Aop: 220

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

Cyp2E1 Activation Leading to Liver Cancer

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
Cyp2E1 Activation Leading to Liver Cancer

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

Francina Webster, Health Canada

Iain B. Lambert, Carleton University

Carole L. Yauk, University of Ottawa 

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
Francina Webster   (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
  • Carole Yauk
  • Francina Webster

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
Open for citation & comment WPHA/WNT Endorsed 1.24 Included in OECD Work Plan
This AOP was last modified on November 02, 2021 14:17
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
Activation of Cyp2E1 June 19, 2021 14:19
Oxidative Stress June 19, 2021 14:42
Hepatocytotoxicity June 19, 2021 14:52
Induction, persistent proliferation/sustained proliferation June 19, 2021 14:56
Liver Cancer June 19, 2021 15:00
Activation of Cyp2E1 leads to Oxidative Stress July 01, 2021 16:05
Oxidative Stress leads to Hepatocytotoxicity June 19, 2021 15:06
Hepatocytotoxicity leads to Sustained proliferation June 19, 2021 15:08
Activation of Cyp2E1 leads to Hepatocytotoxicity December 06, 2018 12:52
Oxidative Stress leads to Liver Cancer June 19, 2021 15:11
Hepatocytotoxicity leads to Liver Cancer May 01, 2020 12:35
Sustained proliferation leads to Liver Cancer October 09, 2020 12:21
>85 known Cyp2E1 substrates June 01, 2017 15:18

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

Cyp2E1 is a cytochrome P450 mono-oxygenase that bioactivates over 85 substrates, thereby creating electrophilic metabolites and oxidative stress. Substrates are low molecular weight compounds that include acetone, acetaminophen, ethanol, chloroform, carbon tetrachloride, furan and molecular oxygen. Mono-oxygenation of these substrates to their reactive metabolites, and the accompanying oxidative stress produced during metabolism, pose health risks because they lead to hepatotoxicity and, often, to liver cancer. Here we describe the AOP for the prolonged activation of Cyp2E1 (MIE) leading to liver cancer (AO). The intervening KEs are oxidative stress (KE1), hepatocytotoxicity (KE2), and sustained/persistent cellular proliferation (KE3). These events occur in the liver, which is the primary site of xenobiotic metabolism in the body. Briefly, the MIE occurs when Cyp2E1 binds a substrate. The Cyp2E1 catalytic cycle is prone to decoupling (adjacent KER1, non-adjacent KER1), which produces oxidative stress (KE1), and mono-oxidation of substrates produces reactive metabolites. Both reactive oxygen species and metabolites cause cytotoxicity (KE2). However, following injury, the liver is able to regenerate itself through an increase in cellular proliferation (KE3). Under conditions of chronic activation of Cyp2E1, excessive chronic increases in levels of reactive oxygen species and cell death, and subsequent dysregulated cellular proliferation, leads to tumour formation (AO). We evaluate the essentiality of the KEs and the biological plausibility of and empirical support for the KERs and report that most are well supported by a large body of scientific literature. Here, we’ve focused on data generated in rodent studies using the Cyp2E1 substrates carbon tetrachloride, chloroform, ethanol and furan. These compounds are all liver carcinogens, but generate negative or equivocal results in short-term genotoxicity tests. In fact, they are widely thought to cause cancer through a cytotoxicity and sustained/persistent proliferation mode of action. We expect that the data and information summarized here will be useful to scientists and regulators that are investigating chemical carcinogens that act through this mechanism. Given the importance of oxidative stress and cytotoxicity in a broad array of toxicological effects, the KE(R)s described should be broadly useful for development of other AOPs. Finally, this AOP describes an important widely acknowledged pathway to toxicity and thus should have many regulatory applications. Further development of the quantitative aspects of this AOP will enable the development of more predictive models of effects resulting from oxidative stress.

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

The subject of this AOP is xenobiotic metabolism by Cyp2E1 (MIE) during prolonged exposures, leading to liver cancer (AO). The intervening KEs are chronic oxidative stress, cytotoxicity, and regenerative proliferation. The setting for these events is the liver, which is the body’s primary venue for chemical detoxification.

Xenobiotic metabolism typically occurs in three phases: (I) the chemical substrate is enzymatically bio-activated to its primary metabolite; (II) the metabolite(s) produced is (are) made less reactive through conjugation; and (III) the modified chemical(s) is (are) excreted. Cyp2E1 is a phase I P450 monooxygenase that bio-activates its substrates through the addition of an oxygen, thereby producing an electrophilic metabolite. Acting as an electrophile following metabolic activation is a key characteristic of a carcinogen (Smith, et al. 2015). While this reactive species often undergoes conjugation (phase II metabolism), sometimes it will react with cellular nucleophiles (e.g., proteins or DNA), which results in formation of adducts that produce cytotoxicity in extreme cases. Another feature of Cyp2E1 is that its catalytic cycle is prone to uncoupling, which leads to the production of reactive oxygen species (ROS). ROS are an important source of cytotoxicity (e.g., via lipid peroxidation) and are a source of oxidative lesions to DNA (which may be a source of cancer-causing mutations) (Caro and Cederbaum 2004).  Redox-sensitive proteins are modified by oxidation; importantly, changes in gene expression are carried out by the redox-sensitive transcription factor Nrf2. Nrf2 increases the expression of genes that encode cyto-protective products, such as anti-oxidants and phase II conjugating enzymes (Furfaro, et al. 2016, Ma and He 2012, Sporn and Liby 2012, Tkachev, et al. 2011). At the same time, dying cells release pro-inflammatory signals and, together, these signals encourage regenerative proliferation of hepatocytes (Brenner, et al. 2013, Luedde, et al. 2014). However, when chronically activated, these molecular signals can produce dysregulated cellular proliferation in which the cytoprotective cellular mechanisms that are intended to promote tissue repair instead may lead to pre-malignant and malignant lesions.

This AOP explores these mechanisms in greater detail. Because exposure to Cyp2E1substrates is relatively common, this AOP will be an important tool for understanding the adverse health impacts of these potentially harmful substances. Cyp2E1 is well studied and is involved in the metabolism of a large number of substrates (Lieber 1997, Tanaka, et al. 2000), so it is impossible to summarize all of the evidence.  Therefore, we report illustrative studies that support each KE and KER. In addition, because no single study has looked at each key event, supporting evidence is gathered from many studies that have used a variety of in vitro and in vivo systems, as well as a collection of Cyp2E1 substrates. We focus on evidence gathered from: furan (a group 2B carcinogen), ethanol (group 1), chloroform (group 2B), and carbon tetrachloride (group 2B). These compounds are established Cyp2E1 substrates that are known to be rodent carcinogens and are (group 1) or are suspected (group 2B) human carcinogens based on their International Agency for Research on Carcinogens (IARC) evaluations.

