API XML

Aop: 316

AOP Title

?


Trypsin inhibition leading to pancreatic acinar cell tumors

Short name:

?

TI-induced AC tumors

Graphical Representation

?

Click to download graphical representation template

W1siziisijiwmjavmdevmdgvn3qwctlzctl6ml9fms5kuecixsxbinailcj0ahvtyiisijuwmhg1mdaixv0?sha=6c6aed5cb33cdb30

Authors

?


Shigeru Hisada(1)
(1) Non-Clinical Evaluation Expert Committee, Drug Evaluation Committee, Japan Pharmaceutical Manufacturers Association, Tokyo, Japan

Point of Contact

?


Shigeru Hisada   (email point of contact)

Contributors

?


  • Shigeru Hisada

Status

?

Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.60 Included in OECD Work Plan


This AOP was last modified on January 09, 2020 07:28

?

Revision dates for related pages

Page Revision Date/Time
Trypsin inhibition January 08, 2020 02:50
Increased intestinal monitor peptide level January 08, 2020 03:06
Increased blood CCK level January 08, 2020 03:30
Increased exocrine secretion from pancreatic acinar cells January 08, 2020 19:49
Acinar cell proliferation January 08, 2020 03:38
Pancreatic acinar cell tumors January 08, 2020 03:43
Inhibition, trypsin leads to Increased monitor peptide January 08, 2020 03:56
Increased monitor peptide leads to Increased blood CCK level January 08, 2020 19:16
Increased blood CCK level leads to Increased acinar cell exocrine secretion January 08, 2020 18:55
Increased acinar cell exocrine secretion leads to Acinar cell proliferation January 08, 2020 04:29
Acinar cell proliferation leads to Acinar cell tumors January 08, 2020 04:36

Abstract

?


Pancreatic exocrine secretion is controlled mainly by the gastrointestinal hormone cholecystokinin (CCK), which is secreted by CCK-producing I cells located in the mucosa of the small intestine. Once the contents in the stomach is transported to the small intestine, I cells are stimulated to release CCK into the bloodstream. Several mechanisms to stimulate CCK release are involved.

In rats, pancreatic acinar cells secrete monitor peptide (MP) into the intestinal lumen as a pancreatic soluble trypsin inhibitor (TI). MP stimulates I cells to release CCK into the bloodstream through their surface MP receptors. Then, the increased blood concentration of CCK induces pancreatic exocrine secretion. When fasting, trypsin–MP complexes are formed to decrease the level of free MP in the small intestinal lumen; thereafter, CCK release is suppressed. Meanwhile, upon feeding, partially ingested proteins in the diet consume trypsin to increase the luminal concentration of free MP followed by stimulation of CCK release.

When soybean powder (raw soya flour) containing trypsin inhibitory molecules or TIs such as camostat are given to rats, the intestinal concentration of free MP is increased due to trypsin–TI complex formation. Then, intestinal I cells are stimulated to release CCK. The resulting increased blood level of CCK stimulates pancreatic exocrine secretion of MP, which induces further CCK release via a positive feedback loop. A sustained increase in the CCK level might induce pancreatic hypertrophy and hyperplasia and ultimately result in acinar cell tumor formation.

This increased blood CCK level induced by trypsin inhibition may also occur in humans and other mammalian species including rats. Luminal CCK-releasing factors (LCRFs) are trypsin-sensitive peptides secreted from small intestinal mucosa that stimulate CCK release by intestinal I cells. Luminal levels of LCRFs are increased after TI ingestion; however, the resultant increase in CCK levels does not stimulate further release of LCRFs, in contrast to MP.

Species differences in CCK-mediated stimulation of pancreatic enzyme secretion have been described in rats and humans. In rats, CCK stimulates pancreatic exocrine secretion and/or proliferation directly via CCK1 receptors expressed on acinar cell surfaces or indirectly via vagal afferent nerves expressing CCK1 receptors, especially at physiological blood CCK concentrations. In contrast to rats, the secretory function of human pancreatic acinar cells is indirectly innervated by vagal afferent nerves expressing CCK1 receptors; however, CCK receptors (mainly CCK2 receptors) expressed on human acinar cell surfaces are not involved in both exocrine secretion and proliferation. These findings suggest that, in humans, innervation of acinar cells in response to elevated CCK blood levels affects mainly secretory functions, with less of an effect on cell proliferation, although the effects of vagal stimulation on acinar cell proliferation are still unclear.

In conclusion, long-term administration of TIs induces pancreatic acinar cell tumors in rats. The main factor contributing to carcinogenesis is a sustained increase in plasma CCK levels mediated by an increased luminal concentration of trypsin-sensitive MP. The risk of trypsin inhibition-induced pancreatic tumors in humans seems to be low or equivocal because of the following reasons:

  1. MP, a pancreatic soluble TI that protects against auto-injury induced by trypsin, stimulates CCK release and thereby pancreatic exocrine secretions containing MP, via a positive feedback loop, in rats only.
  2. An increased CCK level directly stimulates pancreatic acinar cells to proliferate via surface CCK1 receptors in rats but not in humans. It is still unclear whether vagal stimulation of acinar cells promotes proliferation of acinar cells.

     


Background (optional)

?


Raw soy flour and purified trypsin inhibitors (TI) cause pancreatic hypertrophy and hyperplasia in some mammalian species, and prolonged treatment with high levels of TI contained in raw soy induced pancreatic nodular hyperplasia and acinar cell adenoma [Rackis JJ, 1965; McGuinness EE et al, 1984; McGuinness EE et al, 1980; McGuinness EE et al, 1985; McGuinness EE and Wormsley KG, 1986; Gumbmann MR et al, 1986]. TI also promoted nodular hyperplasia and tumor formation in rats treated with low levels of pancreatic carcinogens such as azaserine [McGuinness EE et al, 1984; McGuinness EE et al, 1987; Lhoste EF et al, 1988]. These findings question the safety of TI-containing plant foods, and many different studies and reviews have been published to date. The important factors for TI-induced pancreatic acinar cell tumors seem to be a high level of CCK release and CCK-stimulated acinar cell proliferation. In the present AOP, the pathway progressing from trypsin inhibition to pancreatic acinar cell tumor formation is considered from the viewpoints of such key factors.


