API

Aop: 41

AOP Title

?


Sustained AhR Activation leading to Rodent Liver Tumours

Short name:

?

Sustained AhR Activation leading to Rodent Liver Tumours

Authors

?


Richard A Becker, American Chemical Council (ACC) on behalf of the Business Industry Advisory Committee (BIAC) email:Rick_Becker@americanchemistry.com Contributing authors to the development of this AOP are: Ted Simon (Ted Simon LLC), Robert Budinsky, (The Dow Chemical Company), Grace Patlewicz, (DuPont), Craig Rowlands, (The Dow Chemical Company).

Point of Contact

?


Rick Becker

Contributors

?


  • Rick Becker

Status

?

Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.7 Included in OECD Work Plan


This AOP was last modified on December 02, 2016 11:59

?

Revision dates for related pages

Page Revision Date/Time
N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects November 29, 2016 18:59
Activation, Long term AHR receptor driven direct and indirect gene expression changes December 02, 2016 11:22
Changes/Inhibition, Cellular Homeostasis and Apoptosis September 16, 2017 10:15
Alterations, Cellular proliferation / hyperplasia September 16, 2017 10:15
Formation, Hepatocellular and Bile duct tumors September 16, 2017 10:15
Activation, Long term AHR receptor driven direct and indirect gene expression changes leads to Changes/Inhibition, Cellular Homeostasis and Apoptosis November 29, 2016 20:41
Activation, Long term AHR receptor driven direct and indirect gene expression changes leads to N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects November 29, 2016 19:58
Activation, Long term AHR receptor driven direct and indirect gene expression changes leads to Alterations, Cellular proliferation / hyperplasia November 29, 2016 20:41
Activation, Long term AHR receptor driven direct and indirect gene expression changes leads to Formation, Hepatocellular and Bile duct tumors November 29, 2016 20:41
Changes/Inhibition, Cellular Homeostasis and Apoptosis leads to N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects December 02, 2016 11:47
N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects leads to Alterations, Cellular proliferation / hyperplasia December 02, 2016 11:49
Alterations, Cellular proliferation / hyperplasia leads to Formation, Hepatocellular and Bile duct tumors December 02, 2016 11:49

Abstract

?


An Adverse Outcome Pathway (AOP) represents the existing knowledge of a biological pathway leading from initial molecular interactions of a toxicant and progressing through a series of key events (KEs), culminating with an apical adverse outcome (AO) that has to be of regulatory relevance. An AOP based on the mode of action (MOA) of rodent liver tumor promotion by dioxin-like compounds (DLCs) has been developed and the weight of evidence (WoE) of key event relationships (KERs) evaluated using evolved Bradford Hill considerations. Dioxins and DLCs are potent aryl hydrocarbon receptor (AHR) ligands that cause a range of species-specific adverse outcomes. The occurrence of KEs is necessary for inducing downstream biological responses and KEs may occur at the molecular, cellular, tissue and organ levels. The common convention is that an AOP begins with the toxicant interaction with a biological response element; for this AOP, this initial event is binding of a DLC ligand to the AHR. Data from mechanistic studies, lifetime bioassays and approximately thirty initiation-promotion studies have established a number of substances, including dioxin-like chemicals and indole-3-carbinol from brassica vegetables, as rat liver tumor promoters. Such studies clearly show that sustained AHR activation, weeks or months in duration, is necessary to induce rodent liver tumor promotion; hence, sustained AHR activation is deemed the molecular initiating event (MIE). After this MIE, subsequent KEs are 1) changes in cellular growth homeostasis likely associated with expression changes in a number of genes and observed as development of hepatic foci and decreases in apoptosis within foci; 2) extensive liver toxicity observed as the constellation of effects called toxic hepatopathy; 3) cellular proliferation and hyperplasia in several hepatic cell types. This progression of KEs culminates in the AO, the development of hepatocellular adenomas and carcinomas and cholangiolar carcinomas. A rich data set provides both qualitative and quantitative knowledge of the progression of this AOP through KEs and the KERs. Thus, the WoE for this AOP is judged to be strong. Species-specific effects of dioxins and DLCs are well known -- humans are less responsive than rodents and rodent species differ in sensitivity between strains. Consequently, application of this AOP to evaluate potential human health risks must take these differences into account.

Please also see Becker, R.A., Patlewicz, G., Simon, T.W., Rowlands, J.C., Budinsky, R.A. 2015. The adverse outcome pathway for rodent liver tumor promotion by sustained activation of the aryl hydrocarbon receptor. Regul. Toxicol. Pharmacol. 73, 172-190: PMID: 26145830. The file is open access.


Background (optional)

?



Summary of the AOP

?



Stressors

?


Molecular Initiating Event

?

Title Short name
Activation, Long term AHR receptor driven direct and indirect gene expression changes Activation, Long term AHR receptor driven direct and indirect gene expression changes

Key Events

?

Title Short name
N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects N/A, Hepatotoxicity, Hepatopathy, including a constellation of observable effects
Changes/Inhibition, Cellular Homeostasis and Apoptosis Changes/Inhibition, Cellular Homeostasis and Apoptosis
Alterations, Cellular proliferation / hyperplasia Alterations, Cellular proliferation / hyperplasia

Adverse Outcome

?

Title Short name
Formation, Hepatocellular and Bile duct tumors Formation, Hepatocellular and Bile duct tumors

Relationships Between Two Key Events (Including MIEs and AOs)

?

Network View

?

 

Life Stage Applicability

?


Taxonomic Applicability

?

Term Scientific Term Evidence Link
Rattus sp. ABTC 42503 Rattus sp. ABTC 42503 Strong NCBI
Mus sp. 2000082 Mus sp. 2000082 Strong NCBI

Sex Applicability

?


Graphical Representation

?

Click to download graphical representation template

W1siziisijiwmtyvmtevmjkvnjyzr3jhcghpy2fsx1jlchjlc2vudgf0aw9ux0fiul9bt1aucg5nil0swyjwiiwidgh1bwiilci1mdb4ntawil1d?sha=90e69c5ee113c0ff

Overall Assessment of the AOP

?



The Bradford Hill considerations, which is an approach consistent with the U.S. Environmental Protection Agency’s Guidelines for Carcinogen Risk Assessment available from: http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-25-05.PDF as well as the WHO/IPCS’s human relevance-mode-of-action framework provides the framework for putting the proposed AOP into a weight-of-evidence evaluation. These considerations include dose-response, temporality, strength, consistency, specificity and biological plausibility of the proposed association (the AOP in this case). Alternative AOP propositions must be accounted for and either ruled in or out as part of applying the Bradford Hill considerations. To finalise the Bradford Hill assessment, the AOP template requires an examination of the uncertainties, inconsistencies, data gaps, and the quantitative nature of the KE (as well as the AE and ModFs).

The Bradford Hill (BH) considerations (dose-response, temporality, strength, consistency, specificity and biological plausibility of the proposed association) form the basis for evaluating weight of evidence within the U.S. Environmental Protection Agency's Guidelines for Carcinogen Risk Assessment, the WHO/IPCS human relevance-MOA framework, the key events/dose-response frame- work (KEDRF) (Dellarco and Fenner-Crisp, 2012; Fenner-Crisp, 2012; Julien et al., 2009; Meek et al., 2003; Meek, 2008; OECD, 2013; USEPA, 2005). The BH considerations have recently been updated and additionally tailored for AOPs by the OECD to facilitate evaluations of KEs and KERs as well as the overall AOP (Meek et al., 2013, 2014a, 2014b; OECD, 2014; Becker et al., 2015).

In the tables below, we summarize the weight of evidence evaluation conducted using these AOP-tailored BH considerations of biological plausibility, essentiality and empirical evidence for the sustained AHR activation rodent liver tumor promotion AOP.

Domain of Applicability

?


Essentiality of the Key Events

?

