg) (Sigma Chemical Company, St. Louis, MO) were injected intraperitoneally in six doses 30 min apart. BOP treatment To assess acute dosage effects, hamsters were injected once with 20mg BOP/kg s.c. Pancreatic secretions were collected from 1 hr after BOP treatment. Hamsters, in which long-term effects of BOP were being studied, were given 4 weekly s.c. injections of 10mg BOP/kg. Both treatments have produced pancreatic tumors. However, the latency period is shorter with the latter scheme (Takahashi et al., 1977; Pour et al., 1977). Radioimmunoassays Radioimmunoassays were performed upon aliquots of the collected secretions to determine amounts of insulin and growth hormone present using ~zSI-hGH chromacode and ~251-insulin chromacode radioimmunoassay kits (Bio RIA, Montreal, Quebec. Canada). RESULTS
(A ) Immediate effects Jollowing a single dose o f BOP There were no significant differences in the pH of secretions from BOP-treated hamsters. The flow rate in period (1) was 98.5/~1/100g body weight and remained steady throughout period (2). Protein concentration was highest (83.1 mg/ml) during period (1) and then steadily dropped to a value of 25 mg/ml. There were no significant differences in ion concentrations from previously reported control values (Helgeson et al., 1980a), except for phosphate and sulfate, which were 10-fold higher than in controls, e.g. averages of 1.4mg/liter and 0.3 rag/liter, respectively. (B) Long-term effect o f 4-weekly BOP treatments (BOP-4) There were no differences in pH, flow rate or protein concentration at weeks 0 or 8. By week 16 the flow rate in the BOP-treated hamsters during period (1) (0-3 hr) was 55 pl/hr/100 g body weight compared with
85 ,ul/hr/100 g body weight in the untreated controls. Period (2) BOP-4s had a flow rate 54~o of the period (2) flow rate in UCs (e.g. 56/ll/hr/100 g body weight vs 106.1/.tl/hr/100g body weight). Protein concentrations did not vary significantly between collection periods. There were no differences in pH during week 24. Protein concentrations were higher in U C hamsters than in BOP-4 animals during period (1), and continued to follow this pattern during period (2), although differences between U C and BOP-4 were smaller (i.e. U C 25.9+_ 12.8mg protein/ml BOP-4 12.6 _+ 9.5 mg). Week 32. There were no differences in pH. The flow rate was reduced considerably in BOP-4, e.g. U C values period (2). 64/.L1/hr/100 g body weight vs BOP4, 14/~tl/hr/100 g body weight. By this time, in most cases, BOP-4 had developed advanced pancreatic carcinomas (see histology section). Protein concentration was reduced substantially in all BOP-4s during all periods. Concentration of ions. During week 0 there were no differences in the ion concentrations between BOP-4 and U C hamsters nor were there differences during weeks 8, 16 or 32, except in the case of sulfate, which was not present in detectable amounts in any of the secretions collected during week 32.
(C) 4 Doses o f BOP and stimulation with secretin or pancreozymin Table 1, week O. The pH of pancreatic juice from BOP-pretreated hamsters stimulated with secretin (BOP-4-Sec) was lower than that from pancreozymin-stimulated animals (BOP-4-PZ), e.g. 8.75 avg. vs 9.75 avg. Control animals, which had not been pretreated with BOP (UC), were found to have similar pH differences between UC-Sec and U C - P Z hamsters. Differences between BOP-4-Sec or BOP-4PZ and UC-Sec and U C - P Z animals were minimal in regard to flow rate and protein concentration, with the following exceptions: (1) U C - P Z hamsters during period (3) had a flow rate 40% higher than did BOP-4-PZ hamsters, e.g. 92/~l/hr/100 g body weight vs 55/~l/hr/100g body weight. BOP-4-Secs had a protein concentration twice that of UC-Secs in period (3), 18.8 vs 8.2mg/ml. Week 8. There were again differences in pH between BOP-E-Sec and BOP-4-PZ hamsters, such as an average of 8.85 for the former and 9.85 for the latter. The flow rate was 505o lower on the average in BOP-4-Sec or BOP-4-PZ than in UC-Secs or UCPZs, e.g. avg. 185gl/hr/100g body weight vs 120/.d/hr/100 g body weight. During period (2) flow