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
1 MIE 1391 Activation of Cyp2E1 Activation of Cyp2E1
2 KE 1392 Oxidative Stress Oxidative Stress
3 KE 1393 Hepatocytotoxicity Hepatocytotoxicity
4 KE 1394 Induction, persistent proliferation/sustained proliferation Sustained proliferation
5 AO 1395 Liver Cancer Liver Cancer

Relationships Between Two Key Events (Including MIEs and AOs)

This 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
Title Adjacency Evidence Quantitative Understanding
Activation of Cyp2E1 leads to Hepatocytotoxicity non-adjacent High Not Specified
Oxidative Stress leads to Liver Cancer non-adjacent Moderate Not Specified
Hepatocytotoxicity leads to Liver Cancer non-adjacent Moderate Not Specified
Sustained proliferation leads to Liver Cancer non-adjacent Moderate Not Specified

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
Name Evidence Term
>85 known Cyp2E1 substrates High

Life Stage Applicability

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

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
rodents rodents High NCBI
Homo sapiens Homo sapiens 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

Biological plausibility:

Biological plausibility is based on fundamental understanding of the structural or functional relationship between the key events in the normal biological state. In general, there is high biological plausibility and coherence for the direct and (some of) the indirect relationships in this AOP. It is established that Cyp2E1 is stabilized upon substrate binding and generates ROS during metabolism. The link between ROS-induced lipid and DNA damage has been carefully mapped out, with a broad understanding of the spectrum of damage induced by ROS in a cell, and the signalling cascades induced that lead to cell death. There is extensive understanding that the liver regenerates following injury and cytotoxicity. Chronic toxicity would cause the liver to be undergoing increased cellular proliferation over a prolonged period of time in this tissue that, under normal circumstances, would have a relatively low mitotic index. There is a strong association, with some defined intervening steps, between liver regeneration and the probability of developing hepatocellular carcinoma, which is likely due to increased probability of incurring cancer-driver mutations with more DNA replication [e.g., tissues undergoing more cellular division have higher incidences of cancer (Tomasetti and Vogelstein 2015, Wu, et al. 2016)]. Moreover, chronic inflammation caused by increased and sustained levels of hepatotoxicity also contributes to increased probability of developing hepatocellular carcinoma. It is important to emphasize that the adverse effects observed are the product of chronic activation of Cyp2E1, which leads to sustained production of ROS, cytotoxicity and regenerative proliferation. A case study of this mode of action is presented in Meek et al (2003) using chloroform as an example. The case study describes ‘sustained cytotoxicity and regenerative cell proliferation’ as key events for a range of animal tumors, including for chloroform leading to liver tumours in mice. Thus, the overall biological plausibility for this AOP, especially in rodent models, is strong.

Time- and dose-response concordance:

Time- and dose-response concordance evaluation considers the available empirical data to determine if upstream events occur before downstream, and at lower or the same doses. A major assumption is that all KEs can be measured with equal precision and sensitivity. Overall, there is an extensive database of studies on Cyp2E1 substrates (furan, carbon tetrachloride, ethanol, etc.) that supports that the events occur in the correct order temporally, and that the upstream events occur at lower doses than the downstream events. Within each of the KERs, the example of furan is mapped out in detail, demonstrating the ability to measure increased levels of ROS and hepatotoxicity at lower doses than those causes liver regeneration and cancer.

Essentiality:

Essentiality refers to evidence that supports the idea that if a given KE is blocked or prevented, the downstream events in the sequence represented in the AOP will not occur (unless impacted by another pathway sharing those events).  In this AOP, there is strong evidence of essentiality of activation of Cyp2E1, with knock-out studies demonstrating that the downstream key events do not occur in the absence of this. For example, hydrogen peroxide production and lipid peroxidation are blocked in rat microsomes following inhibition of Cyp2E1 with anti-Cyp2E1 IgG (Ekstrom and Ingelman-Sundberg 1989). Cyp2E1 over-expressing HepG2-E47 cells have higher baseline levels of oxidative stress than wildtype HepG2 cells that do not express Cyp2E1. Moreover, ethanol-dependent lipid peroxidation can be prevented by treatment with Cyp2E1 inhibitors/antioxidants in Cyp2E1 over-expressing human HepG2 cells (Wu and Cederbaum 2005). Cyp2E1-null mice exposed to chloroform do not present with either hepatotoxicity or regenerative proliferation (Constan, et al. 1999). Chloroform-dependent hepatotoxicity and regenerative proliferation do not occur in Cyp2E1-null mice (Constan, et al. 1999). Blocking Cyp2E1 gene transcription (using the drug Bortezomib) blocks acetaminophen-, carbon tetrachloride-, and thioacetamide-dependent hepatotoxicity in a dose and time dependent manner (Park, et al. 2016).

