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Relationship: 351

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Increased, Induced Mutations in Critical Genes leads to Increased, Clonal Expansion / Cell Proliferatin to form Pre-Neoplastic Altered Hepatic Foci

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/b-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression. In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help
Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

AFB1 induces G:C to T:A transversions. The third nucleotide in codon 249 of the p53 gene is a guanine and is a preferred binding target for AFB1 metabolites. In humans, some evidence exists that this codon is a mutational hot spot in liver cancers in people exposed to AFB1. (Puisieux et al., 1991). Although there is a large literature describing mutations in this location in humans and the corresponding p53 codon 247 in rodents, whether these mutations by themselves actually lead to tumors is unknown. (Lee et al., 1998) In vitro data collected from two human cell lines suggest that doses of AFB1 sufficient to induce DNA damage also induce an incomplete cell cycle checkpoint response, likely involving p53; this suggests a potential mechanism that might ultimately lead to tumor formation in vivo.(Gursoy-Yuzugullu et al., 2011). In rats, Ki-ras codon 12 mutations observed after AFB1 dosing may also be associated with hyperplastic foci to a greater extent than in hepatocellular carcinomas (HCCs), suggesting some change in the mutational spectrum during tumor development. (Tokusashi et al., 1994)

In general, the exact origin of cells that form altered hepatic foci (AHF) is not known with certainty. (Sell, 1993, 2002; Sell and Leffert, 2008). Four types of cells in the liver are thought to be capable of becoming tumor cells: 1) mature hepatocytes, 2) bile duct progenitor cells, 3) ductular bipolar progenitor cells, and 4) periductular stem cells. Pre-neoplastic foci and HCCs likely form from the last two cell types. [6] In rats, pre-neoplastic foci expressing the placental form of GST appear to be descended from hepatocytes. (Bralet et al., 2002; Avril et al., 2004; Gournay et al., 2002).

However, Sell (2008) has criticized the hypothesis the HCC arise by de-differentiation of hepatocytes and suggested the cellular origin of HCC may be liver stem cells undergoing an arrest of maturation. Trosko (2014) has considered de-differentiation in light of one of the emerging “Hallmarks of Cancer”—alterations of cellular metabolism—and argues that the likelihood of cancer arising through de-differentiation is vanishingly small. (Hanahan and Weinberg, 2011; Trosko and Kang, 2012; Trosko, 2013, 2014). The questions of the origin of the progenitor cells that replace hepatocytes after liver injury and the origin of liver cancer stem cells are the subjects of intense research. (Gong et al., 2013; Jia et al., 2013; Miyajima et al., 2014; Rehman et al., 2014; Shin and Kaestner, 2014; Tanimizu and Mitaka, 2014). The cyotokine IL-6 may be involved in the transformation of hepatic progenitor cells into cancer stem cells. (Dang et al., 2014; He et al., 2013). In addition, Dragan et al. (1994) observed that the increase in BrdU labeling, indicating cell proliferation, occurred to a greater extent in altered hepatic foci (AHF).

There is no direct evidence addressing AFB1 induced critical gene mutations and the subsequent induction of AHF. However, in general the progression from chemical exposure to pre-neoplastic lesions appears to include six general mechanisms: 1) decrease in the activity of the Keap1/Nrf2/ARE pathway; 2) decrease in p53 function leading to increased survival in the presence of genomic instability; 3) changes in the tumor microenvironment within cells; 4) alterations in apoptosis; 5) changes in Wnt/β-catenin signaling, and 6) gene expression changes that may be related to cancer promotion/progression (Miyajima et al., 2014; Gong et al., 2013; Jia et al., 2013; Dang et al., 2014; He et al., 2013; Caballero et al., 2004; Ma and He, 2012; Gross-Steinmeyer and Eaton, 2012; Liby et al., 2008; Yates et al., 2007; Liby and Sporn, 2012; Ikeda et al., 2004; Haridas et al., 2004). In addition to these six possible mechanisms, epigenetic changes in DNA methylation may occur and may be related to the progression of cancer (Kwak et al., 2001; Honda et al., 2011; Shelton and Jaiswal, 2013; Fabregat, 2009; Zhang et al., 2003).

The following is an overview of these 6 general biological processes that appear to be involved in the progression of mutant cells (whether occurring as the result of direct chemical induction or increased due other events such as cell proliferation and clonal expansion of spontaneously occurring mutant cells) to AHF and pre-neoplastic lesions.