Table 1. Physiologicaleffectsof l0 mg/kg doses of BOP l x weekly for 4 weeks upon exocrine biophysiologicalparameters* of pancreatic secretory protein, pH and flow rate after secretagogues (week 0) Flow rate Protein concentration pH 1/hr/100 g.b.w, mg/ml Control Experimental Control Experimental C o n t r o l Experimental Collection period 8.8+0.04 8.6_+0.11 181.9_+12.7 163.9-+26.4 22.9-+1.8 26.0-+0.69 (1) 0-3 hr Secretin 9.4+0.06 9.5 -+0.04 88.4_+ 13.5 89.9_+ 14.8 35.6_+0.9 27.6_+1.64 Pancreozymin 8.8_+0.03 8.7_+0.10 68.9_+31.5 45.8_+2.1 24.3_+0.1 27.9_+1.24 (2) 3 6hr Secretin 9.3_+0.06 9.6_+0.06 43.7_+8.8 50.8_+9.2 23.1_+0.9 22.6+3.99 Pancreozymin 8.9_+0.05 8.8+_0.08 86.6_+ 1 6 . 6 88.5_+0.3 10.6_+2.1 23.8-+2.50 (3) 6 10hr Secretin 9.5-+0.03 9.4-+0.16 92.6-+9.9 6 8 . 9 - + 1 7 . 4 24.0_+2.2 25.3_+0.40 Pancreozymin *Computed from a mean of at least four animals.
Carcinogen (BOP) affects hamster pancreas
193
Table 2. Physiological effects of 10 mg/kg doses of BOP 1 x weekly for 4 weeks upon exocrine biophysiological parameters* of pancreatic secretory protein, pH and flow rate after secretagogues (week 16)
Collection period (1) 0 3 h r Secretin Pancreozymin (2) 3 6 h r Secretin Pancreoxymin (3) 6 10hr Secretin Pancreozymin
Control
pH Experimental
8.74_+0.04 9.89_+0.09 8.71 -+0.08 9.88+0.01 8.78_+0.07 9.9_+0.04
8.83_+0.06 9,72_+ 0.41 8,79_+0.02 9.66_+0.34 8.81 + 0 . 0 9 9.86_+0.03
Flow rate #l/hr/100 g.b.w, Control Experimental 335.3_+38.2 213.4_+ 1,6 119.7_+ 13.5 86.0_+13.7 8 6 . 6 + 16.6 91.9_+6.5
105.0_+ 15.6 23.2_+ 1.6 42.7_+ 11.2 35.4_+20.7 88.6_+0.3 54.8_+15.7
Protein concentration mg/ml Control Experimental 18.3_+3.5 38.5_+0.6 6.0_+0.3 17.5_+3.5 7.9_+0.2 18.8+0.4
21.9+2.6 33.8-+2.1 13.8-+3.3 20.6_+l.7 7.3_+ 1.2 8.2+0.8
*Computed from a mean of at least four animals.
rate in both BOP-4-Sec and BOP-4-PZ hamsters was 0.33 times as high as in UC-Sec or UC-PZ animals. During period (3) there was little difference in flow rate between BOP-4-PZ or BOP-4-Sec and UC-Sec or UC-PZ animals. During period (1), protein concentration in BOP-4-Sec was 25 mg/ml, twice that in UC-Sec animals, while that of BOP-4-PZ animals was 25.2 mg/ml, a lower protein concentration than UC-PZ animals had at 44.7 mg/ml. BOP-4-Secs had three times the protein concentration of UC-Secs during periods (2) and (3). NOP-4-PZ animals had a slightly lower protein concentration than did UC-PZs through the latter 2 periods. Table 2, week 16. There was no effect upon pH. Flow rate, however, was affected dramatically in the BOP-4-Secs rate that was 33°/0 of the rate in UC-Secs, while the flow rate in BOP-4-PZs was 10~o that of UC-PZs. In period (2), the flow rate in BOP-4-Secs was 40% that of UC-Secs, as was also the case with BOP-4-PZs vs UC-PZs. In period (3) there was no difference between UC-Secs and BOP-4-Secs, while BOP-4-PZs had a flow rate 60?/o of UC-PZs. Protein concentrations were similar in period (1) in all groups. In period (2) BOP-E-Secs had twice the protein concentration of UC-Secs. There were no differences in BOP-4-PZs and UC-PZs during this period. During period 3 BOP-4-Secs and UC-Secs had similar protein concentrations, while BOP-4-PZ animals had 50°Jo of the protein concentration of UC-PZs.
