Aop:107

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AOP Title

Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat
Short name: CAR activation- HCC

Authors

Richard C. Peffer, Syngenta Crop Protection, LLC, Greensboro, NC, USA

Katie Bailey, Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA

Kristin Lichti-Kaiser, Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA

Brian Lake, Centre for Toxicology, University of Surrey, UK.

Corresponding author for wiki entry (richard.peffer@syngenta.com)

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OECD Project 1.29: A catalog of putative AOPs that will enhance the utility of US EPA Toxcast high throughput screening data for hazard identification

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Abstract

The constitutive androstane receptor (CAR) is a nuclear receptor that is involved in the regulation of various cellular processes following its activation by xenobiotics or endogenous ligands. Activation of CAR in the liver of rats and mice leads to altered gene expression, including genes related to Phase 1 and Phase 2 metabolism, transporters, cell cycle control and apoptosis regulation. This AOP describes the sequence of key events and associative events that occur in rats and mice following lifetime exposures to CAR activators, which leads from activation of CAR to an increased incidence of liver hepatocellular adenomas and carcinomas. The molecular initiating event (MIE) is activation of the CAR nuclear receptor, either by direct binding of a ligand or by an indirect mechanism (e.g. as with phenobarbital), both of which allow the CAR protein to migrate to the nucleus and alter expression of CAR target genes. In rats and mice, CAR activation alters the expression of certain genes related to cell cycle control, producing changes resulting in a pro-proliferative and anti-apoptotic environment. These gene expression changes lead to an increase in cell proliferation, a key event (KE), as well as suppressed apoptosis, an associative event (AE). In this proliferative environment, a higher number of spontaneously mutated hepatocytes can form. When present, suppressed apoptosis allows these damaged cells to avoid degradation and survive. With longer time intervals, the mutated hepatocytes clonally expand into pre-neoplastic altered foci. Eventually, under continued CAR activation, the pre-neoplastic foci expand to form hepatocellular adenomas and carcinomas, the adverse outcome (AO). Based on a wide dataset of testing with CAR activators, this AOP is considered to only be operative in mice and rats, and not in other mammalian species including humans. Good dose concordance between the early KEs and the AO (rodent liver tumors) have been demonstrated with example molecules. A potential application of this AOP is to guide future risk assessments, where dose-response values for critical early key events (e.g. NOAEL (no observed adverse effect level) or BMDL (Benchmark Dose Lower Limit) values) may be useful endpoints. Another potential application of this AOP is to highlight how methods for assessment of the MIE and/or the early KEs can be reliably used to demonstrate that a CAR mode of action is operative for a particular molecule, avoiding large scale use of animal testing to demonstrate every KE in the proposed pathway.

Background (optional)

In lifetime carcinogenicity studies conducted in rats and mice as part of the registration process for drugs, agrochemicals and other xenobiotics, a frequent finding after high dose treatments is hepatocellular adenomas and carcinomas in the liver (Cohen, 2010; Gold et al., 2005). An AOP via activation of the CAR nuclear receptor is one well-understood mechanism by which these tumors can occur (Elcombe et al., 2014). Studies comparing the postulated key events in various species (e.g. rat, mouse, hamster, guinea pig, non-human primate) as well as in human hepatocytes or humans on lifetime treatment with CAR activating drugs, have indicated large species differences in the susceptibility to 1) certain key events including KE3 (cell proliferation) and 2) the ability of CAR activators to produce liver tumors. The main purpose of this AOP is to outline the measurable key events and associative events for an AOP via CAR activation that leads to liver tumors in rats and mice. However, by summarizing experimental results across a wider range of mammalian species, the AOP outlines species differences relating to the KEs and the AO. By this approach, it is intended that the AOP can help the wider scientific and regulatory community to recognize the measurable KEs and AEs that would indicate a xenobiotic produces liver effects via this AOP, and the methods typically employed to demonstrate the thresholds in dose-response, below which no KEs and no tumors have been shown to occur in rats and/or mice with model CAR activators.

In the published proceedings of a nuclear receptor workshop on the CAR / PXR MoA (Andersen et al., 2014; Elcombe et al., 2014), the authors could not identify a suitable nongenotoxic PXR activator for which carcinogenicity data were available and hence a MOA was not developed for liver tumor formation by pregnane-X receptor (PXR) activators. CAR and PXR are often cited together regarding potential MoAs for a specific chemical agent, because extensive cross-talk between these two nuclear receptors has been described (Stanley et al., 2006), and some agents can activate both CAR and PXR in a particular species (Elcombe et al., 2014). In fact, PXR is activated by a large array of chemical substances, far more than those that activate CAR (Martin et al., 2010; Timsit and Negishi, 2007; Willson and Kliewer, 2002). PXR has been shown to increase liver weight after activation by a number of substrates, but it does not produce an increase in cell proliferation in the same way that CAR or PPARα cause this key event (Shizu et al., 2013; Thatcher and Caldwell, 1994). PXR activation is classically considered to selectively induce increased expression of Cyp3a isoforms, with lesser induction of Cyp2b isoforms, but again, cross-talk between PXR and CAR receptors upon activation of either nuclear receptor can be part of the altered expression of these Cyp isoforms in vivo. Given the lack of actual tumorigenic key events due to PXR activators alone, the rest of this current AOP will focus on the CAR MoA by itself.

Not all of the possible biochemical steps discussed by Elcombe et al. (2014) or proposed in subsequent literature are shown, in order to refine this AOP to a definitive set of readily measurable endpoints that encompass the KEs that are critical to the progression from CAR activation to liver tumor formation, and differentiate an agent that works via CAR activation from one that operates by alternative MoAs. Specifically, Elcombe et al. (2014) identified altered epigenetic changes specific to CAR activation and inhibition of gap junction intercellular communication (GJIC) as possible additional AEs that may also be present in the CAR MoA in mice and rats. While changes in DNA methylation status or inhibition of GJIC have been shown to occur in rodent liver after treatment with CAR activators such as phenobarbital (Klaunig et al., 1990; Moennikes et al., 2000; Phillips and Goodman, 2008) these were viewed by Elcombe et al. as associative events, since clear demonstrations of essentiality and/or association with CAR activation have not been indicated. More importantly regarding the AOP for CAR activation, these possible associative events require specialized techniques to demonstrate them, such as micro-injection of individual hepatocytes with dye in the case of GJIC (Klaunig et al., 1990). Considering the specialized methods needed and the fact that these AEs are not considered essential to demonstrating the overall AOP, they are not included in the set of key events described in Figure 1 and Table 1.