The defining question contained in the OECD AOP guidance (OECD, 2014) for evaluation of essentiality is “are downstream KEs and/or the AO prevented if an upstream KE is blocked?” Overall, the evidence in support of essentiality for sustained AHR activation, the MIE, is strong. There is direct evidence of essentiality from the stop- exposure group in the cancer bioassay; the 100 ng/kg/d dose of TCDD was stopped after 30 weeks and at the 2-year termination, no statistically significant increase in tumor frequency was observed (NTP, 2006a; NTP, 2006b; NTP, 2006c). This observation also in- dicates that the MIE of sustained AHR activation requires more than 30 weeks of continuous exposure and is consistent with the general onset of hepatopathy around the same time (Hailey et al., 2005). Additional support for essentiality comes from studies that show that KEs fail to occur when AHR activity is lost through mutation, polymorphism or knockdown (Gasiewicz et al., 2008). Further- more, the loss of AHR responsivity to ligand-activation has been confirmed in reduction and/or loss of ligand-mediated gene tran- scription and resistance to TCDD-induced toxicity (Harrill et al., 2013). Conversely, constitutive AHR activity in mice increased the incidence of tumors and hepatotoxicity (Andersson et al., 2002; Brunnberg et al., 2006; Chopra and Schrenk, 2011; Moennikes et al., 2004).

 

Support for Essentiality of KEs Defining Question High (Strong) Moderate Weak
  Are downstream key events and/or the AO prevented if an upstream key event is blocked? [e.g., stop/reversibility studies, antagonism, knock out models, etc.) Multiple lines of experimental evidence illustrating essentiality for several of the key events There is at least one line of experimental evidence indicating essentiality of an important key event Indirect or no experimental evidence of the essentiality of any of the key events
Pre-MIE: Binding of ligands to the AHR Essentiality of the pre-MIE is Strong.

Rationale: Binding to the AHR is a necessary element and downstream KEs do not occur in knock-out animals.

MIE: Sustained AHR Activation Essentiality of the MIE is Strong.

Rationale: Extensive qualitative and quantitative information showing that downstream KEs occur in with increasing time and extent of continued AHR activation

KE#1: Changes in Cellular Homeostasis and Inhibition of Apoptosis Essentiality of the KE1 is Strong

Rationale: Growth of Altered hepatic foci has been explored in many initiation-promotion studies

KE#2: Hepatoxicity, Hepatopathy Essentiality of the KE2 is Strong

Rationale: The regenerative nature of the liver is such that the extensive hepatopathy induced by sustained AHR activation leads to a highly proliferative environment in the liver.

KE#3: Alterations in Cellular Proliferation/Hyperplasia Essentiality of the KE3 is Strong

Rationale: Hyperplasia has been strongly linked to the induction of cancer in many systems.

 


Weight of Evidence Summary

?

This section provides brief descriptions of the essentiality of each of the KEs and the biological plausibility, empirical support and any uncertainties or inconsistencies in the key event relationships (KERs). These descriptions are consistent with the table presented in the AOP User’s Guide from OECD. Whilst the overall confidence in the AOP as a result of these evaluations is high, the challenge is to apply this knowledge for a regulatory purpose. An additional section is included below on the Application of the AOP. This provides a discussion of the possible regulatory uses of this AOP.

Empirical Evidence

The OECD AOP guidance (OECD, 2014) for evaluation of empirical evidence focuses on dose-response, temporality and incidence concordance. Defining questions include: “Does the empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses and earlier time points than KEdown and is the incidence of KEup > than that for KEdown?” Highly consistent dose-response relationships (KERs) along the sequence of KEs in this AOP exhibit dose- and time-concordance. This consistency in the concordance of both temporality and incidence supports a weight of evidence determination of high for the empirical evidence underpinning this AOP.

When the KEs and associative events are placed in sequential order based on dose and temporality, the dose-response slopes from Hill dose-response model fits of the data increase in value. Thus, the later KEs occur with a steeper slope than early KEs and the number of KEs observed increases as a function of dose and time (Simon et al., 2009; Budinsky et al., 2014).

Induction of xenobiotic metabolizing enzymes is one the earliest and most sensitive responses to AHR activation (Budinsky et al., 2010; Silkworth et al., 2005). Xenobiotic metabolizing enzyme induction reflects acute transcriptional and proteomic changes that are more aligned with the concept of a pre-MIE and thus provides an associative event for AHR activation. Since measurable enzyme induction persists for at least one year, we used the AUC of a biomarker for this enzyme induction as a measure of sustained AHR activation. Both Hill equation coefficients and half maximal concentrations increase with increasing values of sustained AHR activation and reflect dose-dependent transitions as KEs occur at the various levels of biological organization (Simon et al., 2009; Budinsky et al., 2014).

The dose-response temporality table above depicts the KEs increasing in frequency in both dose and time (Meek et al., 2013; Simon et al., 2014). This table is an essential requirement of the human relevance MOA framework and is recommended in the OECD AOP guidance (OECD, 2014). Here, the value of the sustained AHR activation index has been provided for each dose-time combination. One can easily see the increase in sustained AHR activation due to the increase in dose going down the table and the increase in duration going across the table.

The need for AHR activation for a sustained period of time, i.e. temporal concordance, is supported by the stop-exposure group in the TCDD cancer bioassay, which showed that when the administration of 100 ng/kg/d TCDD was stopped after 30 weeks, a statistically significant increase in tumor frequency was not observed (NTP, 2006a). This observation also indicates that the AHR activation needs to be sustained for more than 30 weeks for KE#2 to occur (Hailey et al., 2005).

 

Concordance of dose-response relationships

Dose-Time Concordance Table

Empirical evidence: application of the dose and temporal concordance AOP weight of evidence considerations for Key Events (KEs) at dose/time combination. This table is based on NTP (2006a), Teeguarden et al. (1999) and Maronpot et al. (1993). The dose in the left most column shows the range of average liver concentration (ng/kg) from 14 weeks to 2 years from NTP (2006a). The number in parentheses is the administered gavage dose in ng/kg/d. The numerical value of the sustained AHR activation index (ppb-weeks) is shown for each dose/time combination; the calculation of this value is described on the MIE page.

Dose Increasing Time -->
  Weeks to Months (14 wk) Months (31 wk) 1 yr (53 wk) 2 yr (104 wk)
5-20 (0.1)** 0.1 ppb-wk 0.5 ppb-wk 1 ppb-wk 2.4 ppb-wk
100-200 (1)** 1.7 ppb-wk 4.7 ppb-wk 8.8 ppb-wk 19 ppb-wk
450-650 (3)** 4.3 ppb-wk 11 ppb-wk 19 ppb-wk 40 ppb-wk
1500-2000 (10) MIE = 8.3 ppb-wk

Apoptosis Inhibition (KE#1)

MIE = 19 ppb-wk

Apoptosis Inhibition (KE#1)(presumed)

MIE = 34 ppb-wk MIE = 69 ppb-wk

Toxic Hepatopathy (KE#2) Hyperplasia/Proliferation (KE#3)

3500-4200 (22) MIE = 11 ppb-wk

Apoptosis Inhibition (KE#1)(presumed)

MIE = 24 ppb-wk

Apoptosis Inhibition (KE#1)(presumed)

MIE = 42 ppb-wk MIE = 83 ppb-wk

Toxic Hepatopathy (KE#2) Hyperplasia/Proliferation (KE#3)

7000-8000 (46) MIE = 12 ppb-wk

Apoptosis Inhibition (KE#1)(presumed)

MIE = 27 ppb-wk

Apoptosis Inhibition (KE#1)(presumed)

MIE = 47 ppb-wk

Toxic Hepatopathy (KE#2)

MIE = 93 ppb-wk

Toxic Hepatopathy (KE#2) Hyperplasia/Proliferation (KE#3) Cholangiocarcinomas (AO)

15000-17000 (100) MIE = 13 ppb-wk

Apoptosis Inhibition (KE#1)

MIE = 29 ppb-wk

Apoptosis Inhibition (KE#1) Toxic Hepatopathy (KE#2)

MIE = 50 ppb-wk

Toxic Hepatopathy (KE#2) Hyperplasia/Proliferation (KE#3)

MIE = 98 ppb-wk

Toxic Hepatopathy (KE#2) Hyperplasia/Proliferation (KE#3) Cholangiocarcinomas (AO) Hepatic Adenomas (AO)

    • these dose levels are insufficient to induce the degree of sustained AHR activation necessary to exceed the threshold of homeostasis/adaptation; exceeding this threshold is required to trigger the MIE.