(D ) Ion concentration qf~er secretin or pancreozymin There were no effects of BOP treatment which caused deviations in the ion concentrations of BOP4-Secs or BOP-4-PZs from these same concentrations in UC-Secs or UC-PZs during weeks 0, 8 or 16. (E) Pancreas pathology of BOP-treated animals (histology) Week O. Hyperplasia was observed in BOP-4s. Week 8. Ductular adenomas, focal hyperplasia and ductular hyperplasia were seen in BOP-4s. Week l6. Ductular adenomas and ductular dysplasia were seen in all BOP-4 hamsters. Fifty per cent had hyperplasia of the large ducts and 50~o had either adenocarcinomas or microadenocarcinomas. None of the pancreases examined from either group showed conditions which would manifest as physical obstruction of pancreatic secretory flow (R. Runge, personal communication). Week 24. A few tumors were observed macroscopically in BOP-4 hamsters and blockage of ducts CBP 77,1(" M
was evident, as well as invasion and destruction of acinar tissue by ductular adenocarcinoma cells. Week 32. Degenerative changes progressed in BOP-4s with more destruction of acinar tissues, with obvious physical blockage of ducts and many more macroscopic tumors.
(F) Concentration o.1"GHLIR and ILIR in pancreatic secretions Table 3. This table gives values for the amounts of growth hormone, including immunoreactivity (GHLIR) and insulin-like immunoreactivity (ILIR), in secretin (UC-Sec)- or pancreozymin (UC-PZ)-treated control hamsters and those which had been given BOP prior to secretagogue stimulation (BOP-4-Sec) or (BOP-4-PZ). In period (1) (0-3 hr), pancreozymin stimulation of controls resulted in secretion of 7 times the amount of GHLIR that secretin stimulated during week 0. This was also true during period (2) (3-6 hr), at which time 190 microinternational units of GHLIR were secreted per ml of pancreatic fluid after secretin stimulation, while 1640.5 microinternational units of GHLIR were secreted per ml after pancreozymin stimulation. In period (3) (6-10 hr) the animals stimulated with pancreozymin had 10 times the amount of GHLIR in their pancreatic secretions as those stimulated with secretin. Amounts of ILIR in pancreatic juice were similar whether stimulation was by secretin or pancreozymin, except in hamsters during period (3), at which time the amount of ILIR per ml in UC-Secs was 7~o that in UC-PZs. In BOP-4-Secs and BOP-4-PZs almost identical differences can be seen during all collection periods when examining secretion of GHLIR and, unlike the case of ILIR in UC-Secs vs UC-PZs, pancreozymin consistently evoked from 2.4-2.6 times as much ILIR secretion as in animals pretreated with BOP. There was no significant difference in GHLIR secretion during week 0 between UC-Sec and BOP4-Sec animals in periods (1) and (2); however, a slight difference was found during period (3). UC-PZs had no GHLIR differences in weak 0, but BOP-4-PZ hamsters excreted 2.2 2.8 times as much (ILIR) as UC-PZs. Week 8. When values for UC-PZ and UC-Sec are compared at 8 weeks, the differences between GHLIR amounts resulting from stimulation by the two hormones were less extreme during the first 2 periods than during the same periods in week 0, although pancreozymin still caused more GHLIR release than did secretin in period (3). The differences in GH secretions between UC-Sec and UC-PZ hamsters
1800+- 1 0 . 0 1490 +- 14.1 1740 +- 64.9
2575.0 + 106.1 1640.5 ± 225.6 t410.5 ± 409.0
(1) (~3 hr (2) 3 6 h r (3) 6-10 hr
(1) (~3 hr (2) 3-6 hr (3) 6-10 hr
*Computed from a mean of at least four animals.