File:Table 1 AOP107.pdf

In Table 1, the typical data that is generated to demonstrate a molecule produces a liver tumor response in mice or rats via CAR activation AOP is described. However, this should not be viewed as a set of required studies, as other techniques are available that can lead to the same endpoint of demonstrating the CAR AOPs KEs and/or AEs. For example, other useful data that can also be generated (if needed) based on currently available techniques are listed in Table 1, such as the use of CAR reporter assays or microarray datasets where gene pathways as a signature for CAR activation changes in the liver can be generated (Omiecinski et al., 2011; Oshida et al., 2015a).

Summary of the AOP

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Molecular Initiating Event

Molecular Initiating Event Support for Essentiality
Constitutive androstane receptor, Activation

Key Events

Event Support for Essentiality
Mitogenic cell proliferation (hepatocytes), Increase Strong
Preneoplastic foci (hepatocytes), Increase Strong
Hepatocytes, Altered gene expression specific to CAR activation Strong

Adverse Outcome

Adverse Outcome
Adenomas/carcinomas (hepatocellular), Increase

Relationships Among Key Events and the Adverse Outcome

Event Description Triggers Weight of Evidence Quantitative Understanding
Mitogenic cell proliferation (hepatocytes), Increase Directly Leads to Preneoplastic foci (hepatocytes), Increase Strong Strong
Preneoplastic foci (hepatocytes), Increase Directly Leads to Adenomas/carcinomas (hepatocellular), Increase Strong Strong
Constitutive androstane receptor, Activation Directly Leads to Hepatocytes, Altered gene expression specific to CAR activation Strong Moderate
Hepatocytes, Altered gene expression specific to CAR activation Directly Leads to Mitogenic cell proliferation (hepatocytes), Increase Strong Moderate

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Life Stage Applicability

Life Stage Evidence Links
Not Otherwise Specified Strong

Taxonomic Applicability

Name Scientific Name Evidence Links
Rattus norvegicus Rattus norvegicus Strong NCBI
Mus musculus Mus musculus Strong NCBI

Sex Applicability

Sex Evidence Links
Male Strong
Female Strong

Graphical Representation

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Overall Assessment of the AOP

Graphical Representation of AOP

Figure 1. AOP for CAR Activation

1. Concordance of Dose-Response Relationships

Exposure-response relationships for nearly all of the key events have been established in vitro and/or in vivo in both mice and rats. A large and growing number of xenobiotics have been demonstrated to produce rodent liver tumors via the CAR activation AOP (Deguchi et al., 2009; Geter et al., 2014; Huang et al., 2005; LeBaron et al., 2013; Peffer et al., 2007). For ease of presentation, data are provided for three prototypical CAR activators (phenobarbital, TCPOBOP and metofluthrin). There are several variables that can impact dose-response relationships, including the CAR activating compound and the strain, sex, and species of the test system. Therefore, these variables need to be held constant (as much as possible) to evaluate the dose–response and time-response relationships in the section below. With this goal in mind, one exemplar species/strain/sex for each of these three model CAR activators are summarized in the dose-response and time concordance tables (Tables 2-5) that are illustrative for this AOP.

Phenobarbital has produced an increase in liver tumors via the CAR activation AOP in male and female mice, and in certain strains of male and female rats (reviewed in Elcombe et al., 2014). For illustration, Table 2 shows dose-response and time concordance of liver tumors with phenobarbital in male CD-1 mice. In this sex/strain, phenobarbital treatment for 2 years produced increased tumors at 75 and 150 mg/kg/day and no increases in tumors at 10 mg/kg/day (Whysner et al., 1996). As shown in Table 2, very strong dose concordance was observed for each KE or AE (as markers for in vivo CAR activation), with all of these events showing responses at the tumorigenic dose levels of 75 – 150 mg/kg/day. Certain early KEs or AEs produced effects at non-tumorigenic levels of 1.5 – 15 mg/kg/day, with increased PROD activity being the most sensitive overall endpoint (Geter et al., 2014).

Table 3 provides dose-response data for administration of phenobarbital in male mice of the C57BL/10J strain. A recent 2-year (99 week) study in this strain of mice produced a clear increase in liver adenomas and carcinomas at 1000 ppm in the diet, with no increases in tumors at 200 ppm (Jones et al., 2009). In addition, preliminary studies were conducted in male and female C57BL/10J mice fed diets containing 0 (control), 100, 200, 400, 700 and 1000 ppm phenobarbital for periods of 3, 8, 15 and 29 days (Jones et al., 2009), and a short-term study was conducted in C57BL/6J wild type and CAR null mice after 4 daily intraperitoneal (ip) doses of 80 mg/kg/day phenobarbital (Ross et al., 2010), which is similar to the achieved dose level for 700 ppm PB in the diet. As shown in Table 3, the 4-day treatment with 80 mg/kg/day phenobarbital produced large fold-change in markers of CAR activation, including KE1 (increased Cyp2b10 mRNA) and AE1 (increased PROD activity). Other gene expression changes associated with CAR activation (KE1) were also impacted, including increased Ki67 and decreased Tsc22 expression. These genomic markers were unaffected in the CAR null mice, indicating they were a result of CAR activation. The associative events of hepatocyte hypertrophy and increased relative liver weight were increased at all dose levels of 200 ppm and above after 29 days (Jones et al., 2009). After 8 days of treatment in the diet, cell proliferation (KE2) as indicated by BrdU labeling index was increased at 700 and 1000 ppm, but not at lower dose levels. Finally, an increase in eosinophilic and clear cell foci was observed after 99 weeks at the tumorigenic dose level (1000 ppm), but not at the non-tumorigenic dose level (200 ppm).