When the KEs and associative events are placed in sequential order based on dose and temporality, the dose-response slopes from Hill dose-response model fits of the data increase in value. Thus, the later KEs occur with a steeper slope than early KEs and the number of KEs observed increases as a function of dose and time (Table 2) (Simon et al., 2009; Budinsky et al., 2014).

Induction of xenobiotic metabolizing enzymes is one the earliest and most sensitive responses to AHR activation (Budinsky et al., 2010; Silkworth et al., 2005). Xenobiotic metabolizing enzyme in- duction reflects acute transcriptional and proteomic changes that are more aligned with the concept of a pre-MIE and thus provides an associative event for AHR activation. Since measurable enzyme induction persists for at least one year, we used the AUC of a biomarker for this enzyme induction as a measure of sustained AHR activation. Both Hill equation coefficients and half maximal concentrations increase with increasing values of sustained AHR activation and reflect dose-dependent transitions as KEs occur at the various levels of biological organization (Simon et al., 2009; Budinsky et al., 2014).

The table shown above is the dose-response temporality table and depicts the KEs increasing in both dose and time (Meek et al., 2013; Simon et al., 2014). This table is an essential requirement of the human relevance MOA framework and is recommended in the OECD AOP guidance (OECD, 2014). Here, the value of the sustained AHR activation index has been provided for each dose-time combination. One can easily see the increase in sustained AHR activation due to the increase in dose going down the table and the increase in duration going across the table.

The need for AHR activation for a sustained period of time, i.e. temporal concordance, is supported by the stop-exposure group in the TCDD cancer bioassay, which showed that when the administration of 100 ng/kg/d TCDD was stopped after 30 weeks, a statistically significant increase in tumor frequency was not observed (NTP, 2006a). This observation also indicates that the AHR activation needs to be sustained for more than 30 weeks for KE#2 to occur (Hailey et al., 2005).

Support for the Biological Plausibility of the KERs

The OECD AOP guidance (OECD, 2014) for evaluation of biological plausibility of an AOP provides this defining question for evaluating biological plausibility: “is there a mechanistic (i.e., structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?” Under the OECD guidance, a high degree of confidence is afforded when there is an established mechanistic basis and “extensive understanding of the KER based on extensive previous documentation and broad acceptance.” For biological plausibility, for this AOP, the WoE for each KER is judged to be strong, as is the WoE for the overall AOP.

All the elements in this AOP are strongly associated with the biological steps and elements of carcinogenesis (Hanahan and Weinberg, 2011). First, there is extensive body of mechanistic evi- dence in support the biological plausibility of this MOA (see recent review by Budinsky et al., 2014). Further, the relationships between sustained AHR activation and 1) decreased intrafocal apoptosis (KE#1); 2) increased cell proliferation (KE#2); 3) toxic hepatopathy (KE#3); and 4) eventual tumor formation (AO) are evident from a surfeit of published studies (e.g., Fig. 4). Moreover, overall consistency with knowledge of the pathogenesis of liver tumor promo- tion is supported by replication of events related to tumor promotion across different laboratories and the multiple lines of evidence for sustained AHR activation acting as a mechanism of liver tumor promotion. Thus, the AOP is well supported by the KEs, consistent with the biology of carcinogenesis and the events of tumor promotion (Dietrich and Kaina, 2010; Gasiewicz et al., 2008; Roberts et al., 1997).

The unique sensitivity of the female rat response suggests a possible role for estrogen as a modulating factor in the tumorigenic MOA. Estrogen is an established co-promoter of tumorigenesis and thus may play a role in the MOA (Graham et al., 1988; Hiraku et al., 2001; Lucier et al., 1991; Vickers and Lucier, 1996; Vickers et al., 1989). Crosstalk between the AHR pathway and the estrogen receptor pathway may also be a contributing factor (Matthews and Gustafsson, 2006). Such receptor mediated cross talk is consistent with the sustained AHR MOA.

 

Support for Biological Plausibility of KERs Defining Question High (Strong) Moderate Weak
  a) Is there a mechanistic (i.e., structural or functional) relationship between KEup and KEdown consistent with established biological knowledge? Extensive understanding of the KER based on extensive previous documentation and broad acceptance (e.g., mutation leading to tumors)

-Established mechanistic basis

The KER is plausible but scientific understanding is not completely established. Only limited or indirect evidence for KER (i.e., based on empirical support, only (See 3.)
Binding of Ligands to the AHR leading to sustained activation of the AHR Biological Plausibility of the pre-MIE => MIE is Strong

Rationale: Long-established knowledge of and extensive research on dioxin-like chemicals and other AHR ligands.

Sustained AHR Activation directly leading to Changes in Cellular Homeostasis and Inhibition of Apoptosis Biological Plausibility of MIE => KE1 is Strong.

Rationale: Direct empirical evidence showing continued application of AHR activators leads to growth alteration of altered hepatic foci.

Sustained AHR Activation indirectly leading to Hepatoxicity and Hepatopathy Biological Plausibility of MIE => KE2 is Strong

Rationale: Long established knowledge : Empirical data from two-year bioassays using AHR activation and sustained administration

Sustained AHR Activation indirectly leading to Alterations in Cellular Proliferation and Hyperplasia Biological Plausibility of MIE => KE3 is Strong

Rationale: Empirical data from two-year bioassays using AHR activation and sustained administration

Sustained AHR Activation indirectly leading to Hepatocellular Adenomas and Cholangiocarcinomas Biological Plausibility of MIE => AO is Strong

Rationale: Empirical data from two-year bioassays using AHR activation and sustained administration

 

Uncertainties, Inconsistencies and Conflicting Evidence for KERs and the AOP

The evidence supporting the KERs and AOP is strong. Alternative MOA(s) or KEs and KE elements can be examined to help ascertain the confidence in the MOA considered most likely (Boobis et al., 2006, 2008, 2009; Cohen et al., 2003; Cohen et al., 2004; Julien et al., 2009; Meek et al., 2003; Meek, 2008; Seed et al., 2005; Sonich-Mullin et al., 2001; USEPA, 2005).

One such alternative MOA would be that DLCs act to produce liver tumors in rodents by a mutagenic mechanism. However, there is substantial evidence that DLCs are neither mutagenic nor genotoxic compounds and thus do not act by a mutagenic MOA (Bock and Kohle, 2005; Dragan and Schrenk, 2000; Knerr et al., 2006; Poland and Glover, 1979; Randerath et al., 1990; Schwarz et al., 2000; Turteltaub et al., 1990; Wassom et al., 1977; Whysner and Williams, 1996).

Effects on gap junctions or induction of oxidative stress are two other potential mechanisms. TCDD disrupts normal gap junction activity and intercellular communication in rat primary hepatocytes and WB-344 cells (Andrysík et al., 2013; Bager et al., 1997; Herrmann et al., 2002; Weiss et al., 2008). Further research is needed to understand the contribution of this mechanism to DLC- induced rodent liver tumor formation. Oxidative stress appears less likely as an alternative MOA and may be a late-occurring associative event due to continued high activity of phase 1 mixed function oxidases and accompanying cytotoxicity.

In summary, the WoE in support of sustained AHR activation leading to changes in cellular growth homeostasis and eventually promotion of liver tumors in rodents is strong. The WoE supporting the alternative MOAs is much weaker.

Uncertainty and Conflicting Evidence for KERs and AOP Defining Question High (Strong) Moderate Weak
  Are there inconsistencies in empirical support across taxa, species which don’t align with appropriate pattern for hypothesized KERs and AOP?

Are there significant knowledge gaps or uncertainties with regard to the relationship between the KEs and overall AOP?

No (or very few) knowledge gaps or inconsistent / conflicting lines of evidence.) Some inconsistent evidence but which can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.) Contradictory evidence in for which no plausible explanation is known.
Binding of Ligands to the AHR leading to sustained activation of the AHR Inconsistencies / Uncertainties of Pre-MIE => MIE is Strong

Rationale: Highly certain. While a large number of ligands bind to and activate the AHR, only the biologically persistent ligands, such as dioxin-like chemicals produce the MIE, sustained AHR activation

Sustained AHR Activation directly leading to Changes in Cellular Homeostasis and Inhibition of Apoptosis Inconsistencies / Uncertainties of MIE => KE1 is Strong.