177.8±10.3 190.0 ± 28.3 87.5 -!-__58.0
Week 0
(1) (~3 hr (2) 3-6 hr (3) 6-10 hr
195.0 ± 77.8
(3) 6-10 hr
No BOP Control Secretin 25 iu per kg body wt 6 times in 3 hr BOP 10 mg/kg 1 x 4 weeks then Pancreozymin 20 iu per kg body wt 6 times in 3 hr No BOP Control Pancreozymin
200.0±28.3 262.5 _+67.2
Period (1) (~3 hr (2) 3 6 h r
Treatment
BOP 10 mg/kg 1 x 4 weeks then Secretin 25 iu per kg body wt
~iu G H L I R per ml
29.4 ± 3.4 29.3 + 0.6 27.0 ± 5.7
47.5 + 2.1
82.5__+2.1 53.0 + 2.8
28.5+-2.1 37.0 +- 1.4 1.9 ± 0.6
13.5+--2.1
32.0_+2.8 20.5 _+ 5.0
MU ILIR per ml
425.0 +- 106.1 605,0 + 35.4 685.0 ± 91.9
656.3 ± 254.5
608.3_+231.4 882.4 -+ 407.4
370.3+__37.8 227.0 ± 53.7 82.5 ± 3.5
320.9+-165.1
MU ILIR per ml
40 ± 2.8 33 ± 2.8 23.7 ± 0.5
35.8 +- 14.3
52.1 +- 1 6 . 7 46.4 +- 18.0
56±2.83 34.5 ± 2.12 35 +__6.49
30.00+-4.97
71.00+-56.02 38.88 ± 10.6
Week 8
391.9+-218,9 522.6 _+372.6
#iu G H L I R per ml
1175.0 ± 106.1 385.0 + 91,9 337.5 ± 102.5
136.7 ± 56.1
886.7+-61.1 267.7 -+ 39.0
1110.0+268.7 840.0 +- 28.3 585.0 + 63.6
1290.0 ± 1 1 5 . 3
1706.7_+90.2 270.0 +- 268.5
47.0 4-__5.7 88.0 + 5.7 44.5 ± 12,0
25.2 ± 12.3
110.0_+54.7 28.7 +- 2.3
16.0+-5.66 32.0 + 1.41 29.0 ± 0.00
57.7+-1.5
45.4+14.9 42.2 +- 5.4
Week 16 ,uiu G H L I R MU ILIR per ml per ml
Table 3. Concentrations* of growth hormone and insulin in pancreatic secretions collected at least 24 hr following I x 4 weeks of 10 mg BOP/kg hamster body weight
Z
P
;>
4~
Carcinogen (BOP) affects hamster pancreas became much greater, since pancreozymin caused release of 6.6 times the GHLIR as secretin during period (3). There were no significant differences between UC-Sec and UC-PZ hamsters as far as ILIR secretion was concerned. BOP-4-Sec and BOP-4-PZ hamsters exhibited no significant difference in GHLIR secretion or ILIR. When compared with UC animals, BOP-4 animals showed variance in GH secretion after secretin stimulation, although not in period (l). Periods (2) and (3), however, had from 2-8 times as much secreted GHLIR. Secretin caused no differences in insulin nor did pancreozymin stimulation result in a difference for either hormone in BOP-4, as opposed to UC hamsters. W e e k 16. In UC-Sec and UC-PZ hamsters, secretin for the first time caused a greater secretion of GHLIR than did pancreozymin. Pancreozymin caused ILIR secretion, as can be seen when UC-Sec and UC-PZ hamsters are compared during periods (2) and (3). Period (2) UC-Sec's value of 840 iu GHLIR per ml was 2.2 times the value of period (2) UC-PZ, which was 385 iu per ml. The difference was less during period (3). ILIR levels in secretions of UC-Sec hamo/ sters were ~~,,/o those of UC-PZs during all collection periods. Differences in GHLIR secretion for BOP-4Sec and BOP-4-PZ hamsters in periods (1) and (3) were apparently significantly different. BOP-4-Secs secreted 2 times as much GHLIR during period (1) and 5.4 times as much during period (3). There were no apparent differences in GHLIR secretion during period (2). ILIR secretion results were not as readily discernible; however BOP-4-Secs secreted more ILIR than BOP-4-PZs except in period (1). The variability during period (2) in BOP-4-PZ hamsters was too large for a valid statement regarding significance. GHLIR secretions were compared and BOP-4-Secs were found as capable of GHLIR secretion as UCSecs during week 16, although times of maximum secretion differed as UC-Sec GHLIR secretion peaked during period (1), stayed high during period (2), and dropped during period (3). No UC-Sec value was as high as the period (1) and (3) peaks for BOP-4-Secs. ILIR secretion in periods (1) (3) was about 2 times higher in BOP-4-Secs than in UC-Secs. BOP-4-PZ hamsters secreted consistently smaller GHLIR concentrations than did UC-PZ hamsters, while UC-PZ hamsters generally secreted more ILIR per ml of pancreatic fluid, except in period (1), at which time BOP-4-PZ hamsters secreted a greater ILIR concentration than UC-PZs. DISCUSSION