Treatment with anti-oxidants to reduce oxidative stress reduces cytotoxicity and removal of antioxidants has the opposite effect. Key evidence to support the link between oxidative stress and cell death involves glutathione levels. Severity of cytotoxicity and levels of glutathione are inversely related (Smith, et al. 1979). Cyp2E1 over-expressing HepG2-E47 cells have higher baseline levels of oxidative stress than wildtype HepG2 cells that do not express Cyp2E1. For example, increasing cellular ROS through the depletion of thioredoxin or glutathione, or the addition of pro-oxidants in Cyp2E1-over-expressing E47 cells results in cell death (Cederbaum, et al. 2012, Yang, et al. 2011)(Wu and Cederbaum 2008). Ethanol-dependent hepatotoxicity in rats can be prevented by treatment with L-2-oxothiazolidine-4-carboxylic acid (OTC, a cysteine prodrug that maintains glutathione levels and thus reduces ROS) (Iimuro, et al. 2000). Ethanol-dependent lipid peroxidation can be prevented by treatment with antioxidants antioxidants in CYP2E1 over-expressing human HepG2 cells (Wu and Cederbaum 2005). Non-induced or phenobarbital-induced, glutathione-depleted mice treated with 0.5 ml/kg carbon tetrachloride exhibited increases in liver lipid peroxidation and significant elevation of liver-specific serum enzyme activities (Younes and Siegers 1985). In mice, pre-treatment with the iron-chelating agent desferrioxamine (DFO) suppressed lipid peroxidation and inhibited hepatotoxicity; whereas, depletion of glutathione exacerbated it (Younes and Siegers 1985). Primary rat hepatocytes exhibit a dose-dependent increase in thiobarbituric acid reactive substances (TBARS) and increased cytotoxicity following exposure to fumonisin B1 (FB1) (Abel and Gelderblom 1998). However, addition of the antioxidant alpha-tocopherol significantly decreases cytotoxicity and decreases TBARS to basal levels, supporting the essentiality of lipid peroxidation. Carbon tetrachloride is converted by Cyp2E1 to the trichloromethyl radical, which reacts with oxygen to form the trichloromethyl peroxy radical. The latter initiates lipid peroxidation, which is the main cause of carbon tetrachloride-dependent cytotoxicity (Kadiiska, et al. 2005, Manibusan, et al. 2007, Weber, et al. 2003). Lipid peroxidation has been shown to occur before liver injury and necrosis in rats (Hartley, et al. 1999). Inhibition of lipid peroxidation (using desferrioxamine, an iron chelator) prevents the associated hepatotoxicity; whereas, depletion of glutathione exacerbates it in mice (Younes and Siegers 1985). Another study that tested both carbon tetrachloride and chloroform found that cytotoxicity only occurred at doses at which glutathione was depleted in human HepG2 cells (Beddowes, et al. 2003). Male Wistar rats exposed for one month to 35% ethanol had abasic sites, Ogg1-sensitive sites, and increased expression of BER genes in liver DNA; this ROS-dependent DNA damage occurs at earlier time points than the corresponding carcinogenesis. Importantly, when this experiment was repeated in wild type, humanized Cyp2E1 (hCyp2E1), and Cyp2E1-null mice, wild type and hCyp2E1 mice had similar responses to ethanol: increased Cyp2E1 protein levels, increased expression of BER genes, and an increase in abasic sites; whereas, Cyp2E1-null mice had no oxidative or DNA damage phenotype (Bradford, et al. 2005).

Cytototoxicity is known (and needs) to occur before regenerative proliferation. Molecular signals that are released from dying cells trigger regenerative proliferation of existing cells. AP-1 (particularly the c-Jun monomer) and NF-kappaB are important transcription factors for this signaling pathway. Both are up-regulated following partial hepatectomy and are required for hepatic regeneration. Rodents lacking either of these transcription factors display impaired liver regeneration, often leading to death (Behrens, et al. 2002, Schrum, et al. 2000). C-Jun and NF-kappaB have also been shown to be required for normal liver development and loss of function in either molecule is embryonic lethal due to impaired liver development (Hilberg, et al. 1993, Jochum, et al. 2001, Rudolph, et al. 2000).   

Wild type and humanized Cype2E1 knock-in mice have dose-dependent increases in Cyp2E1 protein and activity levels when exposed to ethanol, whereas Cyp2E1 knock-out mice do not. Further, the humanized mice show the largest increases in necrosis, inflammation, Aspartate-aminotransferase (AST), Alanine-aminotransferase (ALT) and TBARS, and the largest decrease in glutathione (GSH) levels of all three groups (Lu, et al. 2010). 

 

Uncertainties, inconsistencies, and data gaps:

The current major uncertainty in this AOP is the mechanistic link between liver regeneration and cancer. There are also agents that are substrates of Cyp2E1 that do not cause liver cancer. For example, acetaminophen is a Cyp2E1 substrate that does not cause cancer (IARC group 3). However, it is a very strong hepatocytotoxicant and oxidant (Hinson, et al. 2010). The extreme cytotoxicity caused by high levels of acetaminophen leads to liver failure and death, which preclude liver regeneration or tumour development. In addition, Cyp2E1 is not uniquely responsible for producing ROS and inflammation in the liver. There are other well-known modes of action that can also produce these effects that may be occurring in parallel with this AOP. For example, ROS production was shown to be driven by inflammatory cytokines (as opposed to Cyp2E1) in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In this case, hepatocellular carcinoma was driven by the production of carcinogenic exocyclic etheno-DNA adducts (Linhart et al., 2015). 

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

This AOP is relevant to animals exposed chronically to chemicals that activate Cyp2E1. Thus, it is relevant during development and through to adulthood. In addition, cancer induced by chemicals thought to operate via this pathway affect both sexes. Studies to support it were conducted primarily in mouse, rat, rabbit, hamster, and immortalized human hepatoma cells.

While this AOP appears to be relevant in both sexes (the Moser et al. 2009 was done in female mice and the NTP 1993 cancer bioassay was done in both genders), a recent study has suggested that male mice might be more sensitive to the Cyp2E1-dependent oxidative stress causing cancer mode of action (Wang, et al. 2015). The ability of estrogen to inhibit IL-6 has been identified as an important factor (Naugler, et al. 2007). Gender differences in furan-dependent gene expression were also reported in furan-exposed rats (Dong, et al. 2015).

The evidence for this AOP is primarily derived from rodent models. Human cells in culture were also used in some investigations, demonstrating a link between ROS, cytotoxicity and genotoxicity. Humanized Cyp2E1 (hCyp2E1) mice have been used to demonstrate relevance to humans for progression from MIE and oxidative stress (Bradford, et al. 2005). There is an association between ROS and liver cancer in humans (Poungpairoj, et al. 2015, Wang, et al. 2016a). A variety of lines of evidence support the relationship between oxidative stress with the development and progression of hepatocellular carcinoma. For example, reduced superoxide dismutase 2 (an antioxidant gene) mRNA and protein expression is associated with mortality of hepatocellular carcinoma patients in a mutant p53-dependent manner (Wang, et al. 2016a). This decrease in expression is accompanied by decreased copy number of the gene in tumours, supporting a genetic basis for the molecular phenotype. Plasma protein carbonyl content (biomarker of oxidative stress) is significantly higher, whereas plasma Total Antioxidant Capacity (TAC) biomarker of antioxidant capacity) is significantly lower in Hepatocellular Carcinoma (HCC) patients than healthy controls (Poungpairoj, et al. 2015).

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

Studies in Cyp2E1 knockout mice include: carbon tetrachloride (Wong, et al. 1998), acetone (Bondoc, et al. 1999), benzene (Powley and Carlson 2001), thioacetamide (Chilakapati, et al. 2007), trichloroethylene (Kim and Ghanayem 2006), acrylonitrile (El Hadri, et al. 2005), urethane (Hoffler, et al. 2003, Hoffler and Ghanayem 2005), acetaminophen (Lee, et al. 1996, Zaher, et al. 1998), and ethanol (Bardag-Gorce, et al. 2000).