TCPOBOP is a very potent direct activator of mouse CAR, and it has been shown to produce tumors in male and female mice of various strains at an experimental dose level of 3 mg/kg/day (ip or oral gavage). For illustration, Table 4 shows time concordance of TCPOBOP effects in male C57BL/6 mice at 3 mg/kg/day, given for 1 day, 3 days, 28 days, or 30 weeks (with sacrifice after 60 weeks), using various dosing methods and frequencies. TCPOBOP is a lipophilic substance that is excreted slowly in mice and is a very potent mouse CAR activator, so dosing regimes often have involved intermittent dosing such as once every 14 days (Huang et al., 2005). Consistent effects on all associative events and key events were observed across the multiple studies summarized in Table 4 at this dose level, including Cyp2b10 mRNA increases (KE1, a marker of CAR activation), altered CAR-mediated gene expression marking a pro-proliferative / anti-apoptosis response (e.g. increased Cdc20, Cdk1 and Gadd45β), increased cell proliferation, suppressed apoptosis, increased altered foci and eventually hepatocellular tumors. The potent response of the mouse liver to TCPOBOP also afforded a useful experimental tool to investigate the CAR-mediated nature of the effects, via studies in CAR knockout mice and other similar transgenic models. Lack of effects on the early KEs and AEs (including the gene expression changes), and a lack of any increase in liver tumors in the CAR null mice demonstrated that these processes and the eventual development of tumors was dependent on activation of CAR (Huang et al., 2005; Yamamoto, Y. et al., 2004).

Metofluthrin has produced an increase in liver tumors via the CAR activation AOP in male Wistar rats (900 and 1800 ppm) and female Wistar rats (1800 ppm), but it did not produce liver tumors in CD-1 mice (Yamada et al., 2009). For illustration, Table 5 shows dose-response and time concordance of liver tumors with metofluthrin in male Wistar rats. Investigative studies were conducted at dose levels from 200 ppm (the tumor NOAEL) up to 3600 ppm (a dose considered in excess of the Maximum Tolerated Dose [MTD]) (Deguchi et al., 2009; Hirose et al., 2009). The key events of CYP2B1/2 and/or CYP3A1 mRNA levels (as markers for CAR activation), increased cell proliferation and increased altered foci were each observed at the tumorigenic dose levels of 900 ppm and above. Associative events of increased PROD activity, increased CYP2B protein levels, hepatocellular hypertrophy and increased liver weight were increased at tumorigenic dose levels, but they were unaffected at 200 ppm, the tumor NOAEL. In isolated rat hepatocyte cultures treated with 50 µm metofluthrin, co-administration of siRNA for CAR (a gene silencing technique) caused a knockdown of CAR and resulting suppression of the response to metofluthrin (50 uM) for CYP2B1 mRNA and CAR mRNA (Deguchi et al., 2009).

File:Table 2 AOP107.pdf

File:Table 3 AOP107.pdf

File:Table 4 AOP107.pdf

File:Table 5 AOP107.pdf

2. Temporal Concordance Among the Key Events and Adverse Outcome

As shown for the 3 example molecules (Table 2-5), empirical evidence shows good time concordance between the MIE (CAR activation at 1 day and continuing throughout), intermediate key events and associative events (days to weeks), leading eventually (months) to a later key event of increased altered foci and then to tumors.

3. Strength, Consistency and Specificity of the Association of Adverse Effect and Initiating Event

The scientific evidence linking the MIE (CAR activation) and the AO (liver tumors in rodents) have been presented for multiple compounds, and the same sequence of key events was observed for each compound. Blocking the initial MIE (via testing in transgenic mice lacking the CAR nuclear receptor) was able to block all of the subsequent key events, including CAR-specific gene expression, cell proliferation and ultimately liver tumor formation, thus demonstrating high specificity of this association.

4. Biological Plausibility, Coherence and Consistency of the Experimental Evidence

There is a high biological plausibility of the proposed AOP, as it matches with known biology. Also, the demonstrated key events were consistent across 3 different compounds and between mice and rats in their responses to a demonstrated CAR activator for that species.

5. Alternative Mechanisms

For the data-rich molecules that were chosen such as phenobarbital and TCPOBOP, whole mouse microarray experiments are available (Currie et al., 2014; Geter et al., 2014; Nesnow et al., 2009; Oshida et al., 2015a; Tojima et al., 2012) that allow an examination for markers of certain alternative liver Modes of Action that have been characterized in rodent studies (Cohen, 2010). No evidence of PPARα activation (as marked by Cyp4a induction) was observed, nor was there evidence of estrogenic effects. AhR activation (as marked by high levels of Cyp1a / Cyp1b induction) was shown not be operative; a minor increase in certain isoforms of Cyp1a or Cyp1b has been shown to occur upon treatment with CAR activators (Oshida et al., 2015a; Oshida et al., 2015b), but the large fold-changes that AhR activators produce are not observed. For all 3 molecules, the wider database indicates they are not genotoxic, they do not produce immediate cytotoxicity and regenerative hyperplasia in the liver, and do not produce histological evidence of liver changes via iron deposition or an infectious process within the liver (Elcombe et al., 2014; Huang et al., 2005; Yamada et al., 2009). Thus, no alternative mechanisms appear to be operative, and these 3 example molecules can be concluded to work via an AOP initiated by CAR activation.

6. Uncertainties, Inconsistencies and Data Gaps

Across the full database for these three example molecular, very few uncertainties or data gaps exist; any data that are missing in one species/strain/test compound are typically present in the others. For example, phenobarbital treatment of male CD-1 mice has been reported in a review article to cause an increase in adenomas and carcinomas of the liver at 75 and 150 mg/kg/day, with a tumor NOAEL at 10 mg/kg/day (Whysner, 1996–via a personal communication), but the actual tumor incidence data for this study have not been published. Accordingly, no data for the KE3 of increased altered foci are available in CD-1 mice. However, tumorigenic responses in various strains of mice after phenobarbital treatment have been described in other studies (reviewed in Elcombe et al., 2014), and studies in C57BL/10J mice have shown a clear increase in both altered foci and liver tumors at 1000 ppm and a NOAEL at 200 ppm (Table 3; Jones et al., 2009).

One other uncertainty is the difficulty with some molecules to demonstrate the associative event of suppression of apoptosis (AE4). For example, Deguchi et al. (2009) examined the cytoplasmic histone-associated DNA fragments in the liver of metofluthrin-treated rats, but were unable to detect a difference in this apoptosis marker vs. controls. In young rats or mice, the background, control level of apoptosis is inherently low, and thus the ability to detect a decrease in vivo is often difficult and is also dependent on the methods selected as well as the potency of the CAR activator and the duration of treatment (Peffer et al., 2007). For TCPOBOP, a very potent mouse CAR activator, Huang et al. (2005) were able to show a statistically significant decrease in apoptosis by TUNEL staining after 60 weeks. They also showed that after 3 days treatments with TCPOBOP, the level of apoptosis induced by UV irradiation or methyl methanesulfonate treatment was suppressed by this CAR activator, and the suppression was absent in hepatocytes from CAR null mice. In hepatocyte studies, James and Roberts (1996) have used hepatocytes stimulated into apoptosis with TGFβ1 to examine the apoptosis suppression properties of different molecules, and have consistently shown an ability of CAR activators such as phenobarbital to lower the induced level of apoptosis. In a longer-term study with phenobarbital in male CD-1 mice, Kolaja et al. (1996b) were able to detect suppression of apoptosis within the altered foci of the liver using immunohistochemical methods after 60 days of treatment. Bursch et al. (2005) have proposed that suppression of apoptosis by CAR activators may be of greater importance to the overall tumor progression in rats than in mice, based on greater evidence of suppressed apoptosis in the altered foci of rats compared to mice (Bursch et al., 2005; Schulte-Hermann et al., 1990). Thus, while suppression of apoptosis is included as an associative event in this AOP for CAR activation, the difficulty with readily detecting and/or quantifying its appearance, particularly in shorter-term mechanistic studies, must be acknowledged.

