This AOP is licensed under the BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

AOP: 220


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the 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 name 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 representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool


The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Francina Webster, Health Canada

Iain B. Lambert, Carleton University

Carole L. Yauk, University of Ottawa 

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Francina Webster   (email point of contact)


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Carole Yauk
  • Francina Webster


This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help


Provides 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. OECD Status - Tracks the level of review/endorsement the AOP has been subjected to. OECD Project Number - Project number is designated and updated by the OECD. SAAOP Status - Status managed and updated by SAAOP curators. More help
Handbook Version OECD status OECD project
v1.0 WPHA/WNT Endorsed 1.24
This AOP was last modified on April 29, 2023 16:03

Revision dates for related pages

Page Revision Date/Time
Activation of Cyp2E1 June 19, 2021 14:19
Oxidative Stress March 21, 2023 15:16
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


A concise and informative summation of the AOP under development that can stand-alone from the AOP page. 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.

AOP Development Strategy


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. 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.


Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

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


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 1391 Activation of Cyp2E1 Activation of Cyp2E1
KE 1392 Oxidative Stress Oxidative Stress
KE 1393 Hepatocytotoxicity Hepatocytotoxicity
KE 1394 Induction, persistent proliferation/sustained proliferation Sustained proliferation
AO 1395 Liver Cancer Liver Cancer

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes 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. 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

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (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

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP 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. More help
Term Scientific Term Evidence Link
rodents rodents High NCBI
Homo sapiens Homo sapiens Moderate NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Mixed High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). 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 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

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. 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

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. 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

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. 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.

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

Degree of Quantitative Understanding of each KER

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

Studies in mouse,

rat and human

hepatocytes d,e



(3 weeks) a


(3 weeks) a



(3 weeks) a


(3 weeks) a


(2 years) a



(3 weeks) a


(3 weeks) a


(2 years) a



(4 days) g



(3 weeks) a


(3 weeks) a


(2 years) a



(4 days) g




(7 days) c


(3 weeks) a


(3 weeks) a



(2 years) b

++ b

+++ a

(2 years)



(4 days) g




(4 days) g

(rat; 16 mkd)


(90 days) b



(2 years) b


(90 days) b



(2 years) b


(2 years) b



(8hr, 1day) f



(8hr) f



(90 days) b


(90 days) b



(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)

Addressess 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. 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).


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

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