Cyp2E1 constitutive activation and inhibition in Sprague-Dawley rat liver in the context of diethylnitrosamine-induced hepatocarcinogenesis (DEN) exposure is described by Gao et al. (2018a, 2018b).

The effects of ethanol exposure on the liver are well studied. The role of chronic alcohol exposure leading to inflammation, oxidative stress and DNA damage, and cancer is reviewed by Song et al. (2019). The role of Cyp2E1 in ethanol metabolism leading to the production of ROS, which contribute to carcinogenesis, is explored in Seitz and Mueller (2019). The associated etheno DNA adducts are described in Mueller et al. (2018) and Peccerella et al. (2018).

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

Extent of Biological Plausibility of each KER

Defining question: Is there a mechanistic (e.g., structural or functional) relationship between KE-up and KE-down consistent with established biological knowledge?

Strong: Extensive understanding of the KER based on extensive previous documentation and broad acceptance (e.g., mutations leads to tumours); Established mechanistic basis Moderate: The KER is plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established Weak: there is empirical support for a statistical association between KEs, but the structural or functional relationship between them is not understood.

Table 1: Support for biological plausibility of KERs

Adjacent KER1 MIE-->KE1: Activation of Cyp2E1 leading to oxidative stress

Level of Support: Strong

Mechanism: It is well known that uncoupling of Cyp2E1 catalytic cycle results in the release of harmful reactive oxygen species in the cell.
Adjacent KER2 KE1-->KE2: Oxidative stress leading to cytotoxicity

Level of Support: Strong.

Mechanism: Cellular oxidative damage, especially by lipid peroxidation, leads to cell death.  The mechanisms linking these events are well defined.
Adjacent KER3 KE2-->KE3: Hepatotoxicity leading to sustained cellular proliferation

Level of Support: Strong.

Mechanism: It is well establised that liver cells will proliferate to replace dead cells following chemical or surgical injury.
Non-adjacent KER1 MIE-->KE2: Activation of Cyp2E1 leading to hepatotoxicity

Level of Support: Strong.

Mechanism: Metabolite-dependent toxicity is a well known side-effect of cytochrome P450 mono-oxygenase metabolism of xenobiotics in the liver.
Non-adjacent KER2 KE1-->AO: Oxidative stress leading to liver cancer

Level of Support: Moderate.

Mechanism: ROS-dependent DNA damage causing harmful mutations is known to occur. It is also well known that DNA mutations can lead to cancer. However, the mechanism by which the specific mutations generated in this context promote malignant transformation is incompletely understood.
Non-adjacent KER3 KE2-->AO: Hepatotoxicity leading to liver cancer

Level of Support: Moderate.

Mechanism: Cell death by necrosis and necroptosis produces damage-associated molecular patterns (DAMPs) that trigger inflammation. Inflammation is widely considered to be an important risk factor that sets the stage for malignant transformation; however, mechanistically, it is unclear how it does so.
Non-adjacent KER4

 KE3-->AO: Sustained cellular proliferation leading to liver cancer

Level of Support: Strong.

Mechanism: Highly dividing cells are at greater risk of obtaining and fixing a mutation. If appropriately placed in the genome, such a mutation can facilitate the malignant transformation of the cell.

Extent of Support for the Essentiality of each KE

Defining question: Are downstream KEs and/or the AO prevented is an upstream KE is blocked?

Strong: Direct evidence from

specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g., stop/reversibility studies, antagonism, knock out models, etc.)
Moderate: Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE (e.g., augmentation of proliferative response (KEup) leading to increase in KEdown or AO).

Weak: No or contradictory experimental evidence of the essentiality of any

of the KEs.

Table 2: Support for essentiality of KEs.

MIE: Activation of Cyp2E1. Strong. Refs.
MIE-->KE1: Hydrogen peroxide production and lipid peroxidation are blocked in rat microsomes following inhibition of Cyp2E1 with an anti-Cyp2E1 antibody. (Ekstrom and Ingelman-Sundberg 1989)
MIE-->KE1,KE2,KE3 (furan): ROS increase following furan exposure, which can be inhibited in a dose-dependent way by apigenin (a secondary plant pigment). Mouse lymphoma cells can tolerate exposure to furan at extremely high doses (up to 3100 uM) without experiencing cytotoxicity; however, cells experiences 50% mortality at much lower concentrations (50 uM) of furan’s primary metabolite, BDA. Therefore, cytotoxicity observed following exposure to furan is caused by BDA (not furan), which is produced by Cyp2E1. In addition, in vivo hepatotoxicity and cellular proliferation following furan exposure can be prevented by treatment with a cytochrome P450 inhibitor (ABT). (Fransson-Steen, et al. 1997, Kellert, et al. 2008, Wang, et al. 2014)
MIE-->KE1, KE2 (carbon tetrachloride): Cytotoxicity and lipid peroxidation are prevented in rats and mice by pre-treatment with cytochrome P450 inhibitors (colchicine or SKF-525A). Cytotoxicity is exacerbated in cell lines that over-express Cyp2E1. Wild-type mice exposed to carbon tetrachloride experience increases in hepatotoxicity and associated liver pathologies; these do not occur in Cyp2E1-null mice. (Bechtold, et al. 1982, Letteron, et al. 1990, Martinez, et al. 1995, Mourelle, et al. 1989, Takahashi, et al. 2002)
MIE-->KE2, KE3 (chloroform): Cyp2E1-null mice do not experience chloroform-dependent hepatotoxicity or subsequent increases in cellular proliferation. (Constan, et al. 1999)
KE1: Oxidative stress. Strong.  
KE1-->KE2 (carbon tetrachloride): Treatment of mice with an anti-oxidant (silymarin) prevents lipid peroxidation. Depletion of glutathione (by dithyl maleate, DEM) leads to an increase in lipid peroxidation in carbon tetrachloride fed rats. (Bechtold, et al. 1982, Letteron, et al. 1990)
KE1-->KE2, AO (ethanol): Levels of glutathione, ROS and lipid peroxidation are higher in HepG2 cells that stably over-express Cyp2E1 compared to wild-type HepG2 cells (that do not express Cyp2E1); glutathione depletion using buthionine sulfoximine (BSO), thioredoxin knock-down, or ethanol exposure in E47 cells results in elevated cytotoxicity, which does not occur in wild-type HepG2 cells. Apoptotic phenotype in ethanol treated HepG2-Cyp2E1 cells can be rescued by treatment with 4-MP (a cyp2e1 inhibitor), trolox (an antioxidant), or a caspase inhibitor. Rats exposed to ethanol present with time-dependent increases in cytotoxicity and inflammation, which can be blocked by treatment with OTC (a compound that sustains glutathione levels). Wild type and hCyp2E1 mice present with oxidative DNA adducts, which do not occur in Cyp2E1-null mice. (Bradford, et al. 2005, Iimuro, et al. 2000, Wu and Cederbaum 1996, Yang, et al. 2011)
KE1-->KE2 (chloroform): A study in rats showed that cytotoxicity only occurs at doses that are sufficient to deplete glutathione. (Beddowes, et al. 2003)
KE2: Hepatotoxicity  
Weak. We are not aware of any experiments that have specifically blocked cytotoxicity following chemical exposure.  
KE3: Sustained or persistent proliferation  
Moderate. It is well understood that cellular proliferation is a precursor to cancer; however, a better understanding of the molecular signals involved is required to experimentally demonstrate this using knock-down or knock-out models.  