Summary of the AOP

?


Events: Molecular Initiating Events (MIE)

?

Key Events (KE)

?

Adverse Outcomes (AO)

?

Sequence Type Event ID Title Short name
MIE 1720 Trypsin inhibition Inhibition, trypsin
KE 1721 Increased intestinal monitor peptide level Increased monitor peptide
KE 1722 Increased blood CCK level Increased blood CCK level
KE 1723 Increased exocrine secretion from pancreatic acinar cells Increased acinar cell exocrine secretion
KE 1724 Acinar cell proliferation Acinar cell proliferation
AO 1725 Pancreatic acinar cell tumors Acinar cell tumors

Relationships Between Two Key Events
(Including MIEs and AOs)

?

Title Adjacency Evidence Quantitative Understanding
Inhibition, trypsin leads to Increased monitor peptide adjacent Moderate Low
Increased monitor peptide leads to Increased blood CCK level adjacent High Moderate
Increased blood CCK level leads to Increased acinar cell exocrine secretion adjacent High High
Increased acinar cell exocrine secretion leads to Acinar cell proliferation adjacent High Moderate
Acinar cell proliferation leads to Acinar cell tumors adjacent High High

Network View

?

 

Stressors

?

Life Stage Applicability

?

Life stage Evidence
All life stages High

Taxonomic Applicability

?

Term Scientific Term Evidence Link
Homo sapiens Homo sapiens Low NCBI
Macaca fascicularis Macaca fascicularis Low NCBI
Rattus norvegicus Rattus norvegicus High NCBI
Mus musculus Mus musculus Moderate NCBI

Sex Applicability

?

Sex Evidence
Mixed High

Overall Assessment of the AOP

?



Long-term treatment with trypsin inhibitors (TIs) such as raw soya flour (RSF) in rats increases the incidence of pancreatic acinar cell tumors [McGuinness EE et al, 1984; Gumbmann MR et al, 1986; McGuinness EE et al, 1987; Woutersen RA et al, 1991]. The causative factors for tumorigenesis are a TI-induced increase in cholecystokinin (CCK) release from small intestinal I cells into the blood and direct stimulation of acinar cell proliferation via surface CCK1 receptors [Watanapa P and Williamson RC, 1993].

Differences in these tumor risk factors between rodents and humans are described below.

In rats, trypsin-sensitive monitor peptide (MP), a pancreatic soluble TI (PSTI) found in pancreatic juice that protects against the auto-injury induced by trypsin [Iwai K et al, 1987; Iwai K et al, 1988; Tsuzuki S et al, 1991; Tsuzuki S et al, 1992], plays a major role in stimulating pancreatic exocrine secretion via CCK release [Miyasaka K et al, 1989; Fushiki T et al, 1989; Miyasaka K and Funakoshi A, 1998]. TIs increase the luminal concentration of MP to stimulate CCK release, which in turn increases the MP level as well as pancreatic enzyme secretion via positive regulation. Moreover, repeated injection of CCK into rats increased the level of MP mRNA in the pancreas [Tsuzuki S et al, 1992]. Therefore, the TI-induced increase in CCK release seems to be robust in rodents compared with other species.

On the other hand, in humans, PSTIs do not directly stimulate CCK release [Miyasaka K et al, 1989]. Furthermore, other trypsin-sensitive CCK-releasing peptides (luminal CCK-releasing factors, LCRFs) secreted by intestinal mucosal cells are found in multiple species including rodents and humans [Spannagel AW et al, 1996; Herzig KH et al, 1996; Tarasova N et al, 1997; Li Y et al, 2000; Owyang C, 1999; Wang Y et al, 2002]. TIs increase luminal concentrations of LCRFs, which stimulate CCK release; however, the increase might be mild compared with that induced by MP, because LCRF release does not increase in response to increased CCK levels.

Regarding mitotic activity, high plasma levels of CCK directly stimulate proliferation of rodent pancreatic acinar cells via their surface CCK1 receptors [Povoski SP et al, 1994; Myer JR et al, 2014]. In humans, surface CCK receptors (mainly CCK2 receptors) are not involved in stimulating pancreatic functions; the secretory functions of human acinar cells are innervated mainly by vagal afferent nerves expressing CCK1 receptors [Dufresne M et al, 2006]. However, the vagal contribution to acinar cell proliferation is controversial. Oral ingestion of raw soya flour containing TIs has been reported to stimulate CCK release in humans [Calam J et al, 1987]. In addition, some epidemiological surveys suggest that long-term ingestion of TI-containing foods does not increase the risk of pancreatic cancer [Miller RV, 1978]. On the other hand, a strong relationship between pancreatic cancer and a history of subtotal gastrectomy [Mack TM et al, 1986], which induced a higher plasma CCK level in response to fat [Hopman WP et al, 1984], was reported.

Therefore, the present AOP supports a pathway from trypsin inhibition to tumor formation originating from pancreatic acinar cells in rodents. The relevance of these findings to humans seems low, although some evidence of a TI-induced increase in blood CCK levels suggests the need for case-by-case risk assessment of pancreatic cancer in humans.

Domain of Applicability

?

Trypsin

Trypsin is a pancreatic digestive enzyme that has been identified in many animals, including insects, fish, and mammals. The natural substrate of trypsin is generally any peptide that contains Lys or Arg. The active site of trypsin, which is composed of a catalytic triad, is fully conserved, with a similar three-dimensional structure among species, although there are species differences in the amino acid sequence of the enzyme [Baird Jr TT and Craik CS, 2013; Baird Jr TT, 2017]. TIs such as soybean flours and camostat suppress the activity of trypsin in animal species, including rats and humans [Savage GP and Morrison SC, 2003].

Monitor peptide and related peptides with trypsin inhibitory activity

Pancreatic soluble trypsin inhibitors (PSTIs) are found in the pancreatic juice of multiple mammalian species, including rodents and humans [Greene LJ et al, 1968; Pubols MH et al, 1974; Eddeland A and Ohlsson K, 1976; Kikuchi N et al, 1985]. Secreted PSTIs bind tightly to trypsin to protect against trypsin-induced self-injury in the pancreas and intestinal tracts [Voet D and Voet JG, 1995].