Weight of Evidence Summary

Summary Table

Support for Biological Plausibility of KERs
Defining Question
High (Strong)
Moderate
Low (Weak)


Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?

Extensive understanding of the KER based on previous documentation and broad acceptance.

KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete.

Empirical support for association between KEs, but the structural or functional relationship between them is not understood.

MIE => KE1

Strong

Activation of the CAR receptor causes changes in expression of CAR-responsive genes such as Cyp2b10, Gadd45beta, Cdc20, and Ki67, and overall genomic pathway changes associated with increased progression through the cell cycle (leading to later KE2) and decreased apoptosis (leading to later AE4) (Geter et al., 2014; Oshida et al., 2015a; Tojima et al., 2012). The KER is highly plausible, since specific CAR recognition elements for genes in these pathways have been identified previously (refs), and absence of the CAR receptor in CAR-null animals blocks these KE1 gene changes.

KE1 => KE2

Strong

The CAR-mediated changes in expression of pro-proliferative genes such as Gadd45beta, Ki67 and Cdc20 have been demonstrated to occur with multiple CAR activators (Oshida et al., 2015a; Ozawa et al., 2011; Tojima et al., 2012). It is highly plausible that these gene expression changes then lead to increased cell proliferation signals in hepatocytes, as genes in the Gadd45 family are known to interact with cyclins, cyclin-dependent kinase inhibitors and p53 to alter progression through the cell cycle (Liebermann and Hoffman, 2008). For most CAR activators, the DNA labeling index as a marker of cell proliferation is maximal after 1-7 days of treatment, and then diminishes back to control levels by approximately 28 days. However, cell proliferation within altered foci in initiation-promotion studies in mice or in F344 rats has been shown to be enhanced by CAR activator treatment at 20 to 76 weeks after initiation in a dose-responsive manner (Bursch et al., 2005; Klaunig, 1993; Kolaja et al., 1996b). The increased proliferation due to CAR activator treatment tends to occur to a greater extent within eosinophilic foci, and has been shown to be greater in tumor-prone strains of mice (C3H > B6C3F1) than in the comparatively tumor-resistant C57BL/6 strain of mice (Bursch et al., 2005; Pereira, 1993).

KE1 => AE4

Moderate

The cellular signals that lead to apoptosis have been studied extensively, and changes that occur in liver cells treated with CAR activators include upregulation of Gadd45beta, and downregulation of Gadd45gamma and Tsc22 (Hino et al., 2002; Iida et al., 2005; Ozawa et al., 2011; Ross et al., 2010), although the specific genes that are differentially expressed can differ depending on the CAR activator and the species/strain tested. These genes can also be altered by other rodent carcinogens that act via different mechanisms (Iida et al., 2005; Oshida et al., 2015b). If the genes involved in control of apoptosis are altered, then it is plausible that these changes would lead to a suppressed signal for apoptosis within the hepatocyte. As discussed, the ability to demonstrate AE4 (suppressed apoptosis) in young rats and mice is often difficult, since normal levels of apoptosis in the liver at this age is already low. Based on the recorded evidence of suppressed apoptotic genes in CAR treated rodent livers and suppressed apoptosis in vivo or in vitro with certain CAR activators, this KER has moderate biological plausibility.

KE2 => KE3

Strong

The increased cell replication rate in the liver due to CAR activation (i.e. via mitogenic signaling) is similar to other well-understood modes of action where an increase in cell proliferation leads to an eventual increase in pre-neoplastic foci, such as PPARalpha activating ligands or partial hepatectomy leading to regenerative proliferation of the liver. Similarly with AhR activating ligands, progressive liver damage and regenerative proliferation of hepatocytes leads to an increase in pre-neoplastic foci via clonal expansion of transformed hepatocytes (Becker et al., 2015; Cohen, 2010; Corton et al., 2014). In a normal liver, expansion of hepatocyte numbers via proliferation is in a constant balance with controlled cell death of damaged hepatocytes via apoptosis, to keep an appropriate critical mass of functional liver cells that can be maintained by the oxygen and nutrient supplies within the liver lobule (Goldsworthy and Fransson-Steen, 2002). In mice lacking the CAR receptor, including initiation-promotion assays, the upstream key events (e.g. CAR activation, gene expression changes, cell proliferation), the associative events (e.g. suppression of apoptosis) and the downstream events (e.g. pre-neoplastic foci) are all blocked, providing strong support for the biological plausibility of this Key Event Relationship (Huang et al., 2005; Tamura et al., 2015; Tamura et al., 2013; Yamamoto, Y. et al., 2004).

KE3 => AO:

Strong

The development of liver tumors in rodents, whether spontaneously or induced by a non-genotoxic carcinogen, has consistently included the development of altered foci as precursor step to hepatocellular adenomas and carcinomas (Goldsworthy and Fransson-Steen, 2002; Haschek and Rousseaux, 1998). These foci are considered pre-neoplastic lesions, and their ability to progress to form adenomas and/or carcinomas in rodents has been previously recognized. In the case of CAR activators, an increased incidence of pre-neoplastic foci has been consistently shown to precede tumor development, and there is a high biological plausibility for this Key Event Relationship (Elcombe et al., 2014; Goldsworthy and Fransson-Steen, 2002).