Rationale: A large number of initiation-promotion studies with TCDD or other dioxin-like chemicals documented changes in both cell proliferation and inhibition of apoptosis within alterered hepatic foci.

Sustained AHR Activation indirectly leading to Hepatoxicity and Hepatopathy Inconsistencies / Uncertainties of MIE => KE2 is 'Strong

Rationale: Although the exact mechanism is not known, sustained AHR activation often leads to increased concentrations of ROS that may be a factor in generating cytotoxicity. Hepatopathy is common effect of sustained adiministration of AHR activators.

Sustained AHR Activation indirectly leading to Alterations in Cellular Proliferation and Hyperplasia Inconsistencies / Uncertainties of MIE => KE3 is Strong

Rationale: All the biologically persistent AHR activators such as DLCs damage the liver to a sufficient extent that a proliferative/regenerative environment is created in the organ.

Sustained AHR Activation indirectly leading to Hepatocellular Adenomas and Cholangiocarcinomas Inconsistencies / Uncertainties of MIE => AO is Strong

Rationale: These tumors are outcomes of the AHF growth in KE#1 and the increased proliferation in KE#3. Both are induced by the MIE, sustained AHR activation. A large number of bioassays have documented the fact that persistent AHR ligands produce liver tumors in rodents.

 


Quantitative Considerations

?

While binding of ligand to the AHR is identified as a pre-MIE, sustained AHR activation by persistent ligands such as DLCs is linked qualitatively and quantitatively to both downstream KEs and the AO. These linkages notwithstanding, additional work is needed to develop and evaluate such a prediction model before the MIE of sustained AHR activation can be used in a quantitative prediction model of the AO. Any prediction model based on this AOP needs to consider the unique aspects of the AHR and its response to DLCs, including the involvement of initiated or partially differentiated stem cells, and such a model would need evaluation/validation for its intended use (Cox et al., 2014; Patlewicz et al., 2015).


Considerations for Potential Applications of the AOP (optional)

?


The OECD guidance for AOP development (OECD, 2013) suggests a number of potential uses for AOPs. These include 1) category formation for read-across, 2) integrated approaches for testing and assessment 3) development or refinement of test methods such as OECD test guidelines and 4) hazard identification (classification/ labeling) and risk assessment. The use of a specific AOP for any one or more of these applications depends on scientific confidence in the AOP for each specific use. An AOP can offer practical utility in certain applications even if confidence is not sufficient to quantitatively predict the AO from the MIE. Application of the sustained AHR activation AOP for several applications was described in brief in Patlewicz et al. (2015); Below the application of and confidence in this AOP is discussed in more detail.

Which Key Events can be used to predict the AO?

To date, no quantitative models have been developed to predict the adverse outcome from AHR activation by ligand binding. With the exception of DLCs, PCDDs, PCDFs and co-planar PCBs, this predictive capability is highly uncertain for the plethora of AHR ligands. For example, indole 3-carbinol is an AHR ligand occurring in cruciferous vegetables and acts as a cancer chemopreventive agent. In the stomach, indole 3-carbinol forms 3,3- diinolylmethane, a potent AHR ligand that has shown promise for preventing tumor reoccurrence in humans (Banerjee et al., 2011). Dietary administration of indole-3-carbinol for 23 weeks inhibited tumor formation in rats initiated with diethylnitrosamine (Tanaka et al., 1990). However, also in diethylnitrosamine-initiated rats, prolonged indole 3-carbinol administration (>26 weeks) increased the progression of altered hepatic foci to hepatocellular adenomas (Yamamoto et al., 2013). A recent NTP 2-year cancer bioassay failed to demonstrate indole 3-carbinol as a liver tumor promoter in fe- male rats (NTP, 2014). Furthermore, endogenous AHR ligands and naturally occurring exogenous ligands occurring in foods have cancer-preventive properties and likely contribute to a relatively high level of AHR activation activity in human blood (Connor et al., 2008; Navarro et al., 2009, 2011; Peterson et al., 2009; Wincent et al., 2009). These naturally occurring and endogenous ligands induce both their own metabolism and that of other AHR ligands through increased induction of xenobiotic metabolizing enzymes. The transient metabolic increase may be one aspect of the pre- ventive effect against sustained AHR activation that would lead to KEs related to liver tumor promotion.

Neither binding of ligand to the AHR nor short-term transcriptional changes and cellular responses are sufficient to produce liver tumors in rats. Strains of rats that show resistance towards the toxic and carcinogenic effects of DLCs express different genomic profiles outside of the conserved core battery response (Boutros et al., 2011; Yao et al., 2012). Acute genomic changes do not appear to be pre- dictive for the cancer endpoint (Fielden et al., 2011; Ovando et al., 2010). AHR activation-induced transcriptional changes occur within hours of ligand activation; yet the subsequent KEs and AO require months of sustained AHR activation for tumors to occur. Hence, the distinction between short-term and sustained activation of the AHR is an important one and AHR activation must be sus- tained for more than 30% of the rodent lifespan to result in tumor promotion.

While binding of ligand to the AHR is identified as a pre-MIE, sustained AHR activation by persistent ligands such as DLCs is linked qualitatively and quantitatively to both downstream KEs and the AO. These linkages notwithstanding, additional work is needed to develop and evaluate such a prediction model before the MIE of sustained AHR activation can be used in a quantitative prediction model of the AO. Any prediction model based on this AOP needs to consider the unique aspects of the AHR and its response to DLCs, including the involvement of initiated or partially differentiated stem cells, and such a model would need evaluation/validation for its intended use (Cox et al., 2014; Patlewicz et al., 2015).

Using this AOP for grouping chemicals into chemical categories for read-across

Without some measure of sustained activation, the use of pre- MIEs for any purpose other than preliminary screening is problematic as a predictive criteria for liver tumor promotion. Evidence clearly shows it is the combination of sustained AHR activation and the subsequent biological changes involving complex parenchymal and non-parenchymal cell interactions that underlie the hepatotoxicity, the increase in cell proliferation and the apical tumor response. Hence, the MIE is defined as sustained AHR activation, and not simply AHR activation. In addition, the promiscuity of the AHR and the species- and strain-specificity of the initial genomic responses suggest that category development may prove a challenge (Denison, 2011; Dere, 2011).

Using this AOP for integrated approaches to testing and assessment (IATA)

The most straightforward use of this AOP within an integrated testing and assessment approach for hazard evaluation would be to determine the potential for a substance to activate the AHR in a sustained manner with long-term changes in gene transcription involving multiple cell types, which leads to increased liver cell proliferation. An IATA decision-tree approach, for illustrative purposes has been already presented by Patlewicz et al. (2015).

The initial steps in the IATA focus on evaluating molecular and cellular events related to AHR binding and transcriptional activation using rapid and cost effective in silico or in vitro assays. Compounds found to be inactive in such assays would not proceed forward into further testing.

At the present time, there is insufficient understanding to permit the use transcription profiling as a metric of sustained AHR activation to quantitatively predict development of rat liver foci and liver tumors. Therefore, the IATA proposes that substances found to be active in the AHR mechanistic assays be subjected to a decision framework for further evaluating the potential to act as rodent liver tumor promoter. For example, a subchronic study, utilizing an appropriate dosing regimen, may be able to rule-in or rule-out the substance's ability to trigger the critical histological components of hepatopathy (Hailey et al., 2005). Or a rodent liver initiation- promotion assay could be considered, though interpretation can be challenging (Tanaka et al., 1990; Yamamoto et al., 2013; NTP, 2014). Patlewicz et al. (2015) also illustrate how exposure information can be used in conjunction with the AOP to inform IATA decisions.