In rats, two types of PSTIs have been isolated: monitor peptide (MP, also known as PSTI-I) and PSTI-II [Tsuzuki S et al, 1991; Tsuzuki S et al, 1992]. Both are similar in amino acid sequence; however, the former directly stimulates CCK release from intestinal I cells via their surface MP receptors, whereas the latter does not [Yamanishi R et al, 1993]. Human PSTIs do not directly stimulate CCK release from intestinal mucosal cells [Miyasaka K et al, 1989].

Species differences in the mechanism of CCK release

Pancreatic exocrine secretion is controlled mainly by CCK released into the blood steam from intestinal mucosal I cells of the small intestine in response to the gastric contents transported to the intestine [Singer MV and Niebergall-Roth E, 2009; Rehfeld JF, 2017]. Peptides released from gastrointestinal digestion, along with fatty acids, are the main stimuli of CCK release involving several direct and indirect pathways [Caron J et al, 2017].

In humans and canines, amino acids and fatty acids in the gastric contents transported to the small intestine play a major role in stimulating CCK release, which regulates pancreatic exocrine secretion, but MP is not involved in exocrine regulation [Wang BJ and Cui ZJ, 2007].

In rats, however, different from other mammalian species, MP secreted by pancreatic acinar cells plays a major role in protein-stimulated CCK release [Iwai K et al, 1988; Fushiki T et al, 1989]. Ingestion of trypsin inhibitors increases the intestinal level of MP, especially in the intestines during fasting, causing a subsequent increase in the blood level of CCK. Increased levels of CCK stimulate pancreatic exocrine secretion of proteins including MP, which in turn further increases the release of CCK. This positive feedback response associated with MP secretion might lead to continuously elevated plasma levels of CCK [Liddle RA, 1995].

Species differences in CCKs

Several isoforms of CCK, including CCK-83, -58, -39, -33, -22, and -8, have been identified, and there are species differences in CCK isoforms (e.g., CCK-33, -22 and -58 are expressed in humans, CCK-58 in dogs, CCK-8, -33 and -58 in cats, CCK-22, -58, -3 and -8 in pigs, CCK-22 and -8 in rabbits, and CCK-58 in rats). All of these isoforms of CCK have a highly conserved region of amino acids, and all are ligands of CCK1 receptors [Wang BJ and Cui ZJ, 2007].

Species differences in pancreatic exocrine secretion

In rats, physiological plasma level of CCK stimulates pancreatic exocrine secretion and acinar cell growth directly via CCK1 receptors expressed on the cell surface, and exocrine secretion is also innervated by vagal afferent nerves expressing CCK1 receptors [Singer MV and Niebergall-Roth E, 2009; Pandiri AR, 2014]. Higher plasma levels of CCK may stimulate acinar cell proliferation only via surface CCK receptors but not by vagal nerve innervation [Yamamoto M et al, 2003].

On the other hand, human pancreatic acinar cells express CCK2 receptors, which are not involved in secretion nor proliferation, and exocrine secretion is regulated exclusively by innervation of vagal nerves expressing CCK1 receptors [Soudah HC et al, 1992; Beglinger C et al, 1992; Singer MV and Niebergall-Roth E, 2009], although there is some evidence of direct stimulation of exocrine secretion of human pancreatic acinar cells [Murphy JA et al, 2008].

Species differences in CCK receptors

Although the distribution of CCK receptors is different between humans and rodents, the structures of CCK1 receptors are highly conserved among mammalian species, and all CCK isoforms function as ligands of CCK1 receptors [Wang BJ and Cui ZJ, 2007].

In rats, CCK1 receptors are expressed in pancreatic acinar cells and sensory vagal afferent nerves, whereas in humans, CCK1 receptors are expressed in vagal afferent nerves but not pancreatic acinar cells. Acinar cells instead express CCK2 receptors; however, these CCK2 receptors are not involved in pancreatic exocrine secretion [Ji B et al, 2001; Dufresne M et al, 2006].

Risk of TI-induced tumor formation from pancreatic acinar cells in humans

The mode of action of TI-induced tumor formation from pancreatic acinar cells in rats is based on a persistent increase in the blood level of CCK, which is induced by an increased intestinal level of MP, resulting from positive regulation of pancreatic exocrine secretion and TI activity.

It was reported that raw soya flour increases CCK release in humans [Calam J et al, 1987]. In addition, the plasma CCK concentration was found to increase after oral administration of fat in patients after subtotal gastrectomy [Hopman WP et al, 1984], and a strong association between pancreatic cancer and a history of subtotal gastrectomy was demonstrated in these patients [Hopman WP et al, 1984].

Therefore, based on the findings from animal studies of persistently increased blood CCK levels accompanied by histopathologic changes in acinar cell proliferation, the tumor risk should be evaluated carefully in humans, despite the lower risk compared with rodents.


Essentiality of the Key Events

?

MP:

Atropine-treated rats with diversion of pancreatic juice were infused with a mixture consisting of MP, purified trypsin, and various food proteins into the small intestine after intraluminal lavage, followed by examination of pancreatic exocrine secretion. Exocrine secretion was fully reconstructed by the constituent, suppressed in the absence of MP, and the treatment with an anti-MP antibody decreased this exocrine secretion [Fushiki T et al, 1989]. These results suggest that MP is an essential factor for regulating pancreatic exocrine secretion.

CCK:

CCK-deficient mice generated by gene targeting in embryonic stem cells showed no abnormalities in body weight or pancreatic weight or histopathology, but they showed protein-induced increases in pancreatic growth and proteolytic enzyme secretion, suggesting that other regulatory pathways are modified to compensate for the CCK deficiency [Lacourse KA et al, 1999]. The TI camostat increased pancreatic wet weight and protein and DNA levels in a time-dependent manner over a 10-day period in normal mice, but not in CCK-deficient mice [Tashiro M et al, 2004]. These results suggest that CCK is needed for TI-induced pancreatic hyperplasia.

CCK receptors:

In an experiment in which CCK1 receptor-deficient rats were fed a diet containing 0.1% TI (camostat, ONO-3403, or soybean TI) for 7 days, the CCK mRNA level increased without any change in the protein level in pancreatic juice in each TI treatment group. These results suggest that TI treatment enhances the release of CCK, and that CCK-induced secretion of pancreatic digestive enzymes is mediated by CCK1 receptors [Kawanami T et al, 1999].