Rodents lacking AP-1 or NF-kappaB display impaired liver regeneration, often leading to death.

In TNF receptor type 1 knockout mice and JNK-1 knockout mice, cellular proliferation was impaired, accompanying by decreased liver carcinogenesis (Knight et al., 2000; Hui et al., 2008).  In the JNK-1 knockout mice, genetic inactivation of p21 restored hepatocyte proliferation and also liver carcinogenesis (Hui et al. 2008). Conversely, there is evidence suggesting that sustained proliferation is not the only mechanism by which prenoplstic cells gain selective growth advantage in ther liver; for example, inhibition of cell loss/cell growth can also contribute to altered homeostasis (e.g., Melnick and Huff (1993)).

(Behrens, et al. 2002, Schrum, et al. 2000)

(Knight et al., 2000; Hui et al., 2008; Melnick and Huff, 1993).

Extent of Empirical Support for each KER

Defining question 1: Does the empirical evidence support that a change in KE-up leads to an appropriate change in KE-down? Does KE-up occur at a lower dose and earlier time-point than KE-down? Is the incidence of KE-up > than KE-down?

Defining question 2: Are there inconsistencies in the empirical support across taxa, species, and stressors that don’t align with the expected pattern for hypothesized AOP?

Strong: multiple studies showing dependent change in both events following exposure to a wide range of specific stressors (extensive evidence for temporal, dose-response, and incidence concordance) and or few critical data gaps or conflicting data

Moderate: Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among labs, etc. Weak: limited or no studies reporting dependent change in both events following exposure to a specific stressor (ie, endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesized AOP

Table 3: Empirical support for KERs.

Adjacent KER1 MIE-->KE1: Activation of Cyp2E1 leading to oxidative stress

Level of Support: Strong.

Defining question 1: There is extensive evidence in hepatic cell lines and rodent models that demonstrates that when Cyp2E1 is active there is an increase in oxidative stress, particularly lipid peroxidation. The doses at which the effects are measured are concordant. Further, when Cyp2E1 substrate is present, Cyp2E1 protein levels increase.

Defining question 2: There are no contradictions to the proposed KER in the literature.
Adjacent KER2 KE1-->KE2: Oxidative stress leading to hepatotoxicity

Level of Support: Strong.

Defining question 1: It is clear that oxidative stress and cytotoxicity are downstream of Cyp2E1 activation (occur later and at higher doses). It is also known that oxidative stress is harmful to cells and, in extreme cases, causes loss of cell viability.

Defining question 2: There are no contradictions to the proposed KER in the literature.
Adjacent KER3 KE2-->KE3: Hepatotoxicity leading to cellular proliferation

Level of Support: Strong.

Defining question 1: That hepatotoxicity leads to cellular proliferation has been demonstrated for a number of liver toxicants (as well as surgical resection of the liver). Increased regenerative proliferation occurs following toxicity, and at higher doses than the cytotoxicity.

Defining question 2: We are not aware of any instance in which an injured liver (that is genetically normal) will not regenerate itself.
Non-adjacent KER1 MIE-->KE2: Activation of Cyp2E1 leading to hepatotoxicity

Level of Support: Strong.

Defining question 1: There is a large amount of published data that demonstrate the cytotoxic effects of Cyp2E1 substrates following metabolic activation.

Defining question 2: While the prevailing opinion in the literature is that the toxicity of these metabolites is the result of non-genotoxic mechanisms, there are studies that argue in favour of direct genotoxic effects. It is widely thought that any observed genotoxicity is actually ‘indirect’ and is the product of oxidative stress.
Non-adjacent KER2 KE1-->AO: Oxidative stress leading to liver cancer

Level of Support: Weak.

Defining question 1: Carcinogens that cause cancer by ‘cytotoxicity and regenerative proliferation’ are generally accepted to be indirectly genotoxic. The most realistic source of indirect genotoxicity for these compounds are reactive oxygen species.

Defining question 2: An alternative mechanism—that is not mutually exclusive to the ‘defining question 1’—is that the transcriptional actions of chronic Nrf2 activation provide a molecular environment that promotes malignant transformation.
Non-adjacent KER3 KE2-->AO: Cytotoxicity leading to liver cancer

Level of Support: Moderate.

Defining question 1: Published studies support the idea that inflammation (caused by cellular necrosis and necroptosis) proceeds and somehow facilitates malignant transformation.

Defining question 2:

>That inflammation precedes liver cancer appears to be consistent across studies. The contradictory nature of NF-kappaB’s role in carcinogenesis remains under active investigation.

>This relationship appears to be valid for toxicants that produce moderate levels of cytotoxicity. Acetaminophen is a Cyp2E1 substrate that produces extremely high levels of hepatotoxicity. Acetaminophen does not cause liver cancer because death by liver failure occurs before cancer can develop.
Non-adjacent KER4 KE3-->AO: Sustained or persistent cellular proliferation leading to liver cancer

Level of Support: Moderate

Defining question 1: There is extensive evdience that an increase in cellular proliferation precedes tumour formation.

Defining question 2: Not all cases of increased cellular proliferation produce tumours (some simply regenerate the liver to its healthy form). Therefore, it is evident that malignant transformation is accompanied by perturbations in cellular signaling that ultimately impair tissue homeostasis and normal regenerative processes.