Using this AOP to inform test method development or refinement

An IATA consisting of a suite of in vitro and in vivo (e.g., sub- chronic) assays to predict hepatopathy, a complex histological response, may be needed to differentiate AHR ligands with and without liver tumor promotion potential. Theoretically, it may be plausible to consider using a combination of AHR-binding, AHR- transcriptional activation and rat liver initiation-promotion assays to develop a prediction model for sustained AHR activation- induced rat liver tumors. Therefore, within the OECD test guidelines program, it may be worthwhile to consider developing performance criteria that could be applied to judge the scientific quality and reliability of in vitro AHR-binding and transactivation assays, liver stem cell assays, as well as a validated test guideline for a rat liver tumor (hepatic foci) initiation-promotion assay.


References

?


Abraham, K., Geusau, A., Tosun, Y., Helge, H., Bauer, S., Brockmoller, J., 2002. Severe 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intoxication: insights into the measurement of hepatic cytochrome P450 1A2 induction. Clin. Pharmacol. Ther. 72, 163-174.

Andersen M.E., Preston R.J., Maier A., Willis A.M., Patterson J. 2014. Dose-response approaches for nuclear receptor-mediated modes of action for liver carcinogenicity: Results of a workshop, Crit. Rev. Toxicol. 44, 50-63.

Andrysík, Z., Prochazkova, J., Kabatkova, M., Umannova, L., Simeckova, P., Kohoutek, J., Kozubík, A., Machala, M., Vondracek, J., 2013. Aryl hydrocarbon receptor-mediated disruption of contact inhibition is associated with connexin43 downregulation and inhibition of gap junctional intercellular communication. Arch. Toxicol. 87, 491-503.

Ankley G.T., Bennett R.S., Erickson R.J., Hoff D.J., Hornung M.W., Johnson R.D., Mount D.R., Nichols J.W., Russom C.L., Schmieder P.K., Serrrano J.A., Tietge J.E., Villeneuve D.L. 2010. Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment, Environ. Toxicol. Chem. 29, 730-741.

Bager, Y., Lindebro, M.C., Martel, P., Chaumontet, C., W€arngård, L., 1997. Altered function, localization and phosphorylation of gap junctions in rat liver epithelial, IAR 20, cells after treatment with PCBs or TCDD. Environ. Toxicol. Pharmacol. 3, 257- 266.

Banerjee, S., Kong, D., Wang, Z., Bao, B., Hillman, G.G., Sarkar, F.H., 2011. Attenuation of multi-targeted proliferation-linked signaling by 3,30 -diindolylmethane (DIM): from bench to clinic. Mutat. Res. 728, 47-66.

Becker, R.A., Patlewicz, G., Simon, T.W., Rowlands, J.C., Budinsky, R.A. 2015. The adverse outcome pathway for rodent liver tumor promotion by sustained activation of the aryl hydrocarbon receptor. Regul. Toxicol. Pharmacol. 73, 172-190.

Black, M.B., Budinsky, R.A., Dombkowski, A., Lecluyse, E.L., Ferguson, S.S., Thomas, R.S., Rowlands, J.C., 2012. Cross-species comparisons of transcriptomic alterations in human and rat primary hepatocytes exposed to 2,3,7,8- tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 127, 199-215.

Boobis, A.R., Cohen, S.M., Dellarco, V., McGregor, D., Meek, M.E.B., Vickers, C., Willcocks, D., Farland, W., 2006. IPCS framework for analyzing the relevance of a cancer mode of action for humans. Crit. Rev. Toxicol. 36, 781-792.

Boobis, A.R., Daston, G.P., Preston, R.J., Olin, S.S., 2009. Application of key events analysis to chemical carcinogens and noncarcinogens. Crit. Rev. Food Sci. Nutr. 49, 690-707.

Boobis, A.R., Doe, J.E., Heinrich-Hirsch, B., Meek, M.E.B., Munn, S., Ruchirawat, M., Schlatter, J., Seed, J., Vickers, C., 2008. IPCS framework for analyzing the rele- vance of a noncancer mode of action for humans. Crit. Rev. Toxicol. 38, 87-96.

Boutros, P.C., Yao, C.Q., Watson, J.D., Wu, A.H., Moffat, I.D., Prokopec, S.D., Smith, A.B., Okey, A.B., Pohjanvirta, R., 2011. Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity. Toxicol. Appl. Pharmacol. 251, 119-129.

Coenraads, P.J., Olie, K., Tang, N.J., 1999. Blood lipid concentrations of dioxins and dibenzofurans causing chloracne. Br. J. Dermatol. 141, 694-697.

Connor, K.T., Harris, M.A., Edwards, M.R., Budinsky, R.A., Clark, G.C., Chu, A.C., Finley, B.L., Rowlands, J.C., 2008. AH receptor agonist activity in human blood measured with a cell-based bioassay: evidence for naturally occurring AH re- ceptor ligands in vivo. J. Expo. Sci. Environ. Epidemiol. 18, 369-380.

Becker et al. 2015. Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence, Regul. Toxicol. Pharmacol. 72, 514-37.

Beebe L.E., Fornwald L.W., Diwan B.A., Anver M.R., Anderson L.M. 1995. Promotion of N-nitrosodiethylamine-initiated hepatocellular tumors and hepatoblastomas by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 in C57BL/6, DBA/2, and B6D2F1 mice, Cancer. Res. 55, 4875-4880.

Beischlag, T.V., Luis Morales, J., Hollingshead, B.D., Perdew, G.H., 2008. The aryl hydrocarbon receptor complex and the control of gene expression. Crit. Rev. Eukaryot. Gene. Expr. 18, 207-250.

Black, M.B., Budinsky, R.A., Dombkowski, A., Lecluyse, E.L., Ferguson, S.S., Thomas, R.S., Rowlands, J.C., 2012. Cross-species comparisons of transcriptomic alterations in human and rat primary hepatocytes exposed to 2,3,7,8- tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 127, 199-215.

Bock K.W., Kohle C. 2005. Ah receptor- and TCDD-mediated liver tumor promotion: clonal selection and expansion of cells evading growth arrest and apoptosis, Biochem. Pharmacol. 69, 1403-1408.

Buchmann A., Stinchcombe S., Korner W., Hagenmaier H., Bock K.W. 1994. Effects of 2,3,7,8-tetrachloro- and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin on the proliferation of preneoplastic liver cells in the rat, Carcinogenesis. 15, 1143-1150.

Budinsky, R.A., LeCluyse, E.L., Ferguson, S.S., Rowlands, J.C., Simon, T., 2010. Human and rat primary hepatocyte CYP1A1 and 1A2 induction with 2,3,7,8- tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8- pentachlorodibenzofuran. Toxicol. Sci. 118, 224-235.

Budinsky R.A., Schrenk D., Simon T., Van den Berg M., Reichard J.F., Silkworth J.B., Aylward L.L., Brix A., Gasiewicz T., Kaminski N., Perdew G., Starr T.B., Walker N.J., Rowlands J.C. 2014. Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study, Crit. Rev. Toxicol. 44, 83-119.

Carlson, E.A., McCulloch, C., Koganti, A., Goodwin, S.B., Sutter, T.R., Silkworth, J.B., 2009. Divergent transcriptomic responses to aryl hydrocarbon receptor agonists between rat and human primary hepatocytes. Toxicol. Sci. 112, 257-272.

Carmichael N., Bausen M., Boobis A.R., Cohen S.M., Embry M., Fruijtier-Pölloth C., Greim H., Lewis R., Bette Meek M.E., Mellor H., Vickers C., Doe J. 2011. Using mode of action information to improve regulatory decision-making: an ECETOC/ILSI RF/HESI workshop overview, Crit. Rev. Toxicol. 41, 175-186.

Cohen, S. M., Ellwein, L. B., 1990. Cell proliferation in carcinogenesis. Science. 249, 1007-11.

Cohen, S.M., Klaunig, J., Meek, M.E., Hill, R.N., Pastoor, T., Lehman-McKeeman, L., Bucher, J., Longfellow, D.G., Seed, J., Dellarco, V., Fenner-Crisp, P., Patton, D., 2004. Evaluating the human relevance of chemically induced animal tumors. Toxicol. Sci. 78, 181-186.