Domain of Applicability

Life Stage Applicability, Taxonomic Applicability, Sex Applicability

Studies in various species, or in isolated hepatocytes from various mammalian species including humans, have demonstrated that CAR activators such as phenobarbital or metofluthrin produce a cell proliferation response that is seen in mice or rats, but not in hamsters, guinea pigs, non-human primates or humans (Hasmall and Roberts, 1999; Hirose et al., 2009; James and Roberts, 1996; Yamada et al., 2009). Also, in a series of experiments in hepatocytes induced into apoptosis with TGFβ1, James and Roberts (1996) showed that phenobarbital treatment suppressed the apoptosis response in rat hepatocytes, but it had no effect on apoptosis in hamster or guinea pig hepatocytes. In accord with the lack of response for these two key events in hamsters, phenobarbital and other CAR activators do not produce liver tumors in long term studies in hamsters (Diwan et al., 1986; Elcombe et al., 2014) . Consistent with the lack of effects on proliferation and on tumor development, Diwan et al. (1986) also reported that phenobarbital treatment at 500 ppm in the drinking water did not produce any increases in preneoplastic foci of cellular alteration in hamsters compared to groups that received an initiator alone. Substances such as phenobarbital have been shown to activate the CAR receptor in mice, rats, hamsters, primates and humans, resulting in altered gene expression for metabolizing enzymes (a subpart of KE1) and increased CYP2B enzyme activity (AE1), hepatocellular hypertrophy (AE2) and increased relative liver weight (AE3) (summarized in Elcombe et al., 2014). However, experimental evidence has demonstrated that while phenobarbital and other CAR activators can produce non-adverse liver changes via these associative events in multiple species, they only produce the key events of increased cell proliferation and increased foci of alteration in rats and mice, but not in other mammalian species including humans.

Multiple epidemiological studies with phenobarbital and other anticonvulsant drugs have been performed (Friedman et al., 2009; IARC, 2001; Lamminpaa et al., 2002; Olsen et al., 1989; Olsen et al., 1995; Selby et al., 1989; White et al., 1979; Whysner et al., 1996) and results of these studies have been reviewed by La Vecchia and Negri (2014) and Elcombe (2014). In only one study, patients treated with various anticonvulsant drugs showed a possible increase in liver tumors (Lamminpaa, 2002), although an outside review indicated that other factors such as alcohol and smoking may have contributed to the increase in liver tumors and that there was no indication of an excess risk attributable to phenobarbital use (La Vecchia and Negri, 2014). In contrast, multiple earlier epidemiological studies in patients that received phenobarbital have demonstrated that phenobarbital did not increase the incidence of liver tumors or of any other tumor type in humans (Friedman et al., 2009; Olsen et al., 1989, 1995; Selby et al., 1989). In their review of the full range of published studies up through 2012, La Vecchia and Negri (2014) concluded that there was no evidence for a specific role of phenobarbital in human liver cancer risk. In the studies that showed no evidence of increased liver tumor risk, subjects received phenobarbital over many years at doses that produced plasma concentrations similar to those that are carcinogenic in rodents. For example, phenobarbital administered at 500 ppm in drinking water achieved blood concentrations of 5–29 ug/ml in mice (three different strains), and 20 – 33 ug/mL in Wistar male and female rats. In human patients receiving phenobarbital at therapeutic doses of 3–6 mg/kg/day, plasma concentrations ranged from 10–25 ug/ml, which reflects the recommended therapeutic range of this anticonvulsant (Benet and Shiner, 1985; Monro and Davies, 1993).

In summary, human epidemiological studies support a conclusion that the AOP for CAR-mediated rodent liver tumors following phenobarbital treatment is not relevant to humans. The lack of effects on causal key events and/or certain associative events (e.g. altered expression of CAR-modulated genes related to cell cycle control, including KE1, KE2, AE4 and KE3) in human hepatocytes further support this conclusion. In vivo and in vitro experimental data indicate the causal key events for this AOP have been demonstrated to occur in certain strains of mice and rats, but not in other mammalian species such as hamsters, guinea pigs and humans. As the generation of tumors in rodents requires lifetime treatment with most CAR activators, there is no known association of differing susceptibility in different life stages for humans. For specific CAR-activating molecules, increases in liver tumor incidences have been seen selectively in one rodent species/sex but not the other (Currie et al., 2014; Deguchi et al., 2009), while other CAR activators produce tumors in both males and females, and in both mice and rats (Elcombe et al., 2014; Whysner et al., 1996). Such species/strain/sex differences may be linked to different CAR binding properties of a particular ligand, differences in pharmacokinetics, or other factors such as a lack of changes in expression for genes related to cell proliferation and apoptosis control. For example, large differences in the ligand binding properties of CAR from mice, rats, dogs and humans have been demonstrated in CAR nuclear translocation assays with a range of molecules (Omiecinski et al., 2011), and these differences have been correlated for some of the studied molecules to differing potential for liver effects in the respective species in vivo (Omiecinski et al., 2011).

Male mice tend to have a higher background incidence of liver tumors spontaneously, compared to female mice or male/female rats (Monro and Davies, 1993), and some compounds that are activators of CAR in rodents may express an increased tumor response only in male mice (Currie et al., 2014). The tendency for greater background incidences of liver tumors, and greater susceptibility to certain CAR activators, in male mice compared to female mice or male/female rats, has been attributed to a number of factors, including:

  • Known sensitivity of male mice to liver effects based on higher background levels of oxidative metabolism and lesser antioxidant defense systems compared to rats or other species. This includes a higher reliance on conjugation via glutathione-S-transferase (GST) and lower activity of epoxide hydrolase and UDP-glucuronyltransferase (UDPGT) in the livers of mice compared to rats (Bachowski et al., 1998; Parke and Ioannides, 1990).
  • Known differences between male and female mice in tumor sensitivity, based on the promoting effects of androgens and protective effects of ovarian hormones (Poole and Drinkwater, 1995; Yamamoto, K. et al., 1991).
  • A greater background load of DNA lesions in the liver of male mice (de Boer et al., 1998; Moore et al., 1999).

These properties related to the background tumor incidence in male mice may alter the potential for liver tumors to occur with greater incidence in this sex and species for some compounds, but there are clear examples (e.g. metofluthrin and pyrethrin liver tumors in rats but not mice) where other factors produce a different pattern of effects in rodent species.

Essentiality of the Key Events

Molecular Initiating Event Summary, Key Event Summary

Support for Essentiality of KEs
Defining Question
High (Strong)
Moderate
Low (Weak)


Are downstream KEs and/or the AO prevented if an upstream KE is blocked?

Direct evidence from experimental studies illustrating essentiality for at least one of the important KEs.

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE.

No or contradictory experimental evidence of the essentiality of any of the KEs.