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

Degree of Quantitative Understanding of each KER

Dose-response, temporal and incidence concordance for furan in mouse (unless otherwise specified).
  MIE KE1 KE2 KE3 AO
Dose (mkd)          
In vitro

Studies in mouse,

rat and human

hepatocytes d,e

       
0.5    

-

(3 weeks) a

-

(3 weeks) a

 
1    

+

(3 weeks) a

-

(3 weeks) a

-

(2 years) a

2    

+

(3 weeks) a

-

(3 weeks) a

-

(2 years) a

4  

+

(4 days) g

(rat)

++

(3 weeks) a

-

(3 weeks) a

+

(2 years) a

8  

++

(4 days) g

(rat)

---------

+

(7 days) c

+++

(3 weeks) a

+

(3 weeks) a

------------

+++

(2 years) b

++ b

+++ a

(2 years)

12  

++

(4 days) g

(rat)

     
15  

++

(4 days) g

(rat; 16 mkd)

+

(90 days) b

-----

+++

(2 years) b

-

(90 days) b

-----

+++

(2 years) b

+++

(2 years) b

30

++

(8hr, 1day) f

(rat)

++

(8hr) f

(rat)

+++

(90 days) b

++

(90 days) b

 
60    

+++

(90 days) b

+++

(90 days) b

 

Studies: a (Moser, et al. 2009); b (NTP 1993); c (Wang, et al. 2014); d (Kedderis, et al. 1993); e (Kedderis and Held 1996); f (Hickling, et al. 2010); g (Ding, et al. 2012).  

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

The events described in this AOP will be useful to scientists and regulators who are interested in non- or indirectly genotoxic compounds that cause liver cancer through the cytotoxicity and sustained/persistent cellular proliferation mode of action. This group of compounds is challenging to assess because they produce negative or equivocal results in short-term genotoxicity tests, which are typically used as a first-pass screen for carcinogenicity. Therefore, this AOP that describes sets of assays that can be used to determine if compound acts via this mode of action and evaluate the weight of evidence. HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumours, combined with other liver tumours, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route (USEPA, 2014).

References

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

Abel, S., Gelderblom, W.C., 1998. Oxidative damage and fumonisin B1-induced toxicity in primary rat hepatocytes and rat liver in vivo. Toxicology 131, 121-131.

Bardag-Gorce, F., Yuan, Q.X., Li, J., French, B.A., Fang, C., Ingelman-Sundberg, M., French, S.W., 2000. The effect of ethanol-induced cytochrome p4502E1 on the inhibition of proteasome activity by alcohol. Biochem. Biophys. Res. Commun. 279, 23-29.

Bechtold, M.M., Gee, D.L., Bruenner, U., Tappel, A.L., 1982. Carbon tetrachloride-mediated expiration of pentane and chloroform by the intact rat: the effects of pretreatment with diethyl maleate, SKF-525A and phenobarbital. Toxicol. Lett. 11, 165-171.

Beddowes, E.J., Faux, S.P., Chipman, J.K., 2003. Chloroform, carbon tetrachloride and glutathione depletion induce secondary genotoxicity in liver cells via oxidative stress. Toxicology 187, 101-115.

Behrens, A., Sibilia, M., David, J.P., Möhle-Steinlein, U., Tronche, F., Schütz, G., Wagner, E.F., 2002. Impaired postnatal hepatocyte proliferation and liver regeneration in mice lacking c-jun in the liver. EMBO J. 21, 1782-1790.

Bondoc, F.Y., Bao, Z., Hu, W.Y., Gonzalez, F.J., Wang, Y., Yang, C.S., Hong, J.Y., 1999. Acetone catabolism by cytochrome P450 2E1: studies with CYP2E1-null mice. Biochem. Pharmacol. 58, 461-463.

Bradford, B.U., Kono, H., Isayama, F., Kosyk, O., Wheeler, M.D., Akiyama, T.E., Bleye, L., Krausz, K.W., Gonzalez, F.J., Koop, D.R., Rusyn, I., 2005. Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver. Hepatology 41, 336-344.

Brenner, C., Galluzzi, L., Kepp, O., Kroemer, G., 2013. Decoding cell death signals in liver inflammation. J. Hepatol. 59, 583-594.

Caro, A.A., Cederbaum, A.I., 2004. Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu. Rev. Pharmacol. Toxicol. 44, 27-42.

Cederbaum, A.I., Yang, L., Wang, X., Wu, D., 2012. CYP2E1 Sensitizes the Liver to LPS- and TNF alpha-Induced Toxicity via Elevated Oxidative and Nitrosative Stress and Activation of ASK-1 and JNK Mitogen-Activated Kinases. Int. J. Hepatol. 2012, 582790.

Chilakapati, J., Korrapati, M.C., Shankar, K., Hill, R.A., Warbritton, A., Latendresse, J.R., Mehendale, H.M., 2007. Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital. Toxicol. Appl. Pharmacol. 219, 72-84.

Constan, A.A., Sprankle, C.S., Peters, J.M., Kedderis, G.L., Everitt, J.I., Wong, B.A., Gonzalez, F.L., Butterworth, B.E., 1999. Metabolism of chloroform by cytochrome P450 2E1 is required for induction of toxicity in the liver, kidney, and nose of male mice. Toxicol. Appl. Pharmacol. 160, 120-126.

Ding, W., Petibone, D.M., Latendresse, J.R., Pearce, M.G., Muskhelishvili, L., White, G.A., Chang, C.-., Mittelstaedt, R.A., Shaddock, J.G., McDaniel, L.P., Doerge, D.R., Morris, S.M., Bishop, M.E., Manjanatha, M.G., Aidoo, A., Heflich, R.H., 2012. In vivo genotoxicity of furan in F344 rats at cancer bioassay doses. Toxicol. Appl. Pharmacol. 261, 164-171.

Dong, H., Gill, S., Curran, I.H., Williams, A., Kuo, B., Wade, M.G., Yauk, C.L., 2015. Toxicogenomic assessment of liver responses following subchronic exposure to furan in Fischer F344 rats. Arch. Toxicol.

Ekstrom, G., Ingelman-Sundberg, M., 1989. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). Biochem. Pharmacol. 38, 1313-1319.

El Hadri, L., Chanas, B., Ghanayem, B.I., 2005. Comparative metabolism of methacrylonitrile and acrylonitrile to cyanide using cytochrome P4502E1 and microsomal epoxide hydrolase-null mice. Toxicol. Appl. Pharmacol. 205, 116-125.

Fransson-Steen, R., Goldsworthy, T.L., Kedderis, G.L., Maronpot, R.R., 1997. Furan-induced liver cell proliferation and apoptosis in female B6C3F1 mice. Toxicology 118, 195-204.