Cohen, S.M., Meek, M.E., Klaunig, J.E., Patton, D.E., Fenner-Crisp, P.A., 2003. The human relevance of information on carcinogenic modes of action: overview. Crit. Rev. Toxicol. 33, 581-589.

Cohen, S. M., Arnold, L. L., 2011. Chemical carcinogenesis. Toxicol Sci. 120 Suppl 1, S76-92.

Connor, K.T., Aylward, L.L., 2006. Human response to dioxin: aryl hydrocarbon re- ceptor (AhR) molecular structure, function, and dose-response data for enzyme induction indicate an impaired human AhR. J. Toxicol. Environ. Health B Crit. Rev. 9, 147-171.

Connor, K.T., Harris, M.A., Edwards, M.R., Budinsky, R.A., Clark, G.C., Chu, A.C., Finley, B.L., Rowlands, J.C., 2008. AH receptor agonist activity in human blood measured with a cell-based bioassay: evidence for naturally occurring AH re- ceptor ligands in vivo. J. Expo. Sci. Environ. Epidemiol. 18, 369-380.

Cox, L.A., Popken, D., Marty, M.S., Rowlands, J.C., Patlewicz, G., Goyak, K.O., Becker, R.A., 2014. Developing scientific confidence in HTS-derived prediction models: lessons learned from an endocrine case study. Regul. Toxicol. Pharmacol. 69, 443-450.

Della Porta G., Dragani T.A., Sozzi G. 1987. Carcinogenic effects of infantile and long-term 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the mouse, Tumori. 73, 99-107.

Dellarco V., Fenner-Crisp P.A. 2012. Mode of Action: Moving toward a More Relevant and Efficient Assessment Paradigm, J. Nutr. 142, 2192S-2198S.

Denison M.S., Nagy S.R. 2003. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals, Annu. Rev. Pharmacol. Toxicol. 43, 309-334.

Denison M.S., Soshilov A.A., He G., DeGroot D.E., Zhao B. 2011. Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor, Toxicol. Sci. 124, 1-22.

Dere, E., Lee, A.W., Burgoon, L.D., Zacharewski, T.R., 2011. Differences in TCDD- elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells. BMC Genomics 12, 193.

Dragan Y.P., Xu X.H., Goldsworthy T.L., Campbell H.A., Maronpot R.R., Pitot H.C. 1992. Characterization of the promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the female rat, Carcinogenesis. 13, 1389-1395.

Dragan, Y.P., Schrenk, D., 2000. Animal studies addressing the carcinogenicity of TCDD (or related compounds) with an emphasis on tumour promotion. Food. Addit. Contam. 17, 289-302.

Fenner-Crisp P.A. 2012. Application of the International Life Sciences Institute Key Events Dose-Response Framework to food contaminants, J. Nutr. 142, 2199S-2206S.

Fielden, M.R., Adai, A., Dunn, R.T., Olaharski, A., Searfoss, G., Sina, J., Aubrecht, J., Boitier, E., Nioi, P., Auerbach, S., Jacobson-Kram, D., Raghavan, N., Yang, Y., Kincaid, A., Sherlock, J., Chen, S.-J., Car, B., Predictive Safety Testing Consortium, Carcinogenicity Working Group, 2011. Development and evaluation of a genomic signature for the prediction and mechanistic assessment of non- genotoxic hepatocarcinogens in the rat. Toxicol. Sci. 124, 54-74.

Flaveny, C.A., Murray, I.A., Perdew, G.H., 2010. Differential gene regulation by the human and mouse aryl hydrocarbon receptor. Toxicol. Sci. 114, 217-225.

Gasiewicz T.A., Henry E.C., Collins L.L. 2008. Expression and activity of aryl hydrocarbon receptors in development and cancer, Crit. Rev. Eukaryot. Gene. Expr. 18, 279-321.

Goodman D.G., Sauer R.M. 1992. Hepatotoxicity and carcinogenicity in female Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): a pathology working group reevaluation, Regul. Toxicol. Pharmacol. 15, 245-252.

Guzelian, P., Quattrochi, L., Karch, N., Aylward, L., Kaley, R., 2006. Does dioxin exert toxic effects in humans at or near current background body levels?: An evidence-based conclusion. Hum. Exp. Toxicol. 25, 99-105.

Hailey J.R., Walker N.J., Sells D.M., Brix A.E., Jokinen M.P., Nyska A. 2005. Classification of proliferative hepatocellular lesions in harlan sprague-dawley rats chronically exposed to dioxin-like compounds, Toxicol. Pathol. 33, 165-174.

Haarmann-Stemmann, T., Bothe, H., Kohli, A., Sydlik, U., Abel, J., Fritsche, E., 2007. Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines. Drug. Metab. Dispos. 35, 2262-2269.

Herrmann S., Seidelin M., Bisgaard H.C., Vang O. 2002. Indolo[3,2-b]carbazole inhibits gap junctional intercellular communication in rat primary hepatocytes and acts as a potential tumor promoter, Carcinogenesis. 23, 1861-1868.

Hill A.B. 1965. The Environment and Disease: Association or Causation? Proc. R. Soc. Med. 58, 295-300.

Hu Q., He G., Zhao J., Soshilov A., Denison M.S., Zhang A., Yin H., Fraccalvieri D., Bonati L., Xie Q., Zhao B. 2013. Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands, PLoS. One. 8, e66258.

Julien E., Boobis A.R., Olin S.S., Ilsi Research Foundation Threshold Working Group 2009. The Key Events Dose-Response Framework: a cross-disciplinary mode-of-action based approach to examining dose-response and thresholds, Crit. Rev. Food. Sci. Nutr. 49, 682-689.

Kim, S., Dere, E., Burgoon, L. D., Chang, C. C., Zacharewski, T. R., 2009. Comparative analysis of AhR-mediated TCDD-elicited gene expression in human liver adult stem cells. Toxicol Sci. 112, 229-44.

Knerr S., Schaefer J., Both S., Mally A., Dekant W., Schrenk D. 2006. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cytochrome P450s alter the formation of reactive oxygen species in liver cells, Mol. Nutr. Food. Res. 50, 378-384.

Kociba R.J., Keyes D.G., Beyer J.E., Carreon R.M., Wade C.E., Dittenber D.A., Kalnins R.P., Frauson L.E., Park C.N., Barnard S.D., Hummel R.A., Humiston C.G. 1978. Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats, Toxicol.Appl.Pharmacol.. 46, 279-303.

Lambert, G.H., Needham, L.L., Turner, W., Lai, T.J., Patterson, D.G.J., Guo, Y.L., 2006. Induced CYP1A2 activity as a phenotypic biomarker in humans highly exposed to certain PCBs/PCDFs. Environ. Sci. Technol. 40, 6176-6180.

Maronpot R.R., Foley J.F., Takahashi K., Goldsworthy T., Clark G., Tritscher A., Portier C., Lucier G. 1993. Dose response for TCDD promotion of hepatocarcinogenesis in rats initiated with DEN: histologic, biochemical, and cell proliferation endpoints 8, Environ. Health. Perspect.. 101, 634-642.

Matthews, J., Gustafsson, J.A., 2006. Estrogen receptor and aryl hydrocarbon receptor signaling pathways. Nucl. Recept. Signal 4, e016.

Matthews, J., Wihlen, B., Thomsen, J., Gustafsson, J.A., 2005. Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. Mol. Cell Biol. 25, 5317-5328.

Meek, M.E., 2008. Recent developments in frameworks to consider human rele- vance of hypothesized modes of action for tumours in animals. Environ. Mol. Mutagen 49, 110-116.

Meek, M.E., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman- McKeeman, L.D., Longfellow, D.G., Pastoor, T., Seed, J., Patton, D.E., 2003. A framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33, 591-653.

Meek M.E., Bolger M., Bus J.S., Christopher J., Conolly R.B., Lewis R.J., Paolini G.M., Schoeny R., Haber L.T., Rosenstein A.B., Dourson M.L. 2013. A framework for fit-for-purpose dose response assessment, Regul. Toxicol. Pharmacol. 66, 234-240.