MIE: CAR Activation

Strong

When activation of CAR was prevented via testing in CAR knockout mice, virtually all downstream key events were prevented, including tumors (Huang et al., 2005; Peffer et al., 2007; Tamura et al., 2015; Tamura et al., 2013; Tojima et al., 2012; Wei et al., 2000; Yamamoto, Y. et al., 2004). In Wistar rats administered metofluthrin, suppression of CAR synthesis via siRNA was shown to suppress the expression of CYP2B1, confirming that this associative event (AE1) was CAR-dependent in rats (Deguchi et al., 2009).

KE1: Altered gene expression specific to CAR activation

Strong

When activation of CAR was prevented via testing in CAR knockout mice, differential expression of critical genes and pathways related to a pro-proliferative and anti-apoptosis response and marker genes of CAR activation (e.g. Cyp2b10 in mice; CYP2B1/2 in rats) was blocked (Oshida et al., 2015a; Peffer et al., 2007; Tojima et al., 2012). In addition, downstream key events or associative events dependent on altered gene expression were also blocked in the CAR null mice, including Cyp2b enzyme activity (AE1), hepatocellular hypertrophy and increased liver weights (AE2, AE3), cell proliferation (KE2) and the long-term histopathology changes (KE3; AO) (Huang et al., 2005; Peffer et al., 2007; Tamura et al., 2015; Tamura et al., 2013; Wei et al., 2000; Yamamoto, Y. et al., 2004).

KE2: Mitogenic cell proliferation (hepatocytes), Increase

Strong

When activation of CAR was prevented via testing in CAR knockout mice, cell proliferation in the liver was prevented at the tumorigenic dose levels (Huang et al., 2005; Peffer et al., 2007; Ross et al., 2010; Tamura et al., 2013).

AE4: Suppression of apoptosis

Moderate

When activation of CAR was prevented via testing in CAR knockout mice, differential expression of mouse anti-apoptosis genes such as Gadd45betawas blocked (Huang et al., 2005; Peffer et al., 2007; Tojima et al., 2012) and the suppression of apoptosis in vivo was blocked (Huang et al., 2005). Similarly, increased expression of Gadd45betaand decreased expression of Gadd45gamma was observed after phenobarbital treatment in male F344 rats, and these directions of change reflect suppression of apoptosis pathways (Liebermann et al., 2011; Ozawa et al., 2011). Four days after discontinuing treatment of mice with phenobarbital, apoptosis level in the livers were significantly increased, indicating a rebound effect after the phenobarbital-induced suppression was removed (Huang et al., 2005). Following a 4-week recovery period, phenobarbital-treated rats demonstrated a significantly higher expression of Gadd45gamma mRNA, which was considered a compensation for the sustained period of liver apoptosis suppression (Ozawa et al., 2011). Overall, the evidence for essentiality of AE4 is moderate, since the ability to quantitatively demonstrate a decrease in apoptosis in the livers of young rodents is technically challenging and likely is dependent on the relative strength of the CAR activator, the species and the strain.

KE3: Preneoplastic foci (hepatocytes), Increase

Strong

When activation of CAR was prevented via testing in CAR knockout mice, foci of cellular alteration in the liver was prevented in an initiation-promotion model using the CAR activators cyproconazole and fluconazole (Tamura et al., 2015). Also, the incidence of adenomas and carcinomas was similarly decreased (Tamura et al., 2015). With phenobarbital treatment of C3H background mice, absence of the CAR receptor in CAR null mice blocked both the increase in eosinophilic foci and the increase in liver adenomas and carcinomas (Yamamoto, Y. et al., 2004). Thus, there is strong evidence for the essentiality of KE3 to the overall progression to liver tumors with CAR activators, and this essentiality is further confirmed by normal biology of the rodent liver (Goldsworthy and Fransson-Steen, 2002; Haschek and Rousseaux, 1998).

AO: Adenomas/carcinomas (hepatocellular), Increase

Strong

When activation of CAR was prevented via testing in CAR knockout mice, virtually all downstream key events were prevented (Huang et al., 2005; Peffer et al., 2007; Ross et al., 2010; Tamura et al., 2015; Tamura et al., 2013; Tojima et al., 2012; Wei et al., 2000; Yamamoto, Y. et al., 2004). In addition, testing in CAR knockout mice prevented the formation of liver tumors in initiation-promotion models or in studies where CAR activator alone was administered (Huang et al., 2005; Tamura et al., 2015; Wei et al., 2000; Yamamoto, Y. et al., 2004).

Quantitative Considerations

Summary Table

Empirical Support for KERs
Defining Question
High (Strong)
Moderate
Low (Weak)


Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses, earlier time points, and higher in incidence than KEdown ? Inconsistencies?

Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data.

Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors.

Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species

MIE => KE1:

Strong

Strong support for this linkage comes from in vivo and in vitro studies with model CAR activators, and via the absence of the same effects including specific gene expression changes in CAR knockout mice lacking the CAR receptor. In a study with TCPOBOP (a direct activator of mouse CAR), these CAR-dependent changes included Gadd45beta (14-fold increase), Cdk1 (11-fold increase) Cdc20 (37-fold increase) and additional cytokines, and these genes were unaffected in CAR knockout mice (Tojima et al., 2012). Gene pathways (by Ingenuity Pathway Analysis, IPA) that were altered by phenobarbital in a dose-responsive manner in CD-1 mice included “Cell cycle of chromosomal replication” and “Cell cycle: G2/M DNA damage checkpoint regulation”; these were only significantly altered above a NOTEL of 15 mg/kg/day (Geter et al., 2014). Finally, Oshida et al. (2015a) have studied microarray data for a wide range of compounds in the NextBio database, including their own studies in WT and CAR null mice, and demonstrated that a CAR biomarker gene expression signature exists which is statistically significantly enriched for known CAR activators, and is non-significant for model compounds that produce liver effects via alternative mechanisms.