Experiments using CCK1-receptor-deficient Otsuka Long-Evans Tokushima Fatty rats showed the following:

  1. The CCK1 receptor plays a role in the increased cell size associated with normal growth of the pancreas [Miyasaka K et al, 1996].
  2. The CCK1 receptor is not an absolute requirement for normal growth of the pancreas but is important for pancreatic regeneration [Miyasaka K et al, 1997].
  3. Absence of the CCK1 receptor did not affect the acute phase of pancreatitis but significantly retarded regeneration of pancreatic tissue [Miyasaka K, Ohta M et al, 1998].

Evidence Assessment

?

Biological Plausibility

KER

KEup-KEdown

Plausibility

Rationale supported by the literature

KER1

Trypsin inhibition increases the luminal concentration of MP

 

Middle

In rodents, a certain level of monitor peptide (MP) is secreted from pancreatic acinar cells, even between meals or under fasting conditions. However, intestinal MP level is maintained at a low level because of its rapid degradation by trypsin and other proteases (or because of MP–trypsin complex formation, which decreases the level of luminal free MP) [Liddle RA, 1995; Miyasaka K and Funakoshi A, 1998]. After ingestion of trypsin inhibitor (TIs), the intestinal content of MP increases rapidly especially in the fasting state [Iwai K et al, 1988; Liddle RA, 1995]. In other species, trypsin-sensitive CCK-releasing peptide (luminal CCK-releasing factor, LCRF) is released from small intestinal mucosal cells [Owyang C, 1999]. TIs increase the luminal concentration of LCRF; however, the increase in LCRF is not as high compared with MP [Liddle RA, 1995].

KER2

The increased luminal concentration of MP increases the blood CCK level

Strong

In rats, CCK release from I cells in the small intestinal mucosa is regulated by trypsin-sensitive MP [Miyasaka K et al, 1989; Cuber JC et al, 1990; Guan D et al, 1990]. In the empty intestine after dietary protein is digested, secreted MP forms complex with trypsin to be degraded, and luminal level of free MP is kept at low levels, during which CCK release is suppressed Once TIs are ingested, the intestinal concentration of free MP is increased due to trypsin–TI interactions [Liddle RA, 1995; Miyasaka K and Funakoshi A, 1998].Then, the increased MP directly stimulates I cells via their surface MP receptors to release CCK into the blood, leading to increased plasma CCK levels [Bouras EP, 1992; Cuber JC, 1990; Guan D, 1990]. The luminal MP level is further increased along with increased pancreatic exocrine secretion due to the increased plasma level of CCK via a positive feedback loop and trypsin inhibition [Liddle RA, 1995].

In other species including rats, TI increases the luminal level of trypsin-sensitive LCRF to stimulate CCK release, but the increase is transient due to the lack of the positive feedback loop between CCK and LCRF [Liddle RA, 1995].

KER3

The increased blood CCK level stimulates exocrine secretion by pancreatic acinar cells

Strong

Pancreatic exocrine secretion is regulated by CCK released from CCK-producing I cells located in the small intestinal mucosa. CCK stimulates exocrine secretion from pancreatic acinar cells directly via surface CCK receptors and indirectly via vagal afferent nerves expressing CCK receptors in rats. However, in humans, pancreatic secretion is innervated by vagal afferent nerves [Pandiri AR, 2014].

Of the two types of CCK receptors (CCK1 and CCK2 receptors), the former show high affinity to CCK and the latter high affinity to both CCK and gastrin [Dufresne M et al, 2006]. In rats, CCK1 receptors are expressed on pancreatic acinar cells and vagal afferent nerves. On the other hand, in humans, CCK1 receptors are expressed on vagal afferent nerves but not on pancreatic acinar cells, on which CCK2 receptors are expressed instead. CCK2 receptors are not involved in acinar cell functions [Pandiri AR, 2014].

KER4

The exocrine secretion induced by pancreatic acinar cells increases proliferation of pancreatic acinar cells

Strong

An increased plasma level of CCK directly induces proliferation of pancreatic acinar cells via surface CCK1 receptors, as well as exocrine secretion, in rats [Yanatori Y and Fujita T, 1976; Folsch UR et al, 1978; Longnecker DS, 1987; Povoski SP et al, 1994; Tashiro M et al, 2004].

However, the involvement of vagal afferent innervation in acinar cell proliferation under an increased blood level of CCK might be low in humans, but this is unclear [Chandra R and Liddle RA, 2009].

KER5

The increased proliferation of pancreatic acinar cells leads to pancreatic acinar cell tumor formation

Strong

A sustained increase in acinar cell proliferation promotes tumor formation [McGuinness EE et al, 1985]. An increased blood CCK level is the main factor involved in sustained acinar cell proliferation, which promotes acinar cell tumor formation [Douglas BR et al, 1989].

 

Empirical Support

KER

Empirical support for KERs

MIE=>KE1:Trypsin inhibition increases the luminal concentration of MP

Empirical support for the MIE => KE1 is strong.

Rationale:

No study has demonstrated a direct relationship between trypsin inhibition and an increased luminal concentration of monitor peptide (MP). However, several studies have reported a relationship between trypsin inhibitor (TI) treatment and an increased plasma CCK level. Considering that MP directly stimulates CCK release from I cells in the small intestine in rodents, increased plasma CCK levels induced by TIs suggest increased luminal MP levels.

The plasma CCK8 level in rats after 18-hour fasting was 0.31 ± 0.05 pM (mean ± SE) and increased to 6.2 ± 1.8 pM 7.5 minutes after feeding and increased to 10.3 ± 1.8 pM 15 minutes after intragastric instillation of a soybean trypsin inhibitor [Liddle RA et al, 1984].

Immediately after oral feeding of camostat at 400 mg/kg in rats, the plasma CCK level increased 10-fold above that in controls, reached a maximum after 90 min, remained elevated for more than 6 h, and then returned to control levels 24 h after administration of camostat [Goke B et al, 1986].

KE1 =>KE2: An increase in the luminal concentration of MP increases the blood CCK level

Empirical support for the KE1 => KE2 AO is strong.