Meek M.E.B., Palermo C.M., Bachman A.N., North C.M., Jeffrey Lewis R. 2014a. Mode of action human relevance (species concordance) framework: Evolution of the Bradford Hill considerations and comparative analysis of weight of evidence, J. Appl. Toxicol. 34, 595-606.

Meek M.E., Boobis A., Cote I., Dellarco V., Fotakis G., Munn S., Seed J., Vickers C. 2014b. New developments in the evolution and application of the WHO/IPCS framework on mode of action/species concordance analysis, J. Appl. Toxicol. 34, 1-18.

Navarro, S.L., Chen, Y., Li, L., Li, S.S., Chang, J.-L., Schwarz, Y., King, I.B., Potter, J.D., Bigler, J., Lampe, J.W., 2011. UGT1A6 and UGT2B15 polymorphisms and acet- aminophen conjugation in response to a randomized, controlled diet of select fruits and vegetables. Drug. Metab. Dispos. 39, 1650-1657.

Navarro, S.L., Peterson, S., Chen, C., Makar, K.W., Schwarz, Y., King, I.B., Li, S.S., Li, L., Kestin, M., Lampe, J.W., 2009. Cruciferous vegetable feeding alters UGT1A1 activity: diet- and genotype-dependent changes in serum bilirubin in a controlled feeding trial. Cancer Prev. Res.. (Phila) 2, 345-352.

NTP 1980. Bioassay of a Mixture of 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin and 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (Gavage) for Possible Carcinogenicity (CAS No. 57653-85-7,CAS No. 19408-74-3), Natl. Toxicol. Program. Tech. Rep. Ser. 198, 1-187.

NTP 1982a. Carcinogenesis Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (CAS No. 1746-01-6) in Osborne-Mendel Rats and B6C3F1 Mice (Gavage Study), Natl. Toxicol. Program.Tech.Rep.Ser.. 209, 1-195.

NTP 1982b. Carcinogenesis Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (CAS No. 1746-01-6) in Swiss-Webster Mice (Dermal Study), Natl. Toxicol. Program.Tech.Rep.Ser.. 201, 1-113.

NTP 2006a. NTP technical report on the toxicology and carcinogenesis studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (CAS No. 1746-01-6) in female Harlan Sprague-Dawley rats (Gavage Studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 4-232.

NTP 2006b. NTP Toxicology and Carcinogenesis Studies of 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) (CAS No. 57117-31-4) in Female Harlan Sprague-Dawley Rats (Gavage Studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 1-198.

NTP 2006c. NTP toxicology and carcinogenesis studies of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) (CAS No. 57465-28-8) in female Harlan Sprague-Dawley rats (Gavage Studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 4-246.

NTP 2006d. NTP technical report on the toxicology and carcinogenesis studies of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) (CAS No. 35065-27-1) in female Harlan Sprague-Dawley rats (Gavage studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 4-168.

NTP 2006e. NTP Toxicology and Carcinogenesis Studies of a Binary Mixture of 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) (CAS No. 57465-28-8) and 2,2',4,4',5,5'-Hexachlorobiphenyl (PCB 153) (CAS No. 35065-27-1) in Female Harlan Sprague-Dawley Rats (Gavage Studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 1-258.

NTP 2006f. NTP Toxicology and Carcinogenesis Studies of a Mixture of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) (CAS No. 1746-01-6), 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) (CAS No. 57117-31-4), and 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) (CAS No. 57465-28-8) in Female Harlan Sprague-Dawley Rats (Gavage Studies), Natl. Toxicol. Program.Tech.Rep.Ser.. 1-180.

NTP 2010. Toxicology and carcinogenesis studies of 2,3',4,4',5-pentachlorobiphenyl (PCB 118) (CAS No. 31508-00-6) in female harlan Sprague-Dawley rats (gavage studies), Natl. Toxicol. Program. Tech. Rep. Ser. 1-174.

NTP, 2014. NTP toxicology studies of indole-3-carbinol (CAS No. 700-06-01) in F344/N rats and B6C3F1/N mice and toxicology and carcinogenesis studies of indole-3-carbinol in Harlan Sprague-Dawley rats and B6C3F1/N mice (Gavage studies). Natl. Toxicol. Program Tech. Rep. Ser. 1-258.

Nguyen L.P., Bradfield C.A. 2008. The search for endogenous activators of the aryl hydrocarbon receptor, Chem. Res. Toxicol. 21, 102-116.

Okey A.B. 2007. An Aryl Hydrocarbon Receptor Odyssey to the Shores of Toxicology: The Deichmann Lecture, International Congress of Toxicology-XI, Toxicol. Sci. 98, 5-38.

Organisation for Economic Cooperation and Development. 2013. Guidance Document on Developing and Assessing Adverse Outcome Pathways. ENV/JM/MONO(2013)6. Paris. At http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2013)6&doclanguage=en

Organisation for Economic Cooperation and Development. 2014. Users' Handbook Supplement to the Guidance Document for Developing and Asessing AOPs. Supplement to ENV/JM/MONO(2013)6. Paris.

Organisation for Economic Cooperation and Development. 2015. Adverse Outcome Pathways, Molecular Screening and Toxicogenomics at http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screening-and-toxicogenomics.htm. Accessed March 17, 2015.

Ovando, B.J., Ellison, C.A., Vezina, C.M., Olson, J.R., 2010. Toxicogenomic analysis of exposure to TCDD, PCB126 and PCB153: identification of genomic biomarkers of exposure to AhR ligands, BMC. Genomics 11, 583. http://dx.doi.org/10.1186/ 1471-2164-11-583, 1471-2164-11-583 [pii].

Patlewicz G., Simon T., Goyak K., Phillips R.D., Rowlands J.C., Seidel S., Becker R.A. 2013. Use and validation of HT/HC assays to support 21st century toxicity evaluations, Regul. Toxicol. Pharmacol. 65, 259-268.

Patlewicz, G., Simon, T. W., Rowlands, J. C., Budinsky, R. A., Becker, R. A., 2015. Proposing a scientific confidence framework to help support the application of adverse outcome pathways for regulatory purposes. Regul Toxicol Pharmacol. 71, 463-477.

Peterson, S., Schwarz, Y., Li, S.S., Li, L., King, I.B., Chen, C., Eaton, D.L., Potter, J.D., Lampe, J.W., 2009. CYP1A2, GSTM1, and GSTT1 polymorphisms and diet effects on CYP1A2 activity in a crossover feeding trial. Cancer Epidemiol. Biomarkers Prev. 18, 3118-3125.

Petkov P.I., Rowlands J.C., Budinsky R., Zhao B., Denison M.S., Mekenyan O. 2010. Mechanism-based common reactivity pattern (COREPA) modelling of aryl hydrocarbon receptor binding affinity, SAR. QSAR. Environ. Res. 21, 187-214.

Pintilie D.G., Shupe T.D., Oh S.-H., Salganik S.V., Darwiche H., Petersen B.E. 2010. Hepatic stellate cells' involvement in progenitor-mediated liver regeneration, Lab. Invest. 90, 1199-1208.

Poland, A., Glover, E., 1979. An estimate of the maximum in vivo covalent binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to rat liver protein, ribosomal RNA, and DNA. Cancer. Res. 39, 3341-3344.

Randerath, K., Putman, K.L., Randerath, E., Zacharewski, T., Harris, M., Safe, S., 1990. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on I-compounds in hepatic DNA of Sprague-Dawley rats: sex-specific effects and structure-activity relationships. Toxicol. Appl. Pharmacol. 103, 271-280.

Schrenk, D., Schmitz, H.J., Bohnenberger, S., Wagner, B., Worner, W., 2004. Tumor promoters as inhibitors of apoptosis in rat hepatocytes. Toxicol. Lett. 149, 43-50.

Schwarz, M., Buchmann, A., Stinchcombe, S., Kalkuhl, A., Bock, K., 2000. Ah receptor ligands and tumor promotion: survival of neoplastic cells. Toxicol. Lett. 112e113, 69-77.

