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Regulatory Toxicology and Pharmacology 73 (2015) 172e190

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier .com/locate/yrtph

The adverse outcome pathway for rodent liver tumor promotion bysustained activation of the aryl hydrocarbon receptor

Richard A. Becker a, *, Grace Patlewicz b, 1, Ted W. Simon c, J. Craig Rowlands d,Robert A. Budinsky d

a Regulatory and Technical Affairs Department, American Chemistry Council (ACC), Washington, DC 20002, USAb DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, DE 19711, USAc Ted Simon LLC, 4184 Johnston Road, Winston, GA 30187, USAd The Dow Chemical Company, Toxicology & Environmental Research & Consulting, 1803 Building Washington Street, Midland, MI 48674, USA

a r t i c l e i n f o

Article history:Received 7 April 2015Received in revised form19 June 2015Accepted 22 June 2015Available online 2 July 2015

Keywords:Adverse outcome pathwayAryl hydrocarbon receptorLiver tumor promotionMode of action

* Corresponding author. American Chemistry Couington, DC 20002, USA.

E-mail address: rick_becker@americanchemistry.c1 Present address: US EPA, Office of Research and D

for Computational Toxicology, RTP, NC 27711, USA.

http://dx.doi.org/10.1016/j.yrtph.2015.06.0150273-2300/© 2015 The Authors. Published by Elsevier

a b s t r a c t

An Adverse Outcome Pathway (AOP) represents the existing knowledge of a biological pathway leadingfrom 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 onthe mode of action (MOA) of rodent liver tumor promotion by dioxin-like compounds (DLCs) has beendeveloped and the weight of evidence (WoE) of key event relationships (KERs) evaluated using evolvedBradford Hill considerations. Dioxins and DLCs are potent aryl hydrocarbon receptor (AHR) ligands thatcause a range of species-specific adverse outcomes. The occurrence of KEs is necessary for inducingdownstream 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 responseelement; for this AOP, this initial event is binding of a DLC ligand to the AHR. Data from mechanisticstudies, lifetime bioassays and approximately thirty initiation-promotion studies have established dioxinand DLCs as rat liver tumor promoters. Such studies clearly show that sustained AHR activation, weeks ormonths in duration, is necessary to induce rodent liver tumor promotion e hence, sustained AHRactivation is deemed the molecular initiating event (MIE). After this MIE, subsequent KEs are 1) changesin cellular growth homeostasis likely associated with expression changes in a number of genes andobserved as development of hepatic foci and decreases in apoptosis within foci; 2) extensive liver toxicityobserved as the constellation of effects called toxic hepatopathy; 3) cellular proliferation and hyperplasiain several hepatic cell types. This progression of KEs culminates in the AO, the development of hepa-tocellular adenomas and carcinomas and cholangiolar carcinomas. A rich data set provides both quali-tative and quantitative knowledge of the progression of this AOP through KEs and the KERs. Thus, theWoE for this AOP is judged to be strong. Species-specific effects of dioxins and DLCs are well known e

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 differ-ences into account.© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The source-to-outcome pathway sequence consists of release,transport, contact, absorption, dose, molecular interactions, cellular

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responses, tissue and organ changes, culminating in an adverseeffect (Sobus et al., 2011). An adverse outcome pathway (AOP)(Ankley et al., 2010) is a subset of the source to outcome sequence.An AOP, by convention, starts at the molecular interaction step, inwhich a xenobiotic chemical moiety interacts with biologicalmolecule and proceeds to an adverse outcome (AO) via a series ofKey Events (KEs). The sequence of the hierarchical AOP framework(see SF1 in Supplemental Material), and guidance on the develop-ment and evaluation of AOPs has been developed (OECD, 2013;OECD, 2014). Ideally, an AOP will be applicable to a broad

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

R.A. Becker et al. / Regulatory Toxicology and Pharmacology 73 (2015) 172e190 173

spectrum of chemicals that have the potential to operate via acommon pathway or to have KEs in common. However, the OECDAOP program (OECD, 2013) permits submission of an AOP casestudy applicable to a single chemical, or a very limited number ofchemicals, recognizing that, data permitting, such an AOP may beexpanded in the future to cover a category of chemicals.