Decrease in p53 function

A defective p53 gene may contribute to telomere dysfunction. Continuous hepatocyte renewal may be associated with telomere shortening and chromosomal instability [42-44]. In transgenic mice, telomere dysfunction enhanced tumor initiation but interfered with tumor progression/promotion. However, p53 mutation leading to inactivation of the gene, in combination with telomere dysfunction and increased hepatocyte turnover, may increase the risk of HCC (Zhang et al., 2006, 2012; Wu et al., 2013; Pogribny et al., 2011; Farazi et al., 2003).

The Tumor Microenvironment

Intracellular signaling mechanisms are involved in the creation of tumor microenvironment. (Hanahan and Weinberg, 2011). These include alterations in cellular circuits governing proliferation, differentiation, motility, and viability. The x gene is one of the four genes in the Hepatitis B Virus (HBV) genome that is capable of trans-activating cellular genes to contribute to the development of a tumor microenvironment (Brechot et al., 2000, 2010). The x gene can be expressed through stable transfection into LE/6 cells, a cell line derived from hepatic oval cells harvested from rats fed a choline-deficient diet. When LE/6 cells that were transfected with HBx and injected into nude mice administered a single dose of 240 μg/kg AFB1 by IP injection, or in the drinking water continuously for 16 weeks, the mice developed HCC from these rat liver stem cells, indicative of the stem cell-origin of HCC and suggesting that HBV and x expression may foster a tumor microenvironment.

TGF-β has a dual effect on hepatocytes—inhibiting proliferation and inducing apoptosis. Experiments using TGF-β null mutant mice show that this signal is required for normal development and appears to be involved in destruction of damaged cells in the liver. TGF-β 1 also mediates interferon-induced apoptosis in AHF in rats. In rats, AFB1 altered biological processes in hepatic stem-like cells related to cell motion, cell adhesion, immune response, and signal transduction (Yang et al., 2014). Reduced levels of TGF-β produce an increase in E-cadherin and reduce cellular motility, one of the intracellular networks associated with the cancer hallmark of invasion and metastasis (Fransvea et al., 2008).

Effects on Apoptotic Signaling

TGF-β normally inhibits growth and proliferation and induces apoptosis of damaged cells. In primary rat hepatocytes and a cell line (NRML) derived from these primary rat cells, TGF-β inhibited cell proliferation measured by DNA synthesis and the ability to form colonies. In vitro treatment of NRML cells with AFB1 blocked the TGF-β-mediated inhibition of cell growth and proliferation (McMahon et al., 1986). AFB1 transformed cells were also resistant to the growth-inhibiting effects of tumor necrosis factor-alpha (TNF-α) (Chapekar et al., 1989). However, BL9 cells, derived from rat hepatocytes transformed with reactive metabolites if activated AFB1, retained the sensitivity to the inhibitory effects of TGF-β on cell growth.

The product of the p53 gene mutated at codon 249 may itself impair the apoptotic process. In Hep3B human hepatoma cells, transfection with mutant p53 (p53mt249) increased transcription of insulin-like growth factor II (IGF-II). Wild-type p53 inhibited binding of the TATA-box binding protein (TBP) and the Sp1 transcription factors to the IGF-II promoter, possibly by binding to this promoter. However, p53mt249 increased the formation of transcription complexes, possibly due to lack of binding to the promoter. Chang liver cells show characteristics of hepatocytes; transfection with p53mt249 or addition of IGF-II blocked apoptosis mediated by TNF-α or the HBV x gene (Lee et al., 2000).

Adult tree shrews were administered AFB1 at 150 μg/kg/d five times weekly for 105 weeks. Interim sacrifices were performed every 30 weeks to observe liver histopathology, specifically immunohistochemical staining for p53, bcl-2, and bax, proteins in the p53 pathway that leads to apoptosis (Duan et al., 2005). Immunohistochemistry was also conducted to visualize survivin, a protein that acts directly on caspases to inhibit the activity of caspase-3 and caspase-7 to block the terminal pathway of apoptosis (Cheung et al., 2013). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptotic cells.