Seed, J., Carney, E.W., Corley, R.A., Crofton, K.M., DeSesso, J.M., Foster, P.M., Kavlock, R., Kimmel, G., Klaunig, J., Meek, M.E., Preston, R.J., Slikker, W.J., Tabacova, S., Williams, G.M., Wiltse, J., Zoeller, R.T., Fenner-Crisp, P., Patton, D.E., 2005. Overview: using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit. Rev. Toxicol. 35, 664-672.

Silkworth, J.B., Koganti, A., Illouz, K., Possolo, A., Zhao, M., Hamilton, S.B., 2005. Comparison of TCDD and PCB CYP1A induction sensitivities in fresh hepato- cytes from human donors, sprague-dawley rats, and rhesus monkeys and HepG2 cells. Toxicol. Sci. 87, 508-519.

Simon T.W., Simons S.S., Preston R.J., Boobis A.R., Cohen S.M., Doerrer N.G., Fenner-Crisp P.A., McMullin T.S., McQueen C.A., Rowlands J.C., RISK21 Dose-Response Subteam 2014. The use of mode of action information in risk assessment: Quantitative key events/dose-response framework for modeling the dose-response for key events, Crit. Rev. Toxicol. 44 Suppl 3, 17- 43.

Sobus, J. R., Tan, Y.-M., Pleil, J. D., Sheldon, L. S., 2011. A biomonitoring framework to support exposure and risk assessments. Sci Total Environ. 409, 4875-84.

Sonich-Mullin, C., Fielder, R., Wiltse, J., Baetcke, K., Dempsey, J., Fenner-Crisp, P., Grant, D., Hartley, M., Knaap, A., Kroese, D., Mangelsdorf, I., Meek, E., Rice, J.M., Younes, M., International Programme on Chemical Safety, 2001. IPCS conceptual framework for evaluating a mode of action for chemical carcinogenesis. Regul. Toxicol. Pharmacol. 34, 146-152.

Stinchcombe S., Buchmann A., Bock K.W., Schwarz M. 1995. Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver, Carcinogenesis. 16, 1271-1275.

Sutter, C.H., Bodreddigari, S., Sutter, T.R., Carlson, E.A., Silkworth, J.B., 2010. Analysis of the CYP1A1 mRNA dose response in human keratinocytes indicates that relative potencies of dioxins, furans, and PCBs are species and congener specific. Toxicol. Sci. 118, 704-715.

Tanaka M., Itoh T., Tanimizu N., Miyajima A. 2011. Liver stem/progenitor cells: their characteristics and regulatory mechanisms, J. Biochem. 149, 231-239.

Tang, N.J., Liu, J., Coenraads, P.J., Dong, L., Zhao, L.J., Ma, S.W., Chen, X., Zhang, C.M., Ma, X.M., Wei, W.G., Zhang, P., Bai, Z.P., 2008. Expression of AhR, CYP1A1, GSTA1, c-fos and TGF-alpha in skin lesions from dioxin-exposed humans with chlor- acne. Toxicol. Lett. 177, 182-187.

Teeguarden J.G., Dragan Y.P., Singh J., Vaughan J., Xu Y.H., Goldsworthy T., Pitot H.C. 1999. Quantitative analysis of dose- and time-dependent promotion of four phenotypes of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats, Toxicol. Sci. 51, 211-223.

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.

Turteltaub, K.W., Felton, J.S., Gledhill, B.L., Vogel, J.S., Southon, J.R., Caffee, M.W., Finkel, R.C., Nelson, D.E., Proctor, I.D., Davis, J.C., 1990. Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA. Proc. Natl. Acad. Sci. U. S. A. 87, 5288-5292.

United States Environmental Protection Agency (USEPA). 2005. Guidelines for Carcinogen Risk Assessment.

Uno, S., Endo, K., Ishida, Y., Tateno, C., Makishima, M., Yoshizato, K., Nebert, D.W., 2009. CYP1A1 and CYP1A2 expression: comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines. Toxicol. Appl. Pharmacol. 237, 119-126.

Van den Berg M., Birnbaum L.S., Denison M., De V.M., Farland W., Feeley M., Fiedler H., Hakansson H., Hanberg A., Haws L., Rose M., Safe S., Schrenk D., Tohyama C., Tritscher A., Tuomisto J., Tysklind M., Walker N., Peterson R.E. 2006. The 2005 World Health Organization reevaluation of human and Mammalian toxic equivalency factors for dioxins and dioxin-like compounds, Toxicol.Sci.. 93, 223-241.

Villeneuve, D. L., Crump, D., Garcia-Reyero, N., Hecker, M., Hutchinson, T. H., LaLone, C. A., Landesmann, B., Lettieri, T., Munn, S., Nepelska, M., Ottinger, M. A., Vergauwen, L., Whelan, M., 2014a. Adverse outcome pathway (AOP) development I: strategies and principles. Toxicol Sci. 142, 312-20.

Villeneuve, D. L., Crump, D., Garcia-Reyero, N., Hecker, M., Hutchinson, T. H., LaLone, C. A., Landesmann, B., Lettieri, T., Munn, S., Nepelska, M., Ottinger, M. A., Vergauwen, L., Whelan, M., 2014b. Adverse outcome pathway development II: best practices. Toxicol Sci. 142, 321-30.

Wang X., Foster M., Al-Dhalimy M., Lagasse E., Finegold M., Grompe M. 2003. The origin and liver repopulating capacity of murine oval cells, Proc. Natl. Acad. Sci. U. S. A. 100 Suppl 1, 11881-11888.

Wang Y.-J., Chang H., Kuo Y.-C., Wang C.-K., Siao S.-H., Chang L.W., Lin P. 2011. Synergism between 2,3,7,8-tetrachlorodibenzo-p-dioxin and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumor incidence in mice, J. Hazard. Mater. 186, 869-875.

Wassom, J.S., Huff, J.E., Loprieno, N., 1977. A review of the genetic toxicology of chlorinated dibenzo-p-dioxins. Mutat. Res. 47, 141-160.

Weiss, C., Faust, D., Schreck, I., Ruff, A., Farwerck, T., Melenberg, A., Schneider, S., Oesch-Bartlomowicz, B., Zatloukalova, J., Vondracek, J., Oesch, F., Dietrich, C., 2008. TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD and cyclin A. Oncogene 27, 2198-2207.

Westerink, W.M., Stevenson, J.C., Schoonen, W.G., 2008. Pharmacologic profiling of human and rat cytochrome P450 1A1 and 1A2 induction and competition. Arch. Toxicol. 82, 909-921.

Whysner, J., Williams, G.M., 1996. 2,3,7,8-Tetrachlorodibenzo-p-dioxin mechanistic data and risk assessment: gene regulation, cytotoxicity, enhanced cell proliferation, and tumor promotion. Pharmacol. Ther. 71, 193-223, Wincent, E., Amini, N., Luecke, S., Glatt, H., Bergman, J., Crescenzi, C., Rannug, A., Rannug, U., 2009. The suggested physiologic aryl hydrocarbon receptor acti- vator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is pre- sent in humans. J. Biol. Chem. 284, 2690-2696.

Wolfle D., Becker E., Schmutte C. 1993. Growth stimulation of primary rat hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin, Cell. Biol. Toxicol. 9, 15-31.

Xu, L., Li, A. P., Kaminski, D. L., Ruh, M. F., 2000. 2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes 1. Chem Biol Interact. 124, 173-189

Yamamoto, R., Shimamoto, K., Ishii, Y., Kimura, M., Fujii, Y., Morita, R., Suzuki, K., Shibutani, M., Mitsumori, K., 2013. Involvement of PTEN/Akt signaling and oxidative stress on indole-3-carbinol (I3C)-induced hepatocarcinogenesis in rats. Exp. Toxicol. Pathol. 65, 845-852.

Yao, C.Q., Prokopec, S.D., Watson, J.D., Pang, R., P'ng, C., Chong, L.C., Harding, N.J., Pohjanvirta, R., Okey, A.B., Boutros, P.C., 2012. Inter-strain heterogeneity in rat hepatic transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 260, 135-145.

Zhao, Y.-Y., Tao, F.-M., Zeng, E.Y., 2008. Theoretical study of the quantitative structure-activity relationships for the toxicity of dibenzo-p-dioxins. Chemosphere 73, 86-91.