Themolecular initiating event (MIE) is a fundamental concept ofall AOPs (Ankley et al., 2010). The MIE has been operationallydefined as the first key event (KE) and necessary but not sufficientto produce the AO. Other KEs within a Mode of Action (MOA) aredefined similarlydnecessary but not sufficient for the AO to occur(Julien et al., 2009; Simon et al., 2014; USEPA, 2005). Although itcan be argued that initial molecular interactions such as absorptionor parent compound interaction with an enzyme that leads toproduction of a toxicologically-active moiety meet the definition ofan MIE, these events are considered to be upstream of an MIE. TheMIE, by convention (OECD, 2014), “involves a chemical interaction(e.g., a reaction, covalent binding, hydrogen bonding, electrostaticinteraction, etc.) between a chemical stressor and chemicallydefined biomolecules within an organism.” Identifying the MIE asthe first KE within the AOP and thus distinguishing it from earlyupstream events within the source-to-outcome pathway isimportant, especially when adverse outcomes require chronicexposure and dose-dependent transitions (Patlewicz et al., 2013).Hence, Patlewicz et al. (2013) introduced the idea of the “InitialMolecular Event” (IME) to capture necessary biological responsesthat are chemically induced early on in the pathway but may not besufficient to “initiate” an adverse outcome. Drewe et al. (2014) usedthe term pre-MIE to make the same distinction. These alternativeconstructs reflect concern that some would infer certainty of anadverse outcome based solely on responses from assays associatedwith an MIE. In this AOP, we use the term pre-MIE.

Within an AOP, the MIE is followed by one or more intermediateKEs, which are connected in a sequential and integrated manner,culminating in the AO (Andersen et al., 2014). A Key Event Rela-tionship (KER) “connects one key event to another, defines adirected relationship between the two (i.e., identifies one as up-stream and the other as downstream), and facilitates inference orextrapolation of the state of the downstream key event from theknown, measured, or the predicted state of the upstream key event”(OECD, 2014). In other words, a KER captures the knowledge of thetoxicodynamic relationship between KEn and KEnþ1, and thisknowledge may be sufficient to develop a prediction model. KERsnecessarily include homeostatic mechanisms, and therefore, un-derstanding dose-dependent transitions and tipping points alongthe sequence of KEs is important when developing, evaluating andapplying AOPs. Further, responses can be influenced by co-exposureto other substances, and these components are referred to asmodulating factors when considered within a MOA (Andersen et al.,2014); modulating factors affect the nature of KE doseeresponserelationships. Associative events serve as reliable biomarkers orindicators of KE(s) but may not be causal themselves. Thus, oneneeds to consider exposure pathways, chemical properties, ADME,modulating factors, and associative events when developing andapplying AOPs (Carmichael et al., 2011; Dellarco and Fenner-Crisp,2012; Fenner-Crisp, 2012; Julien et al., 2009). Benefiting fromexperience gained from over the last 2e3 years, largely through theOECDAOP program (OECD, 2014) and the AOPWiki (USEPA, 2014), acomponent of the OECD-sponsored AOP Knowledgebase, recentpublications provide greater clarity with respect to the concepts ofKEs, KERs, (Villeneuve et al. (2014a, 2014b), strategies and practicesto use when developing AOPs. Scientific confidence in the identifi-cation of KEs and in KERs may be judged using the evolved BradfordHill (BH) considerations (Hill, 1965; Meek et al., 2013, 2014a, 2014b;OECD, 2014; Patlewicz et al., 2015; Becker et al., 2015).

In constructing AOPs, OECD recognizes the importance ofinitially drawing upon case examples applicable to a single chem-ical or a limited number of chemicals (OECD, 2015). The goal ofthese case examples is to expand the AOP concept to a broaderchemical domain as experience and knowledge is gained. Consis-tent with this goal, we have chosen to draw from the MOA of arylhydrocarbon receptor (AHR) activation causing liver tumors in ro-dents, including the extensive of knowledge of the biological pro-cess of rodent liver tumorigenesis (Budinsky et al., 2014). In thefollowing case study, we describe the AOP of sustained AHR acti-vation leading to rodent liver tumor promotion. This AOP casestudy forms a project within the OECD AOP work program and isalso being summarized for inclusion into the AOP Wiki. The sus-tained AHR activation rodent liver tumor promotion AOP is shownin Fig. 1, organized according to the OECD AOP template.