Immediate B O P effects
The effects of BOP administered at 20 mg/kg are similar to effects from the action of pancreozymin, e.g. a small increase in flow rate and a large increase in protein concentration. We considered that the increased protein concentration might be due to a toxic reaction from BOP and a sloughing off of dead cells, but if this was the case there should have been large amounts of cellular debris in the exocrine fluid, which we did not find. Although Reber et al. (1978) believed that carcinogen had a toxic effect on duct ceils, since his hamsters secreted less HCO~, indi-
195
cating that duct cells were damaged, our hamsters had no such decrease in secretory rate for HCO3. The latter finding indicated that the HCO~--secreting mechanisms of ductular cells were intact and hence the ductular cell toxicity of 20 mg/kg BOP was minimal. Further evidence for the intact nature of the duct cells is provided by the unaltered calcium secretion, which paralleled protein secretion, thereby indicating that duct and acinar cells were intact. Calcium is not only secreted as an ion, but also excreted with zymogens from acinar cells where it helps to regulate stimulus secretion coupling (Sarles, 1977). BOP may stimulate extrusion of zymogens (pro enzymes found in zymogen granules of acinar cells) as does pancreozymin (Mutt, 1980), serving as a stimulus-secretion activator of acinar cells by the mechanism described by Kanno and Yamamoto (1977) or Mutt (1980). Since BOP probably acts in a manner akin to this because it does have an effect similar to secretagogues, we think the compound has a specific affinity for some component of its target cells. This component may be a secretagogue receptor such as that for pancreozymin. Information supporting this process is given by Hahne et al. (1981), who have shown proglumide to have a strong affinity for the pancreozymin receptor on ductular and acinar cells (Christophe et al., 1980). Proglumide has a side chain which is a keto analog of the dipropylnitrosamine group of compounds (which includes BOP). Alternatively, BOP or its metabolites may bind to receptors on chromatin as do steroid hormones (Eisenfield et al., 1980). Long-term B O P effects
The physiological effects of pancreatic carcinoma induction by 4 weekly BOP doses were minimal until 24-32 weeks past the last injection of BOP. Between weeks 24 and 32, hamsters given BOP 4 times (BOP4) failed to secrete as much protein as untreated control hamsters (UC) of the same age. This was probably not due to physical duct blockage as there was no evidence of extensive physical blockage until 32 weeks after the final BOP treatment. The characteristics of the secretions of BOP-4 and UC animals were similar in flow rate, protein, pH and ion content. Long-term effects on response to secretagogues pancreozymin or secretin
At 8 weeks after the final BOP injection there was a pronounced difference in the secretory characteristics of untreated control hamsters stimulated during collection of pancreatic fluid with secretin or pancreozymin, and BOP-treated animals stimulated in an identical fashion. According to our previous results (Helgeson et al., 1980b), we would have expected to find the same degree of flow rate increase caused by secretin administration in "Normal" (see Helgeson et al., 1980) animals as in BOP-treated animals, since base-line values of animals not stimulated with secretagogues were virtually identical to those of BOP treated animals not given secretagogues. Since the secretory mechanism in these hamsters appears intact, the difference between the parameters in both UC hamsters and BOP-4 hamsters given secretin or