Rationale:

MP at concentrations ranging from 3 x 10-12 to 3 x 10-8 M stimulated CCK release from isolated mucosal cells of the rat duodenum in a dose-dependent manner with highest level at 15 minutes after stimulation [Bouras EP et al, 1992].

MP at a concentration range of 2–12 µg/mL induced within a few minutes a dose-dependent transient increase in portal CCK-like immunoreactivity in isolated vascularly perfused rat duodenum/jejunum [Cuber JC et al, 1990].

In rats with biliary and pancreatic fistulas, duodenal infusion of MP at 0.9 µg/rat increased pancreatic secretion and the plasma CCK level [Miyasaka K et al, 1989].

Sorted CCK-positive rat intestinal mucosal cells stimulated with 30 nM MP increased the secretion of CCK in a time-dependent manner as soon as 5 min after the start of stimulation [Liddle RA et al, 1992].

KE2 =>KE3: An increase in the blood CCK level induces exocrine secretion by pancreatic acinar cells

Empirical support of the KE2 => KE3 is strong.

Rationale:

In rats, diversion of bile pancreatic juice induced more than ten-times increase in plasma concentration of CCK at the end of two hours and caused rapid and sustained increase in pancreatic protein secretion with more than two folds at 60 minutes of diversion compared with the basal levels [Li Y and Owyang C, 1994].

Repeated injections of cholecystokinin (CCK) at 130 IU s.c. for 3 weeks significantly increased the pancreatic content and secretion of amylase and trypsin during stimulation with 60 IU/kg-hour of CCK. Peak secretion rates of the enzymes were obtained 45 minutes after the start of the stimulation [Folsch UR et al, 1978].

CCK-mediated feedback control of pancreatic enzyme secretion is also observed in humans. Intraduodenal perfusion of phenylalanine at 10mM, 5mL/min induced a several times increase in the plasma level of CCK within 15 minutes and a four-times increase in one-hour pancreatic outputs of trypsin and chymotrypsin. Simultaneous intraduodenal perfusion of trypsin with phenylalanine lowered plasma CCK level at 24% and pancreatic output of chymotrypsin at 63% compared with the perfusion of phenylalanine alone. Moreover, intravenous infusion of CCK-8 at 20 and 40 ng/kg/h for 60 minutes showed a dose-dependent increase in pancreatic output of chymotrypsin [Owyang C et al, 1986].                                                         

KE3 =>KE4: Induction of exocrine secretion by pancreatic acinar cells increases proliferation of pancreatic acinar cells

Empirical support for the KE2 => KE3 is strong.

Rationale:

KE3/KE4: In rats fed 20, 40, and 100% RSF-containing diet for up to 36 weeks, pancreatic hypertrophy was found in all RSF-fed groups, and hyperplasia was found only in the 40 and 100% RSF-fed groups [Crass RA and Morgan RG, 1982].

KE3: Intraduodenal administration of 30 mg RSF increased the total amount of 1-hour pancreatic protein output at 2.2 ± 1.1 mg/h (mean ± SE) in rats in which bile and pancreatic juice were returned to the duodenum [Jordinson M et al, 1996].

KE4: In rats, administration of TIs in drinking water (“Trypsin soybean inhibitor” (Miles), 400mg/100mL) or injection of CCK (CCK-PZ or CCK-33,400 Ivy Dog unit) for 7 days increased acinar cell proliferation as well as acinar cell hypertrophy [Yanatori Y and Fujita T, 1976], and RSF feeding at libitum increased acinar cell proliferation from 7 to 28 days of treatment leading to hypertrophy and hyperplasia [Oates PS and Morgan RG, 1984].

These results show that trypsin inhibition-induced acinar cell proliferation (hyperplasia) developed at higher doses of RSF compared with those of pancreatic hypertrophy caused by increased secretion, or that pancreatic exocrine secretion and increased acinar cell proliferation were detected after 1 h and 7 days, respectively, after the start of TI or CCK treatment.

KE4 =>AO: Increased proliferation of pancreatic acinar cells induces pancreatic acinar cell tumors

Empirical support for the KE4 => AO is strong.

Rationale:

Rats were fed a diet containing 100 or 200 mg TI concentrates prepared from RSF or potato juice.
KE4: After 28 days of feeding, both sources of TI induced pancreatic hypertrophy.
AO: After 95 weeks of feeding, both TIs induced dose-related pancreatic changes in terms of nodular hyperplasia and acinar adenoma [Gumbmann MR et al, 1989].

Rats continuously fed a diet containing 5% or more RSF developed pancreatic micro/macroscopic nodules and stimulated azaserine-induced nodular hyperplasia and tumorigenesis, and those fed a diet containing 25, 50 and 100% RSF 2 days per week developed pancreatic macro/microscopic nodules, and 100% RSF-fed rats developed pancreatic cancer [McGuinness EE and Wormsley KG, 1986].

Rats fed a diet containing as little as 0.02% camostat 3 days per week developed pancreatic hypertrophy and hyperplasia [Lhoste EF et al, 1988].

F344 rats injected s.c. twice with azaserine at 30 mg/kg BW and treated with camostat at 200 mg/kg BW by gavage 5 days a week for 18 weeks developed azaserine-induced pancreatic preneoplastic lesions. In azaserie-treated Lewis rats, treatment with camostat in diet at 0.5 g/kg diet for 4 weeks and then 0.2 mg/kg diet 3 consecutive days per week for 8 or 16 weeks also promoted the growth of azaserine-induced neoplastic lesions [Lhoste EF et al, 1988].

 


Quantitative Understanding

?

KER1:

No study has shown a dose–response relationship between trypsin inhibition and the luminal concentration of MP in rodents. (further research is needed).

KER2:

MP at concentrations ranging from 3 x 10-12 to 3 x 10-8 M stimulated CCK release within 5 minutes from isolated mucosal cells from the rat duodenum in a dose-dependent manner [Bouras EP et al, 1992].

MP at a concentration range of 2–12 µg/mL induced a dose-dependent transient (within several minutes) increase in portal CCK-like immunoreactivity in isolated vascularly perfused rat duodeojejunum. MP at 36 µg/mL showed lower CCK release [Cuber JC et al, 1990].

