API

Aop: 220

AOP Title

?


Chronic Cyp2E1 Activation Leading to Liver Cancer

Short name:

?

Chronic Cyp2E1 Activation Leading to Liver Cancer

Authors

?


Francina Webster, Health Canada

Iain B. Lambert, Carleton University

Carole L. Yauk, Health Canada

Point of Contact

?


Carole Yauk

Contributors

?


  • Carole Yauk

Status

?

Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.24 Included in OECD Work Plan


This AOP was last modified on November 17, 2017 05:20

?

Revision dates for related pages

Page Revision Date/Time
Activation of Cyp2E1 in the liver May 30, 2017 13:52
Oxidative Stress May 30, 2017 14:02
Hepatocytotoxicity May 30, 2017 14:55
Hepatocellular Regenerative Proliferation May 30, 2017 14:56
Liver Cancer May 30, 2017 14:42
Activation of Cyp2E1 in the liver leads to Oxidative Stress May 30, 2017 15:01
Oxidative Stress leads to Hepatocytotoxicity May 30, 2017 15:04
Hepatocytotoxicity leads to Hepatocellular Regenerative Proliferation May 30, 2017 15:09
Activation of Cyp2E1 in the liver leads to Hepatocytotoxicity May 30, 2017 15:13
Oxidative Stress leads to Liver Cancer May 30, 2017 15:17
Hepatocytotoxicity leads to Liver Cancer May 30, 2017 15:21
Hepatocellular Regenerative Proliferation leads to Liver Cancer May 30, 2017 15:24
>85 known Cyp2E1 substrates June 01, 2017 15:18

Abstract

?


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 chronic activation of Cyp2E1 (MIE) leading to liver cancer (AO). The intervening KEs are oxidative stress (KE1), cytotoxicity (KE2), and regenerative 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, 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 regenerative 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)

?


The subject of this AOP is xenobiotic metabolism by Cyp2E1 (MIE) during chronic 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

?



Stressors

?

Name Evidence Term
>85 known Cyp2E1 substrates Strong

Molecular Initiating Event

?

Title Short name
Activation of Cyp2E1 in the liver Activation of Cyp2E1 in the liver

Key Events

?

Title Short name
Oxidative Stress Oxidative Stress
Hepatocytotoxicity Hepatocytotoxicity
Hepatocellular Regenerative Proliferation Hepatocellular Regenerative Proliferation

Adverse Outcome

?

Title Short name
Liver Cancer Liver Cancer

Relationships Between Two Key Events (Including MIEs and AOs)

?

Title Directness Evidence Quantitative Understanding
Activation of Cyp2E1 in the liver leads to Oxidative Stress Directly leads to Strong Not Specified
Oxidative Stress leads to Hepatocytotoxicity Directly leads to Strong Not Specified
Hepatocytotoxicity leads to Hepatocellular Regenerative Proliferation Directly leads to Strong Not Specified
Activation of Cyp2E1 in the liver leads to Hepatocytotoxicity Indirectly leads to Strong Not Specified
Oxidative Stress leads to Liver Cancer Indirectly leads to Moderate Not Specified
Hepatocytotoxicity leads to Liver Cancer Indirectly leads to Moderate Not Specified
Hepatocellular Regenerative Proliferation leads to Liver Cancer Indirectly leads to Moderate Not Specified

Network View

?

 

Life Stage Applicability

?


Taxonomic Applicability

?


Sex Applicability

?


Graphical Representation

?

Click to download graphical representation template

W1siziisijiwmtcvmduvmzavodbinzvmc2pzy19bt1aymjbfr3jhcghpyy5qcgcixsxbinailcj0ahvtyiisijuwmhg1mdaixv0?sha=801441b2cccb47d4

Overall Assessment of the AOP

?



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. 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. Ethanol-dependent lipid peroxidation can be prevented by treatment with Cyp2E1 inhibitors (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 OTC (a prodrug that maintains glutathione levels and thus reduces ROS) (Iimuro, et al. 2000). Ethanol-dependent lipid peroxidation can be prevented by treatment with antioxidants (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). 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 TBARS and increased cytotoxicity following exposure to 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 (Hartley, et al. 1999). Inhibition of lipid peroxidation (using desferrioxamine, an iron chelator) prevents the associated hepatotoxicity; whereas, depletion of glutathione exacerbates it (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 (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).   

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 .  

Domain of Applicability

?

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 TAC (biomarker of antioxidant capacity) is significantly lower in HCC patients than healthy controls (Poungpairoj, et al. 2015).


Essentiality of the Key Events

?

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).


Weight of Evidence Summary

?

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

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.
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.
KER3 KE2-->KE3: Cytotoxicity leading to regenerative proliferation

Level of Support: Strong.

Mechanism: The liver is well known to regenerate itself following chemical or surgical injury.
iKER1 MIE-->KE2: Activation of Cyp2E1 leading to cytotoxicity

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.
iKER2 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.
iKER3 KE2-->AO: Cytotoxicity leading to liver cancer

Level of Support: Weak.

Mechanism: Cell death by necrosis and necroptosis produces 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.
iKER4

 KE3-->AO: Regenerative proliferation leading to liver cancer

Level of Support: Moderate

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. 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 regenerative 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 regenerative 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 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: Cytotoxicity  
Weak. We are not aware of any experiments that have specifically blocked cytotoxicity following chemical exposure.  
KE3: Regenerative Proliferation  
Weak. 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. (Behrens, et al. 2002, Schrum, et al. 2000)

 

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.

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 ‘defining question1’ in the literature.
KER2 KE1-->KE2: Oxidative stress leading to cytotoxicity

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 ‘defining question 1’ in the literature.
KER3 KE2-->KE3: Cytotoxicity leading to regenerative proliferation

Level of Support: Strong.

Defining question 1: That hepatotoxicity leads to regenerative 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.
iKER1 MIE-->KE2: Activation of Cyp2E1 leading to cytotoxicity

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.
iKER2 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.
iKER3 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.
iKER4 KE3-->AO: Regenerative proliferation leading to liver cancer

Level of Support: Strong

Defining question 1: That an increase in cellular proliferation precedes tumour formation is a universal occurrence.

Defining question 2: Not all cases of regenerative proliferation produce tumours (some simply regenerate the liver to its healthy form). Therefore, it is clear that malignant transformation is accompanied by some sort of abnormal cellular signaling or impaired homeostasis.

 


Quantitative Considerations

?

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)

?


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 regenerative 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, an AOP describing these KEs and KERs might be used as a screening tool for compounds that act via this mode of action. 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

?


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.

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.

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.

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.

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.

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.

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.

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.

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.

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.