Furfaro, A.L., Traverso, N., Domenicotti, C., Piras, S., Moretta, L., Marinari, U.M., Pronzato, M.A., Nitti, M., 2016. The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance. Oxid Med. Cell. Longev 2016, 1958174.

Gao J, Wang Z, Wang GJ, Zhang HX, Gao N, Wang J, Wang CE, Chang Z, Fang Y, Zhang YF, Zhou J, Jin H, Qiao HL. 2018a. Higher CYP2E1 Activity Correlates with Hepatocarcinogenesis Induced by Diethylnitrosamine. J Pharmacol Exp Ther. 365(2):398-407.

Gao J, Wang Z, Wang GJ, Gao N, Li J, Zhang YF, Zhou J, Zhang HX, Wen Q, Jin H, Qiao HL. 2018b. Hepatofibrosis to hepatocarcinogenesis: Higher cytochrome P450 2E1  activity is a potential risk factor. Mol Carcinog. 57(10):1371-1382.

Hartley, D.P., Kolaja, K.L., Reichard, J., Petersen, D.R., 1999. 4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: immunochemical detection and lobular localization. Toxicol. Appl. Pharmacol. 161, 23-33.

Hickling, K.C., Hitchcock, J.M., Oreffo, V., Mally, A., Hammond, T.G., Evans, J.G., Chipman, J.K., 2010. Evidence of oxidative stress and associated DNA damage, increased proliferative drive, and altered gene expression in rat liver produced by the cholangiocarcinogenic agent Furan. Toxicol. Pathol. 38, 230-243.

Hilberg, F., Aguzzi, A., Howells, N., Wagner, E.F., 1993. c-Jun is essential for normal mouse development and hepatogenesis. Nature 365, 179-181.

Hinson, J.A., Roberts, D.W., James, L.P., 2010. Mechanisms of acetaminophen-induced liver necrosis. Handb. Exp. Pharmacol. (196):369-405. doi, 369-405.

Hoffler, U., El-Masri, H.A., Ghanayem, B.I., 2003. Cytochrome P450 2E1 (CYP2E1) is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-null and wild-type mice. J. Pharmacol. Exp. Ther. 305, 557-564.

Hoffler, U., Ghanayem, B.I., 2005. Increased bioaccumulation of urethane in CYP2E1-/- versus CYP2E1+/+ mice. Drug Metab. Dispos. 33, 1144-1150.

Hui L, Zatloukal K, Scheuch H, Stepniak E, Wagner EF. 2008. Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation. J Clin Invest. 118(12):3943-53.

Iimuro, Y., Bradford, B.U., Yamashina, S., Rusyn, I., Nakagami, M., Enomoto, N., Kono, H., Frey, W., Forman, D., Brenner, D., Thurman, R.G., 2000. The glutathione precursor L-2-oxothiazolidine-4-carboxylic acid protects against liver injury due to chronic enteral ethanol exposure in the rat. Hepatology 31, 391-398.

Jochum, W., Passegué, E., Wagner, E.F., 2001. AP-1 in mouse development and tumorigenesis. Oncogene 20, 2401-2412.

Kadiiska, M.B., Gladen, B.C., Baird, D.D., Germolec, D., Graham, L.B., Parker, C.E., Nyska, A., Wachsman, J.T., Ames, B.N., Basu, S., Brot, N., Fitzgerald, G.A., Floyd, R.A., George, M., Heinecke, J.W., Hatch, G.E., Hensley, K., Lawson, J.A., Marnett, L.J., Morrow, J.D., Murray, D.M., Plastaras, J., Roberts, L.J.,2nd, Rokach, J., Shigenaga, M.K., Sohal, R.S., Sun, J., Tice, R.R., Van Thiel, D.H., Wellner, D., Walter, P.B., Tomer, K.B., Mason, R.P., Barrett, J.C., 2005. Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic. Biol. Med. 38, 698-710.

Kedderis, G.L., Carfagna, M.A., Held, S.D., Batra, R., Murphy, J.E., Gargas, M.L., 1993. Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro. Toxicology and applied pharmacology 123, 274-282.

Kedderis, G.L., Held, S.D., 1996. Prediction of furan pharmacokinetics from hepatocyte studies: comparison of bioactivation and hepatic dosimetry in rats, mice, and humans. Toxicology and applied pharmacology 140, 124-30.

Kellert, M., Brink, A., Richter, I., Schlatter, J., Lutz, W.K., 2008. Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells. Mutation research 657, 127-32.

Kim, D., Ghanayem, B.I., 2006. Comparative metabolism and disposition of trichloroethylene in Cyp2e1-/-and wild-type mice. Drug Metab. Dispos. 34, 2020-2027.

Knight B, Yeoh GC, Husk KL, Ly T, Abraham LJ, Yu C, Rhim JA, Fausto N. 2000. Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice. J Exp Med. 192(12):1809-18.

Lee, S.S., Buters, J.T., Pineau, T., Fernandez-Salguero, P., Gonzalez, F.J., 1996. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J. Biol. Chem. 271, 12063-12067.

Letteron, P., Labbe, G., Degott, C., Berson, A., Fromenty, B., Delaforge, M., Larrey, D., Pessayre, D., 1990. Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant. Biochem. Pharmacol. 39, 2027-2034.

Lieber, C.S., 1997. Cytochrome P-4502E1: its physiological and pathological role. Physiol. Rev. 77, 517-544.

Linhart KB, Glassen K, Peccerella T, Waldherr R, Linhart H, Bartsch H, Seitz HK. The generation of carcinogenic etheno-DNA adducts in the liver of patients with nonalcoholic fatty liver disease. Hepatobiliary Surg Nutr. 2015 Apr;4(2):117-23. doi: 10.3978/j.issn.2304-3881.2015.01.14. PMID: 26005678; PMCID: PMC4405412.

Luedde, T., Kaplowitz, N., Schwabe, R.F., 2014. Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterology 147, 765-783.e4.

Ma, Q., He, X., 2012. Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2. Pharmacol. Rev. 64, 1055-1081.

Manibusan, M.K., Odin, M., Eastmond, D.A., 2007. Postulated carbon tetrachloride mode of action: A review. Journal of Environmental Science and Health - Part C Environmental Carcinogenesis and Ecotoxicology Reviews 25, 185-209.

Martinez, M., Mourelle, M., Muriel, P., 1995. Protective effect of colchicine on acute liver damage induced by CCl4. Role of cytochrome P-450. J. Appl. Toxicol. 15, 49-52.

Meek, M,.E., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman-McKeeman, L.D., Longfellow, D.G., Pastoor, T., Seed, J., Patton, D.E. 2003, A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action, Critical Reviews in Toxicology, 33:6, 591-653.