KER3:

The effect of CCK on the stimulation of pancreatic secretion is dose dependent.

Intravenous infusion of CCK-8 at 20 and 40 pM/kg/hour or high affinity CCKR agonist CCK-JMV-189 at 22, 44 and 88 μg/kg/hour in rats induced dose-dependent increases in pancreatic protein secretion from 15 minutes of infusion [Li Y et al, 1997].

Physiological plasma CCK doses (up to ~10 pM) stimulate the vagal afferent pathway, whereas supraphysiological CCK doses stimulate intrapancreatic neurons and pancreatic acini to secret pancreatic protein [Owyang C, 1996].

KER4:

In rats injected subcutaneously with CCK at 7.5 or 30 Ivy dog units (IU) twice daily for 20 days, pancreatic wet weight and DNA content / 100g BW increased with a same manner compared with saline-treated rats, however, pancreatic output of amylase and trypsin in response to submaximal intravenous stimulation with CCK at 15 IU/kg/hour increased with dose-dependent manner [Folsch UR et al, 1978].

Rats were fed diets consisting of four concentrations of purified soybean TIs (93, 215, 337, and 577 mg/100 g diet) and three protein concentrations (10%, 20%, and 30%) and were then sacrificed at 3-month intervals starting at 6 months [Rackis JJ et al, 1985]. Trypsin and chymotrypsin activities per 100g BW, RNA and DNA contents of pancreas indicative of pancreatic hypertrophy and hyperplasia, respectively, were already increased in all of the TI and protein-fed animals after 6-month dosing, although pancreatic nodules were increased in number at 15 months of dosing or later at 215 mg TI/100 g diet or higher [Liener IE et al, 1985].

KER5:

Rats were fed diets consisting of four concentrations of purified soybean TIs (93, 215, 337, and 577 mg/100 g diet) and three protein concentrations (10%, 20%, and 30%) and were then sacrificed at 3-month intervals starting at 6 months [Rackis JJ et al, 1985]. RNA and DNA contents of pancreas indicative of pancreatic hypertrophy and hyperplasia, respectively, were already increased in all of the TI- and protein-fed animals after 6-month dosing. Pancreatic nodules were increased in number at 15 months of dosing or later and at 215 mg TI/100 g diet or higher[Liener IE et al, 1985].


Considerations for Potential Applications of the AOP (optional)

?


TBD


References

?


 1.    Baird Jr TT, Craik CS: Trypsin. Academic Press, Cambridge, Massachusetts (pp)2594-2600,2013

 2.    Baird Jr TT: Trypsin. Elsevier,2017

 3.    Beglinger C, Hildebrand P, Adler G, Werth B, Luo H, Delco F, Gyr K: Postprandial control of gallbladder contraction and exocrine pancreatic secretion in man. Eur J Clin Invest 22:827-834,1992

 4.    Bouras EP, Misukonis MA, Liddle RA: Role of calcium in monitor peptide-stimulated cholecystokinin release from perfused intestinal cells. Am J Physiol 262:G791-6,1992

 5.    Calam J, Bojarski JC, Springer CJ: Raw soya-bean flour increases cholecystokinin release in man. Br J Nutr 58:175-179,1987

 6.    Caron J, Domenger D, Dhulster P, Ravallec R, Cudennec B: Protein digestion-derived peptides and the peripheral regulation of food intake. Front Endocrinol (Lausanne) 8:85,2017

 7.    Chandra R, Liddle RA: Neural and hormonal regulation of pancreatic secretion. Curr Opin Gastroenterol 25:441-446,2009

 8.    Crass RA, Morgan RG: The effect of long-term feeding of soya-bean flour diets on pancreatic growth in the rat. Br J Nutr 47:119-129,1982

 9.    Cuber JC, Bernard G, Fushiki T, Bernard C, Yamanishi R, Sugimoto E, Chayvialle JA: Luminal CCK-releasing factors in the isolated vascularly perfused rat duodenojejunum. Am J Physiol 259:G191-197,1990

10.    Douglas BR, Woutersen RA, Jansen JB, de Jong AJ, Rovati LC, Lamers CB: Modulation by CR-1409 (lorglumide), a cholecystokinin receptor antagonist, of trypsin inhibitor-enhanced growth of azaserine-induced putative preneoplastic lesions in rat pancreas. Cancer Res 49:2438-2441,1989

11.    Dufresne M, Seva C, Fourmy D: Cholecystokinin and gastrin receptors. Physiol Rev 86:805-847,2006

12.    Eddeland A, Ohlsson K: Purification of canine pancreatic secretory trypsin inhibitor and interaction in vitro with complexes of trypsin-alpha-macroglobulin. Scand J Clin Lab Invest 36:815-820,1976

13.    Folsch UR, Winckler K, Wormsley KG: Influence of repeated administration of cholecystokinin and secretin on the pancreas of the rat. Scand J Gastroenterol 13:663-671,1978

14.    Fushiki T, Kajiura H, Fukuoka S, Kido K, Semba T, Iwai K: Evidence for an intraluminal mediator in rat pancreatic enzyme secretion: reconstitution of the pancreatic response with dietary protein, trypsin and the monitor peptide. J Nutr 119:622-627,1989

15.    Goke B, Printz H, Koop I, Rausch U, Richter G, Arnold R, Adler G: Endogenous CCK release and pancreatic growth in rats after feeding a proteinase inhibitor (camostate). Pancreas 1:509-515,1986

16.    Greene LJ, DiCarlo JJ, Sussman AJ, Bartelt DC: Two trypsin inhibitors from porcine pancreatic juice. J Biol Chem 243:1804-1815,1968

17.    Guan D, Ohta H, Tawil T, Liddle RA, Green GM: CCK-releasing activity of rat intestinal secretion: effect of atropine and comparison with monitor peptide. Pancreas 5:677-684,1990

18.    Gumbmann MR, Spangler WL, Dugan GM, Rackis JJ: Safety of trypsin inhibitors in the diet: effects on the rat pancreas of long-term feeding of soy flour and soy protein isolate. Adv Exp Med Biol 199:33-79,1986

