Aop: 316

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Trypsin inhibition leading to pancreatic acinar cell tumors

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
TI-induced AC tumors

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

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

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Arthur Author   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Shigeru Hisada
  • Arthur Author

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
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 April 05, 2021 18:16
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

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

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

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)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

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

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
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)

TESTINGThis table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
All life stages High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
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

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Mixed High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

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

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

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

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

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

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

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

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

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)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

TBD

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

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