The MIE is identified as sustained activation of the AHR pro-duced by biologically persistent dioxin-like chemicals (DLCs). DLCsare potent AHR ligands that cause a range of species-specificadverse outcomes (Okey, 2007). The role of the AHR in carcinoge-nicity has been extensively studied and reviewed (Beebe et al.,1995; Bock and Kohle, 2005; Gasiewicz et al., 2008; Goodmanand Sauer, 1992; Hailey et al., 2005; Knerr et al., 2006; Kocibaet al., 1978; Stinchcombe et al., 1995). A number of cancer bio-assays demonstrate that such compounds can induce hepatocel-lular adenomas/carcinomas and cholangiomas/carcinomas invarious test species (Table 1) (Della Porta et al., 1987; Kociba et al.,1978; NTP, 1980; NTP, 1982a; NTP, 1982b; NTP, 2006a; NTP, 2006b;NTP, 2006c; NTP, 2006d; NTP, 2006e; NTP, 2006f; NTP, 2010).Approximately thirty initiation-promotion studies provide a com-plement to these cancer bioassays and, together have establishedthe mechanism by which dioxin and DLCs act as rodent liver tumorpromoters. Initiation-promotion studies rely on an initiating agent,generally diethylnitrosamine, to increase the population of initiatedliver cells. Following initiation, treatment with DLCs or other tumorpromoters, with or without partial hepatectomy, promotes thedevelopment and growth of pre-neoplastic altered hepatic foci.When treatment with promoters is continued for a sufficient timeand dose, tumor formation occurs (Buchmann et al., 1994; Draganet al., 1992; Maronpot et al., 1993; Teeguarden et al., 1999).

The mechanistic causal link between earlier histopathologicalKEs in liver and tumor development likely resides in the prolifer-ative potential of the liver and the ability of this organ to regeneratefollowing cytotoxicity (e.g., Cohen and Ellwein, 1990; Cohen andArnold, 2011; Tomasseti and Vogelstein, 2015). Replacement ofdamaged hepatocytes may occur through replication of neigh-boring hepatocytes or, if damage is extensive, through recruitmentof liver stem cells (Alison, 2005; Pintilie et al., 2010; Tanaka et al.,2011; Wang et al., 2003; Wolfle et al., 1993). Although DLCs havealso been reported to cause tumors of the lung and oral mucosa inrodents, these tumor types are not considered further since theyare outside the scope of this AOP (NTP, 2006a; NTP, 2006b; NTP,2006c; NTP, 2006d; NTP, 2006e; NTP, 2006f; NTP, 2010; Wanget al., 2011).

Each component and KE of the AOP is discussed below in greaterdetail and in temporal sequence. As is the case for all AOPs, asfurther research increases knowledge of the mechanism of action(detailed molecular events and cellular processes), the KEs andKERs for sustained AHR activation leading to rodent liver tumorpromotion are likely to evolve; the online AOPWiki is well suited toaccommodate such an evolution.

2. Toxicant (chemical properties)

Co-planar halogenated polyaromatic hydrocarbon structureswith halogens in specific locations bind to and activate the AHR,

Fig. 1. Schematic of the sustained AHR activation rat liver tumor promotion AOP showing the molecular initiating event (MIE), intermediate key events (KE) and the adverseoutcome (AO).

Table 1Summary of key chronic tumor bioassays in rodents treated with TCDD.

Study Dose (ng/kg-day) and liver tumors

RatsKociba et al., 1978; Spartan Sprague Dawleya 0 1 10 100Female 2/86 1/50 9/50 18/45Male 2/85 0/50 0/50 1/50

NTP, 1982; Osborne Mendelb 0 1.4 7.1 71Female 5/75 1/49 3/50 14/49Male 0/74 0/50 0/50 3/50

NTP, 2006; Harlan Sprague Dawley 0 2.1 7.1 16 33 71Female OnlyCholangiolarcarcinoma 0/53 0/54 0/53 1/53 4/53 25/53Adenoma 0/53 0/54 0/53 0/53 1/53 13/53Hepatocholangioma 0/53 0/54 0/53 0/53 0/53 2/53

MiceNTP, 1982; B6C3F1b 0 6.7 67 330Female 3/73 6/50 6/48 11/47

0 1.7 8.3 83Male 15/73 12/49 13/49 27/50

Della Porta et al., 1987; B6C3 0 150 360Femalec 3/49 16/42 20/48

0 160 360Malec 15/43 26/51 43/50

a Number of animals with tumors/total number of animals in the study.b Number of tumor-bearing animals/number of animals examined at site; neoplastic nodule or hepatocellular carcinoma.c Combined liver carcinoma and adenoma.