Melnick RL, Huff J. 1993. Liver carcinogenesis is not a predicted outcome of chemically induced hepatocyte proliferation. Toxicol Ind Health. 9(3):415-38.

Moser, G.J., Foley, J., Burnett, M., Goldsworthy, T.L., Maronpot, R., 2009. Furan-induced dose–response relationships for liver cytotoxicity, cell proliferation, and tumorigenicity (furan-induced liver tumorigenicity). Experimental and Toxicologic Pathology 61, 101-111.

Mourelle, M., Fraginals, R., Rodriguez, L., Favari, L., Perez-Alvarez, V., 1989. Protective effect of colchiceine against acute liver damage. Life Sci. 45, 891-900.

Mueller S, Peccerella T, Qin H, Glassen K, Waldherr R, Flechtenmacher C, Straub BK, Millonig G, Stickel F, Bruckner T, Bartsch H, Seitz HK. 2018. Carcinogenic Etheno DNA Adducts in Alcoholic Liver Disease: Correlation with Cytochrome P-4502E1 and Fibrosis. Alcohol Clin Exp Res. 42(2):252-259.

Naugler, W.E., Sakurai, T., Kim, S., Maeda, S., Kim, K., Elsharkawy, A.M., Karin, M., 2007. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 317, 121-124.

Park, W.J., Kim, S.Y., Kim, Y.R., Park, J.W., 2016. Bortezomib alleviates drug-induced liver injury by regulating CYP2E1 gene transcription. Int. J. Mol. Med. 37, 613-622.

Peccerella T, Arslic-Schmitt T, Mueller S, Linhart KB, Seth D, Bartsch H, Seitz HK. 2018. Chronic Ethanol Consumption and Generation of Etheno-DNA Adducts in Cancer-Prone Tissues. Adv Exp Med Biol. 1032:81-92.

Poungpairoj, P., Whongsiri, P., Suwannasin, S., Khlaiphuengsin, A., Tangkijvanich, P., Boonla, C., 2015. Increased Oxidative Stress and RUNX3 Hypermethylation in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma (HCC) and Induction of RUNX3 Hypermethylation by Reactive Oxygen Species in HCC Cells. Asian Pac. J. Cancer. Prev. 16, 5343-5348.

Powley, M.W., Carlson, G.P., 2001. Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice. Toxicology 169, 187-194.

Rudolph, D., Yeh, W.-., Wakeham, A., Rudolph, B., Nallainathan, D., Potter, J., Elia, A.J., Mak, T.W., 2000. Severe liver degeneration and lack of NF-κB activation in NEMO/IKK γ- deficient mice. Genes and Development 14, 854-862.

Schrum, L.W., Black, D., Iimuro, Y., Rippe, R.A., Brenner, D.A., Behrns, K.E., 2000. c-Jun does not mediate hepatocyte apoptosis following NFκB inhibition and partial hepatectomy. J. Surg. Res. 88, 142-149.

Seitz HK, Mueller S. 2019. [The role of Cytochrom P4502E1 in Alcoholic Liver Disease and alcohol mediated carcinogenesis]. Z Gastroenterol. 57(1):37-45.

Smith, M.T., Guyton, K.Z., Gibbons, C.F., Fritz, J.M., Portier, C.J., Rusyn, I., DeMarini, D.M., Caldwell, J.C., Kavlock, R.J., Lambert, P., Hecht, S.S., Bucher, J.R., Stewart, B.W., Baan, R., Cogliano, V.J., Straif, K., 2015. Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis. Environ. Health Perspect.

Smith, M.T., Loveridge, N., Wills, E.D., Chayen, J., 1979. The distribution of glutathione in the rat liver lobule. Biochem. J. 182, 103-108.

Sporn, M.B., Liby, K.T., 2012. NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer. 12, 564-571.

Song BJ, Abdelmegeed MA, Cho YE, Akbar M, Rhim JS, Song MK, Hardwick JP. 2019. Contributing Roles of CYP2E1 and Other Cytochrome P450 Isoforms in Alcohol-Related Tissue Injury and Carcinogenesis. Adv Exp Med Biol. 1164:73-87.

Takahashi, S., Takahashi, T., Mizobuchi, S., Matsumi, M., Morita, K., Miyazaki, M., Namba, M., Akagi, R., Hirakawa, M., 2002. Increased cytotoxicity of carbon tetrachloride in a human hepatoma cell line overexpressing cytochrome P450 2E1. J. Int. Med. Res. 30, 400-405.

Tanaka, E., Terada, M., Misawa, S., 2000. Cytochrome P450 2E1: its clinical and toxicological role. J. Clin. Pharm. Ther. 25, 165-175.

Tkachev, V.O., Menshchikova, E.B., Zenkov, N.K., 2011. Mechanism of the Nrf2/Keap1/ARE signaling system. Biochemistry (Mosc) 76, 407-422.

Tomasetti, C., Vogelstein, B., 2015. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 347, 78-81.

Wang, E., Chen, F., Hu, X., Yuan, Y., 2014. Protective effects of apigenin against furan-induced toxicity in mice. Food Funct. 5, 1804-1812.

Wang, S., Sugamori, K.S., Tung, A., McPherson, J.P., Grant, D.M., 2015. N-Hydroxylation of 4-Aminobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse Model of Chemically Induced Liver Cancer. Toxicol. Sci.

Wu, D., Cederbaum, A.I., 2008. Development and properties of HepG2 cells that constitutively express CYP2E1. Methods Mol. Biol. 447, 137-150.

Wu, D., Cederbaum, A.I., 2005. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1. Toxicol. Appl. Pharmacol. 207, 70-76.

Wu, D., Cederbaum, A.I., 1996. Ethanol cytotoxicity to a transfected HepG2 cell line expressing human cytochrome P4502E1. J. Biol. Chem. 271, 23914-23919.

Wu, S., Powers, S., Zhu, W., Hannun, Y.A., 2016. Substantial contribution of extrinsic risk factors to cancer development. Nature 529, 43-47.

Yang, L., Wu, D., Wang, X., Cederbaum, A.I., 2011. Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells. Free Radic. Biol. Med. 51, 185-196.

Younes, M., Siegers, C.P., 1985. The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity. Chem. Biol. Interact. 55, 327-334.

Zaher, H., Buters, J.T., Ward, J.M., Bruno, M.K., Lucas, A.M., Stern, S.T., Cohen, S.D., Gonzalez, F.J., 1998. Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol. Appl. Pharmacol. 152, 193-199.