19.    Gumbmann MR, Dugan GM, Spangler WL, Baker EC, Rackis JJ: Pancreatic response in rats and mice to trypsin inhibitors from soy and potato after short- and long-term dietary exposure. J Nutr 119:1598-1609,1989

20.    Herzig KH, Schon I, Tatemoto K, Ohe Y, Li Y, Folsch UR, Owyang C: Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine. Proc Natl Acad Sci U S A 93:7927-7932,1996

21.    Hopman WP, Jansen JB, Lamers CB: Plasma cholecystokinin response to oral fat in patients with Billroth I and Billroth II gastrectomy. Ann Surg 199:276-280,1984

22.    Iwai K, Fukuoka S, Fushiki T, Tsujikawa M, Hirose M, Tsunasawa S, Sakiyama F: Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice. Its homology with pancreatic secretory trypsin inhibitor. J Biol Chem 262:8956-8959,1987

23.    Iwai K, Fushiki T, Fukuoka S: Pancreatic enzyme secretion mediated by novel peptide: monitor peptide hypothesis. Pancreas 3:720-728,1988

24.    Ji B, Bi Y, Simeone D, Mortensen RM, Logsdon CD: Human pancreatic acinar cells lack functional responses to cholecystokinin and gastrin. Gastroenterology 121:1380-1390,2001

25.    Jordinson M, Deprez PH, Playford RJ, Heal S, Freeman TC, Alison M, Calam J: Soybean lectin stimulates pancreatic exocrine secretion via CCK-A receptors in rats. Am J Physiol 270:G653-9,1996

26.    Kawanami T, Suzuki S, Yoshida Y, Kanai S, Takata Y, Shimazoe T, Watanabe S, Funakoshi A, Miyasaka K: Different effects of trypsin inhibitors on intestinal gene expression of secretin and on pancreatic bicarbonate secretion in CCK-A-receptor-deficient rats. Jpn J Pharmacol 81:339-345,1999

27.    Kikuchi N, Nagata K, Yoshida N, Ogawa M: The multiplicity of human pancreatic secretory trypsin inhibitor. J Biochem 98:687-694,1985

28.    Lacourse KA, Swanberg LJ, Gillespie PJ, Rehfeld JF, Saunders TL, Samuelson LC: Pancreatic function in CCK-deficient mice: adaptation to dietary protein does not require CCK. Am J Physiol 276:G1302-1309,1999

29.    Lhoste EF, Roebuck BD, Longnecker DS: Stimulation of the growth of azaserine-induced nodules in the rat pancreas by dietary camostate (FOY-305). Carcinogenesis 9:901-906,1988

30.    Li Y, Owyang C: Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats. Gastroenterology 107:525-531,1994

31.    Li Y, Hao Y, Owyang C: High-affinity CCK-A receptors on the vagus nerve mediate CCK-stimulated pancreatic secretion in rats. Am J Physiol 273:G679-85,1997

32.    Li Y, Hao Y, Owyang C: Diazepam-binding inhibitor mediates feedback regulation of pancreatic secretion and postprandial release of cholecystokinin.. J Clin Invest 105:351-359,2000

33.    Liddle RA, Goldfine ID, Williams JA: Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology 87:542-549,1984

34.    Liddle RA, Misukonis MA, Pacy L, Balber AE: Cholecystokinin cells purified by fluorescence-activated cell sorting respond to monitor peptide with an increase in intracellular calcium. Proc Natl Acad Sci U S A 89:5147-5151,1992

35.    Liddle RA: Regulation of cholecystokinin secretion by intraluminal releasing factors. Am J Physiol 269:G319-27,1995

36.    Liener IE, Nitsan Z, Srisangnam C, Rackis JJ, Gumbmann MR: The USDA trypsin inhibitor study. II. Timed related biochemical changes in the pancreas of rats. Qual Plant Foods Hum Nutr 35:243-257,1985

37.    Longnecker DS: Interface between adaptive and neoplastic growth in the pancreas. Gut 28 Suppl:253-258,1987

38.    Louie DS, May D, Miller P, Owyang C: Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats. Am J Physiol 250:G252-9,1986

39.    Mack TM, Yu MC, Hanisch R, Henderson BE: Pancreas cancer and smoking, beverage consumption, and past medical history. J Natl Cancer Inst 76:49-60,1986

40.    McGuinness EE, Morgan RG, Levison DA, Frape DL, Hopwood D, Wormsley KG: The effects of long-term feeding of soya flour on the rat pancreas. Scand J Gastroenterol 15:497-502,1980

41.    McGuinness EE, Morgan RG, Wormsley KG: Effects of soybean flour on the pancreas of rats. Environ Health Perspect 56:205-212,1984

42.    McGuinness EE, Morgan RG, Wormsley KG: Trophic effects on the pancreas of trypsin and bile salt deficiency in the small-intestinal lumen. Scand J Gastroenterol Suppl 112:64-67,1985

43.    McGuinness EE, Wormsley KG: Effects of feeding partial and intermittent raw soya flour diets on the rat pancreas. Cancer Lett 32:73-81,1986

44.    McGuinness EE, Morgan RG, Wormsley KG: Fate of pancreatic nodules induced by raw soya flour in rats. Gut 28 Suppl:207-212,1987

45.    Miller RV: Epidemiology. Alan R. Liss, New York (pp) 39-57,1978

46.    Miyasaka K, Nakamura R, Funakoshi A, Kitani K: Stimulatory effect of monitor peptide and human pancreatic secretory trypsin inhibitor on pancreatic secretion and cholecystokinin release in conscious rats. Pancreas 4:139-144,1989

47.    Miyasaka K, Ohta M, Kanai S, Sato Y, Masuda M, Funakoshi A: Role of cholecystokinin (CCK)-A receptor for pancreatic growth after weaning: a study in a new rat model without gene expression of the CCK-A receptor. Pancreas 12:351-356,1996

48.    Miyasaka K, Ohta M, Masuda M, Funakoshi A: Retardation of pancreatic regeneration after partial pancreatectomy in a strain of rats without CCK-A receptor gene expression. Pancreas 14:391-399,1997

49.    Miyasaka K, Ohta M, Tateishi K, Jimi A, Funakoshi A: Role of cholecystokinin-A (CCK-A) receptor in pancreatic regeneration after pancreatic duct occlusion: a study in rats lacking CCK-A receptor gene expression. Pancreas 16:114-123,1998a