KE1 => KE2:

Strong

Strong support for this linkage comes from in vivo and in vitro studies with model CAR activators, and via the absence of the same effects including specific gene expression changes and cell proliferation changes (by BrdU, PCNA or Ki67 labeling index) in CAR knockout mice lacking the CAR receptor. In a study with TCPOBOP (a direct activator of mouse CAR) in mice, increases in gene expression of Cyp2b10 and Mdm2 were associated with increases in cell proliferation after 3 days or 30 weeks, and both effects were absent in CAR knockout mice (Huang et al., 2005; Wei et al., 2000). In Fischer 344 rats, Ozawa (2011) showed that phenobarbital at 500 ppm in the diet produced a sustained increase in Gadd45beta expression and a decrease in Gadd45gamma expression; these directions of change have been previously associated with enhanced cell proliferation and suppression of apoptosis. Rats treated with 500-1000 ppm phenobarbital in diet or in drinking water exhibit an increase in cell proliferation after 1-7 days of treatment (Deguchi et al., 2009) and eventually liver tumors (Rossi, 1977; Ward, 1983), but the tumor response in rats is variable and dependent on strain, age of rats and dosing regime (Elcombe et al., 2014). However, certain compounds such as metofluthrin and pyrethrins have been shown to produce liver tumors in rats, but not mice, and these long-term effects are preceded by appropriate CAR-mediated gene changes and cell proliferation in the liver (Osimitz and Lake, 2009; Yamada et al., 2009).

KE1 => AE4:

Moderate

Methods for quantification of the number of cells undergoing apoptosis include antibody-based assays (TUNEL) (Kolaja et al., 1996b), assay for cytoplasmic histone-associated DNA fragments (Deguchi et al., 2009), detection of DNA fragments via electrophoresis (Huang et al., 2005) and microscopic examination of H&E stained slides for apoptotic cells based on the shape of their nuclei (Goldsworthy and Fransson-Steen, 2002). However, the success of each method can be highly variable and should be considered when evaluating the reported outcomes. Also, the ability to measure a decrease in hepatocyte apoptosis in CAR activation mode of action studies in vivo or in vitro is often limited by the very low background levels of apoptosis that exist in young rodents (i.e. in the control group). Considering the difficulties in routine measurement of the suppression of apoptosis response in young rats and mice, this event is considered an associative event in the AOP leading to rodent liver tumors.

Moderate support for this linkage comes from in vivo and in vitro studies with some model CAR activators. In studies with potent CAR activators (e.g. TCPOBOP), altered gene expression related to a suppressed apoptosis response has been demonstrated (Huang et al., 2005; Tojima et al., 2012), and the following step of suppressed apoptosis has been demonstrated with TCPOBOP in mice after 3 days or 30 weeks of dosing (Huang et al., 2005) and with phenobarbital in mice (Hasmall and Roberts, 1999; Kolaja et al., 1996b). Also, an absence of the same effects including specific gene expression changes and apoptosis suppression (by TUNEL staining) was demonstrated in CAR knockout mice lacking the CAR receptor (Huang et al., 2005; Tojima et al., 2012). However, some attempts to measure a suppression of apoptosis in young rodents in vivo with less potent CAR activators than TCPOBOP (Deguchi et al., 2009) were not able to show a consistent quantitative difference. Overall, there is moderate support for this KER of altered CAR-mediated gene expression leading to a suppression of apoptosis in hepatocytes.

KE2, => KE3:

Strong

The observed increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined. This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992). With well-studied CAR activators such as phenobarbital, TCPOBOP, pyrethrins, cyproconazole and metofluthrin, increased cell proliferation has been detected at similar dose levels where increased altered foci are seen (Geter et al., 2014; Huang et al., 2005; Kolaja et al., 1996a; Kolaja et al., 1996b; Osimitz and Lake, 2009; Peffer et al., 2007; Tamura et al., 2015; Yamada et al., 2009). In addition, studies in WT and CAR null mice have shown that in the absence of CAR receptor, neither cell proliferation (KE1) nor the development of increased altered foci (KE2) and liver tumors (AO) occurs (Huang et al., 2005; Tamura et al., 2015; Tamura et al., 2013; Yamamoto, Y. et al., 2004). Therefore, there is strong support for the linkage of this earlier key event with CAR activators leading to an increase in pre-neoplastic foci.

KE3 => AO:

Strong

There is a strong empirical data demonstrating that this sequence of events occurs with CAR activators. An increase in numbers of preneoplastic foci, usually with eosinophilic staining properties, is observed with great regularity in mode of action work of CAR activating xenobiotics where histopathology at later times has been examined (Elcombe et al., 2014). This increase in foci (mixed or eosinophilic) after 2 years was observed at tumorigenic dose levels with metofluthrin in male rats (Deguchi et al., 2009), and at tumorigenic dose levels in mice treated with phenobarbital (Jones et al., 2009). With TCPOBOP in mice, multiple eosinophilic foci were reported to co-occur along with an increased incidence of eosinophilic adenomas and carcinomas after 60 weeks of treatment (Diwan et al., 1992).


There is a rich dataset that demonstrates the quantitative relationships between exposure and the key events and/or associative events in this AOP. These quantitative comparisons are summarized in Tables 2-5 for the example molecules phenobarbital, TCPOBOP and metofluthrin, and are described in the accompanying text and footnotes.

The Guidance for describing AOPs in the AOPwiki emphasizes that the AOP and its KERs should be evaluated with empirical data that is available in a Table format that shows: 1) Dose-response; 2) Temporality; and 3) Incidence/strength of response. Because the AOP for rodent liver tumors includes early key events (Immediate and for several days), intermediate key events (days to months) and long-term key events/AO (pre-neoplastic foci and tumors after 18-24 months), obtaining one complete set of data for one molecule at consistent dose levels in a defined sex, strain and species is highly unlikely. However, a very detailed dose-response relationship of early key events for phenobarbital, which produces tumors in male CD-1 mice, has been published by Geter et al. (2014). Therefore, Table 2 assesses phenobarbital in the manner requested by the AOPwiki Guidance, after incorporating data from other longer-term studies that provide suitable dose-response data for additional key events or associative events. The reader is referred to the original paper by Geter et al. (2014), which has calculated Benchmark Dose (BMD) values and 95% lower confidence limit (BMDL) values for a number of measured early endpoints for both male and female CD-1 mice treated with phenobarbital, which may be suitable for risk assessment applications. In addition, Table 3 provides dose-response and time concordance data for the key events and associative events following phenobarbital treatment in a different strain of mice (C57BL/10J), and this data set includes published data showing the long-term effects of increased altered foci (KE3) and increased liver adenomas and carcinomas (AO) (Jones et al., 2009).