Bcl-2 and bax were observed in the livers of less than 10% of treated animals at 30 weeks. This result seems somewhat paradoxical because these proteins are intermediates in the p53 apoptotic pathway; however, the determinant of apoptotic progression may be the bcl-2 / bax ratio (Korsmeyer et al., 1993). The actions of survivin occur much later in the apoptotic pathway and this protein was detected in 40% of livers. Also, the apoptotic index measured by TUNEL was increased significantly at 30 weeks (Duan et al., 2005).

Wnt/β-catenin Signaling

Wnt/β-catenin signaling is a key determinant of liver cell proliferation and eventual differentiation to mature hepatocytes or cholangiocytes. This signaling cascade is generally inactive in healthy adult liver save for a small periportal region of a hepatic lobule. Hepatocytes in this region have constitutively active β-catenin that in turn regulates expression of genes associated with ammonia and xenobiotic metabolism (Monga, 2014). An increase in Wnt/β-catenin signaling appears to play a role in the development of hepatocellular cancers, and mutations in the β-catenin gene have been observed in HCCs from humans (Laurent-Puig and Zucman-Rossi, 2006; Devereux et al., 2001). In areas of high AFB1 exposure in China, β-catenin was overexpressed in a higher proportion of liver samples from HCC patients than in Malaysia where AFB1 exposure is lower (Chen Ban et al., 2004).

Paradoxically, AFB1 appears to increase microRNA-33a, which in turn down-regulates β-catenin (Fang et al., 2013). However, this study was conducted in human cancer cell lines and the signaling pathways in such cells may be considerably different than they are in vivo.

Genomic Changes in Response to AFB1

Administration of AFB1 to rat hepatic stem-like WB-F344 cells derived from oval cells produced changes in pathways related to TGF-β signaling, Wnt signaling, MAPK signaling, regulation of the cytoskeleton, cell adhesion, changes in energy metabolism, and ECM-receptor interaction (Yang et al., 2014). A number of these can be conceptually linked to the hallmarks of cancer (Hanahan and Weinberg, 2011). In another study, the AFB1-induced transcriptome, measured by RNA-Seq with increased resolution and sensitivity was compared to microarrays for pathways with differentially expressed genes, such as glutathione metabolism, cell cycle dysregulation, cancer pathways, Nrf2-mediated oxidative stress, and ten various intermediary and amino acid metabolism pathways (Merrick et al., 2013).

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

There is no direct information as to whether AFB1 induced mutations in critical genes leads to cellular proliferation and clonal expansion of mutant cells. It is known, however that the Hepatitis B virus plays a large role in the development of HCC in many endemic AFB1 regions, and viral infection is thought to interact with AFB1 exposure increasing the possibility that individual cells progress down the pathway to tumor formation. It seems reasonable that this infection may contribute to increased cell proliferation which could contribute to more clonal expansion (and, therefore, an increase in tumor formation) of AFB1-induced critical cancer gene mutant cells. It might also contribute to inflammation and increased cell proliferation which could result in an increased number of spontaneously mutant cells containing mutants in cancer critical genes (such as p53) thus also resulting in HCC (via a non-mutagenic MOA). This interaction between AFB1 and viral infection has motivated much research (Chittmittrapap et al., 2013; Farazi et al., 2006; Gouas et al., 2010; Kew 2003).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Time-scale
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help
Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

In mammals, the identification of AHF as a pre-neoplastic lesion has been recognized for many years as part of the general etiology of HCC (Beer and Pitot, 1987; Dragan et al., 1995; Goldsworthy et al., 1986; Pitot et al., 1990; Sargent et al., 1989). In rats, the presence of AHF with altered growth characteristics has been observed in a number of studies (Bannasch et al., 1985; Fischer, 1986; Fischer et al., 1987; Gil et al., 1988; Godlewski et al., 1985; Manson et al., 1984; Newberne, 1976; Nishizumi et al., 1977; Roebuck et al., 1991; Youngman and Campbell, 1992). The mechanisms involved in the formation of AHF appear to be generalizable across animals and likely apply to fish and birds as well as mammals.

References

List of the literature that was cited for this KER description. More help

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Brechot C., Kremsdorf D., Soussan P., Pineau P., Dejean A., Paterlini-Brechot P., Tiollais P.2010. Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC): molecular mechanisms and novel paradigms.. Pathol Biol (Paris). 58(4), 278-87 http://10.1016/j.patbio.2010.05.00

Caballero F, Meiss R, Gimenez A, Batlle A, and Vazquez E. Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol. 2004, Oct;85(4):213-22.

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