196
A. SCOTT HELGESONet al.
pancreozymin must lie in an inability of the BOP-4Sec or PZ pancreases to respond to these two hormones. The actual mechanism responsible for these differences is open to conjecture, but one possible explanation is that the "neoplastic" pancreatic cells do not bind secretin and pancreozymin effectively. Cells can lose their ability to bind hormones after transformation (Delarco and Todaro, 1980). However, this explanation presupposes a large percentage of pancreatic cells are transformed, which is not usually the case, since the loci of cancer development are generally few (R. Runge, personal communication). Although possible explanation is that BOP binds to the receptors for pancreozymin [especially in light of Hahne's results (1981)] and secretin, and either destroys or down regulates them permanently. The findings of Warren (1982) suggest that alterations in the acinar cell population in rats results in a diminished response to the effect of secretagogues because the diminutive responses of acinar cells to secretagogues reflect an insensitivity of acinar cell carcinoma secretion to extracellular Ca 2+. In cases of pancreatic acinar cell carcinomas, Warren also postulates an inability of c - A M P to stimulate protein secretion by acinar carcinoma cells due to a deficiency of the c - A M P dependent protein kinase system. To postulate a similar occurrence in the case of ductular cell carcinoma is logical since acinar cells and ductular cells are hypothesized to have the same stem cell (Warren, 1982). It is significant that pancreatic secretions from Syrian hamsters contain both insulin-like and growth hormone-like immunoreactivity. If the insulin-like or the growth hormone-like immunoreactivity are insulin and growth hormone then they could affect the induction and development of pancreas cancer. Both hormones stimulate D N A synthesis by forcing the cell into the S phase, and when cells are in S phase they are usually vulnerable to transformation by external effectors, such as viruses or cancer-causing chemicals (Grisham et al., 1980). BOP is a mitogen for ductular cells (P. Pour, personal communication) and consequently the chance of finding cells in S phase could be a combination of mitogenic effects of BOP plus those of insulin and growth hormone. Insulin levels in the blood and pancreatic fluid increase after secretin administration (Mutt, 1980). Pancreozymin increases plasma and pancreatic fluid insulin levels (Mutt, 1980). An important clinical aspect of these findings is their possible use as an early detection system for pancreatic cancer. Table 3 reflects large differences at various times in hormone content between BOPtreated and non-BOP-treated animals. With a more extensive study of this system or of a similar one, a workable system could be developed to detect pancreatic cancer based solely on exocrine secretion of endocrine hormones and abnormalities of their levels in duodenal aspirates. The latter would be a less traumatic way to examine pancreatic fluid than cannulation of the c o m m o n duct. Altered levels of endocrine hormone should be readily detectable. Hopefully, if cancer would be found, it will be in a state whereby conventional treatment and therapy would be adequate to control or eliminate it.
SUMMARY
Pancreatic exocrine secretion was studied in Syrian hamsters immediately following administration of 20mg/kg N-nitrosobis (2-oxopropyl)amine (BOP) and in hamsters which had been given 4 weekly doses of l0 mg BOP/kg. The chronically treated hamsters consisted of 2 experimental groups. G r o u p (1) was given BOP or used as controls without BOP treatment. G r o u p (2) animals were treated in the same manner, with experimental and control hamsters subdivided into secretin-treated or pancreozymintreated groups. Flow rate, pH, protein concentration, total protein secretion, and ionic concentrations of Na 4 , K + , C a 2 + , M g 2 + , H C O ~ , C I ,So~ and PO~ were measured. Acute effects of BOP are simi.ar to those of pancreozymin. Chronically BOP-treated hamsters showed little alteration of any of the abovementioned parameters until 24~32 weeks after carcinogen treatment, at which time 85% had pancreatic adenocarcinomas. Ductular blockage did not occur until 24~32 weeks, although a decreased flow rate and protein concentration in secretions were measured before this time. BOP-treated hamsters, stimulated with secretin and pancreozymin, had a reduced response to these secretagogues as early as week 8, with the reduction becoming quite obvious during week 16. Flow rate in these animals was one-third to one-tenth that of controls, protein concentration in response to pancreozymin was one-half to one-fourth that in controls. Ionic concentrations were unaffected by any of these treatments. The decreased response to secretagogue stimulation appears to be a receptormediated phenomenon. Insulin-like and growth hormone-like immunoreactivities were detected in the collected exocrine pancreatic secretions. Acknowledgements--This study was supported by a grant
from the National Cancer Institute National Pancreatic Cancer Project No. 1 R26 CA20198. Special thanks to Ed Bass for technical aid and to Thorkatla Donnelly and Rita MacCauley for surgical expertise, as well as to Mardelle Susman for editorial assistance. REFERENCES
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