Examining the dose-response and temporal concordance data in Table 2, the empirical data on dose-response and temporality is Moderate or Strong for each Key Event Relationship. For most early events, an increased response was seen with increasing dose, and at the tumorigenic dose levels (75 and 150 mg/kg/day), all of the preceding key events (if measured at that dose) were observed. For KE1 (altered gene expression specific to CAR activation), the gene pathways in Geter et al. (2014; by Ingenuity Pathway Analysis, IPA) that were altered by phenobarbital in a dose-responsive manner in CD-1 mice included “Cell cycle of chromosomal replication” and “Cell cycle: G2/M DNA damage checkpoint regulation”; these were only significantly altered above a NOTEL (no observed transcriptional effect level) of 15 mg/kg/day (Geter et al., 2014). Examining some specific genes that are part of these cell replication pathways, Ki-67 (a marker of cell proliferation) was increased in a dose-responsive manner at 75 and 150 mg/kg/day on Day 2; this gene signal was back to baseline on Day 7 in male mice. Tsc22 was also differentially expressed only on Day 2, at dose levels ≥15 mg/kg/day; suppression of this gene is a marker of suppressed apoptosis. The CAR marker gene Cyp2b10 was greatly increased in its expression by phenobarbital at ≥15 mg/kg/day, and this metabolizing enzyme signal tends to stay elevated as long as treatment continues with a CAR activating compound (Osimitz and Lake, 2009; Peffer et al., 2007). Other genes related to cell cycle control that were differentially expressed in the profiles of TCPOBOP in mice (Table 4) were Gadd45β, Cdc20 and Cdk1 (Tojima et al., 2012). Comparing the genes related to cell proliferation and cell cycle control with phenobarbital and with TCPOBOP, it is clear that individual genes may be differentially expressed in one species/strain/test compound, but not in exactly the same manner with another. As a possible approach to identify a reliable set of gene expression changes that define a CAR activating compound in mice or rats, a pathway-based approach to characterize suspected CAR activators in rodents has advantages over the reliance on a small number of genes. Oshida et al. (2015a) have developed a CAR biomarker gene expression signature that is statistically significantly enriched for known CAR activators, and is non-significant for model compounds that produce liver effects via alternative mechanisms such as AhR activation or PPARα activation (Oshida et al., 2015b; Oshida et al., 2015c).

The key event of increased cell proliferation (KE2) was assessed in the experiment of Geter et al. (2014), both by BrdU labeling index and based on the incidence of “increased mitotic figures”. These two different approaches gave similar dose-response results, with lesser or equivocal increases at 15 mg/kg/day, and more robust changes at the tumorigenic dose levels of 75 and 150 mg/kg/day.

The associative event (AE4) of suppressed apoptosis was not assessed directly in the study of Geter et al. (2014), but data are available in male B6C3F1 mice at dietary treatments with phenobarbital that are estimated to be equivalent to 2, 20 and 100 mg/kg/day (Kolaja et al., 1996b). Results after 60 days are included in Table 2, and showed a clear increase at a dose in the tumorigenic range (100 mg/kg/day), no effects at 2 mg/kg/day, and a numerical suppression that was not statistically significant at an intermediate dose of 20 mg/kg/day. Similar to CD-1 mice, male B6C3F1 mice have been reported to display an increase in liver tumors with phenobarbital treatment (Whysner et al., 1996).

For KE3 (increases in altered foci), an increased incidence was observed in studies in C57BL/10J mice after 99 weeks of phenobarbital treatment (Table 3) at the tumorigenic dose level of 113 mg/kg/day, but not at the tumor NOEL of 22 mg/kg/day (Jones et al., 2009). Overall, the compiled data displayed in Tables 2 and 3 for phenobarbital demonstrate good dose concordance of the key events with the adverse outcome (AO). They also support a logical temporal relationship, with earlier key events preceding later ones in a manner consistent with the known biology.

Data for metofluthrin (Table 5) provides a similar dose-concordance assessment in male rats, and data for TCPOBOP (Table 4) provides a compact summary of multiple key event measurements in male mice with a very potent mouse CAR activator. Specific aspects of these results are described in the assessment of each Key Event Relationship, where appropriate.

Considerations for Potential Applications of the AOP (optional)

This AOP outlines a set of key events that have strong empirical data with several different molecules supporting the dose-concordance, temporal relationships and essentiality of the key events to causing the Adverse Outcome. Accordingly, this AOP provides a mechanistic basis for the development of Integrated Approaches to Testing and Assessment (IATA; Tollefsen et al., 2014). More specifically, the good dose concordance between the early key events and the final AO (rodent liver tumors) can support future risk assessments by demonstrating that derived endpoints (e.g. BMDL values or NOAELs) based on critical early key events are well correlated to later, downstream effects. See Geter et al. (2014) for examples of BMDL values of transcriptomic changes as well as early apical key events/associative events in mice treated with phenobarbital.

Another area of potential applications of this AOP is to illustrate the extent of data needed to establish that the AOP is operative for a regulated chemical, and to assess the lack of human relevance of liver tumors that were produced by that chemical. Table 1 illustrates the MIE, KEs, AEs and AO for this AOP, plus some examples of the typical data that can be generated based on today’s (2016) technology to demonstrate these key events and associative events. This listing in Table 1 should not be viewed as an absolute set of requirements, but as a helpful guide. In particular, as technological advances come into common usage and as our understanding of this AOP progress with time, the typical set of studies and collected data will undoubtedly be refined.

Strong data are provided that indicate the critical key events have been shown to occur in rats and mice, but not in other mammalian species including hamsters, guinea pigs and humans. As regulatory and scientific acceptance of the lack of human relevance for multiple agents that produce rodent liver tumors via this AOP increases with time, the opportunity to reduce animal use by only assessing a limited, critical set of key events (perhaps via in vitro methods) is a potential future application of the AOP. Such approaches could avoid the need for expensive, large-scale mode of action research studies. Some specific key events that may lend themselves to these alternative approaches are:

  • CAR activation assays: possibly via CAR transactivation assays for the different species of interest (see Currie et al., 2014; Omiecinski et al., 2011)
  • Excluding alternative modes of action: via commercially available PPARα, AhR and similar nuclear receptor transactivation assays across species
  • Transcriptomic approaches to demonstrating a CAR signature, and deriving benchmark doses from these endpoints (see Geter et al., 2014; Oshida et al., 2015a; Oshida et al., 2015b; Oshida et al., 2015c)
  • Measuring additional key events as an add-on to existing subchronic and chronic rodent studies, by analysis of formalin-fixed paraffin embedded tissues for important markers such as Ki-67 (a marker of cell proliferation that can be quantified via IHC), or transcriptomic changes in key genes or pathways by use of laser-capture microdissection (LCM) followed by microarray or RT-PCR analysis of gene expression profiles (Coudry et al., 2007; Muskhelishvili et al., 2003).

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