50.    Miyasaka K, Funakoshi A: Luminal feedback regulation, monitor peptide, CCK-releasing peptide, and CCK receptors. Pancreas 16:277-283,1998b

51.    Murphy JA, Criddle DN, Sherwood M, Chvanov M, Mukherjee R, McLaughlin E, Booth D, Gerasimenko JV, Raraty MG, Ghaneh P, Neoptolemos JP, Gerasimenko OV, Tepikin AV, Green GM, Reeve JR Jr, Petersen OH, Sutton R: Direct activation of cytosolic Ca2+ signaling and enzyme secretion by cholecystokinin in human pancreatic acinar cells.. Gastroenterology 135:632-641,2008

52.    Myer JR, Romach EH, Elangbam CS: Species- and dose-specific pancreatic responses and progression in single- and repeat-dose studies with GI181771X: a novel cholecystokinin 1 receptor agonist in mice, rats, and monkeys.. Toxicol Pathol 42:260-274,2014

53.    Oates PS, Morgan RG: Short-term effects of feeding raw soya flour on pancreatic cell turnover in the rat. Am J Physiol 247:G667-73,1984

54.    Owyang C, Louie DS, Tatum D: Feedback regulation of pancreatic enzyme secretion. Suppression of cholecystokinin release by trypsin.. J Clin Invest 77:2042-2047,1986

55.    Owyang C: Physiological mechanisms of cholecystokinin action on pancreatic secretion. Am J Physiol 271:G1-7,1996

56.    Owyang C: Discovery of a cholecystokinin-releasing peptide: biochemical characterization and physiological implications. Chin J Physiol 42:113-120,1999

57.    Pandiri AR: Overview of exocrine pancreatic pathobiology. Toxicol Pathol 42:207-216,2014

58.    Povoski SP, Zhou W, Longnecker DS, Jensen RT, Mantey SA, Bell RH Jr: Stimulation of in vivo pancreatic growth in the rat is mediated specifically by way of cholecystokinin-A receptors. Gastroenterology 107:1135-1146,1994

59.    Pubols MH, Bartelt DC, Greene LJ: Trypsin inhibitor from human pancreas and pancreatic juice. J Biol Chem 249:2235-2242,1974

60.    Rackis JJ: Physiological properties of soybean trypsin inhibitors and their relationship to pancreatic hypertrophy and growth inhibition of rats. Fed Proc 24:1488-1493,1965

61.    Rackis JJ, Gumbmann MR, Liener IE: The USDA trypsin inhibitor study. I. Background, objectives, and procedural details. Qual Plant Foods Hum Nutr 35:213-24,1985

62.    Rehfeld JF: Cholecystokinin-from local gut hormone to ubiquitous messenger. Front Endocrinol (Lausanne) 8:47,2017

63.    Savage GP, Morrison SC: Trypsin inhibitors. Elsevier (pp) 5878-5884,2003

64.    Singer MV, Niebergall-Roth E: Secretion from acinar cells of the exocrine pancreas: role of enteropancreatic reflexes and cholecystokinin. Cell Biol Int 33:1-9,2009

65.    Soudah HC, Lu Y, Hasler WL, Owyang C: Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway. Am J Physiol 263:G102-107,1992

66.    Spannagel AW, Green GM, Guan D, Liddle RA, Faull K, Reeve JR Jr: Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. Proc Natl Acad Sci U S A 93:4415-4420,1996

67.    Tarasova N, Spannagel AW, Green GM, Gomez G, Reed JT, Thompson JC, Hellmich MR, Reeve JR Jr, Liddle RA, Greeley GH Jr: Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract. Endocrinology 138:5550-5554,1997

68.    Tashiro M, Samuelson LC, Liddle RA, Williams JA: Calcineurin mediates pancreatic growth in protease inhibitor-treated mice. Am J Physiol Gastrointest Liver Physiol 286:G784-790,2004

69.    Tsuzuki S, Fushiki T, Kondo A, Murayama H, Sugimoto E: Effect of a high-protein diet on the gene expression of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas. Eur J Biochem 199:245-252,1991

70.    Tsuzuki S, Kondo A, Fushiki T, Sugimoto E: Monitor peptide gene expression is increased by exogenous CCK in the rat pancreas and in a rat pancreatic acinar cell line (AR4-2J). FEBS Lett 307:386-388,1992

71.    Tsuzuki S, Miura Y, Fushiki T, Oomori T, Satoh T, Natori Y, Sugimoto E: Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI). Biochim Biophys Acta 1132:199-202,1992

72.    Voet D, Voet JG: Biochemistry (2nd ed.). John Wiley & Sons (pp) 396-400,1995

73.    Wang BJ, Cui ZJ: How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to humans. Am J Physiol Regul Integr Comp Physiol 292:R666-78,2007

74.    Wang Y, Prpic V, Green GM, Reeve JR Jr, Liddle RA: Luminal CCK-releasing factor stimulates CCK release from human intestinal endocrine and STC-1 cells. Am J Physiol Gastrointest Liver Physiol 282:G16-22,2002

75.    Watanapa P, Williamson RC: Experimental pancreatic hyperplasia and neoplasia: effects of dietary and surgical manipulation. Br J Cancer 67:877-884,1993

76.    Woutersen RA, van Garderen-Hoetmer A, Lamers CB, Scherer E: Early indicators of exocrine pancreas carcinogenesis produced by non-genotoxic agents. Mutat Res 248:291-302,1991

77.    Yamamoto M, Otani M, Jia DM, Fukumitsu K, Yoshikawa H, Akiyama T, Otsuki M: Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats. Am J Physiol Gastrointest Liver Physiol 285:G681-687,2003

78.    Yamanishi R, Kotera J, Fushiki T, Soneda T, Iwanaga T, Sugimoto E: Characteristic and localization of the monitor peptide receptor. Biosci Biotechnol Biochem 57:1153-1156,1993

79.    Yanatori Y, Fujita T: Hypertrophy and hyperplasia in the endocrine and exocrine pancreas of rats fed soybean trypsin inhibitor or repeatedly injected with pancreozymin. Arch Histol Jpn 39:67-78,1976