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e.g., DLCs such as polychlorinated dibenzo-p-dioxins (PCDDs),polychlorinated dibenzofurans (PCDFs), polychlorinated naphtha-lenes (PCNs), co-planar polychlorinated biphenyls (PCBs) and pol-yaromatic hydrocarbons (PAHs). A few structure-activity modelshave been published for PCDDs/PCDF/dioxin-like PCB interactions(Petkov et al., 2010; Zhao et al., 2008). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent AHR agonist in the group of thebicyclic halogenated dioxins and furans. Several other DLCs possesspotency within an order of magnitude of TCDD; these include otherPCDDs, PCDFs and certain PCBs (Van den Berg et al., 2006). Thisstructural requirement for co-planarity with halogens in the2,3,7,8-position is an important component of the toxic equivalencyfactor (TEF) approach for risk assessment for mixtures of PCDDsand PCDFs and reflects the relative binding affinity of these com-pounds to the AHR (Van den Berg et al., 2006). A number of PCDD,PCDF and PCB congeners have been shown to promote clonalexpansion of altered hepatic foci in rodent liver standard initiation-promotion protocols or to induce liver tumors in 2-year cancerbioassays in both mice and rats (reviewed in Budinsky et al., 2014).

Because the AHR is a highly promiscuous receptor,

understanding the structural features associated with potency ofAHR activation is, at best, rudimentary. AHR ligands include aplethora of chemically divergent anthropogenic, natural andendogenous molecules with widely diverse structural features(Denison and Nagy, 2003; Denison et al., 2011; Hu et al., 2013;Nguyen and Bradfield, 2008). Extant knowledge of the ligand-binding domain of the AHR is not yet sufficient to predictwhether a ligand acts an agonist, antagonist, or a modulator.Therefore, except for the DLCs, predicting whether an AHR ligandwill act as a rodent liver tumor promoter is not yet possible.

Summarizing the chemical properties shared by the plethora ofAHR ligands presents a challenge e the large diversity of AHR li-gands complicates the development of predictive structural alerts.Hence, using a QSAR approach to understand the biological activityof AHR ligands is currently not possible, other than for PCDDs,PCDFs and coplanar PCB congeners which share obvious structuralfeatures. In time, structural alerts for the tumor promoting poten-tial of the constellation of AHR ligands may be identified (ST1Supplemental Material)). However, because of the promiscuity ofthe AHR, structural features alone may be insufficient to rule in or

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rule out the potential for a substance to activate the AHR, and theseligands may not be correctly identified in a QSAR screen (Denisonet al., 2011; Herrmann et al., 2002).

3. Macromolecular interactions and ligand-activation oftranscription: Pre-MIE and MIE events

The AHR is a ligand-activated transcription factor that belongsto the Per-Arnt-Sim (PAS) family of proteins (Kumar et al., 2001;Partch and Gardner, 2010; Soshilov and Denison, 2011). The AHRand a number of other nuclear receptor proteins control a widerange of cellular responses through alterations in gene expression.AHR-directed transcription (F2 Supplemental Material) has beenextensively investigated (Abel and Haarmann-Stemmann, 2010;Dietrich and Kaina, 2010; Furness and Whelan, 2009; Gasiewiczet al., 2008).

In the absence of ligand binding and activation, the AHR residesin the cytosol complexed to its chaperone proteins, Hsp90, AIP andp23 proteins. The AHR interacts with other transcription factors,including the estrogen receptor, the glucocorticoid receptor, themitochondrial protein ATP5a1 (Denison et al., 2011; Matthews andGustafsson, 2006; Sato et al., 2013; Tappenden et al., 2013).Following ligand binding, the AHR sheds the chaperones and bindsto the aryl hydrocarbon receptor nuclear translocator or ARNT,another protein in the PAS family. The liganded AHR-ARNT dimerbinds to dioxin response elements on DNA. A number of co-regulatory proteins bind to the transcription complex to provideunique cell and ligand-specific transcriptional outcomes (Beischlaget al., 2008; George et al., 2011; Kumar et al., 2004; Lee and LeeKraus, 2001; Lees and Whitelaw, 1999; Perissi and Rosenfeld,2005; Reen et al., 2002; Tsai and Fondell, 2004). Ubiquitin-mediated proteolysis of the AHR serves to inactivate the receptorcomplex.

Gene expression changes induced by AHR activation occurwithin hours (Matthews et al., 2005). However, AHR activationmust be sustained for a significant portion of the rodent lifespan fortumor formation to occur (NTP, 2006a; NTP. 2006b; NTP, 2006c).Therefore, what may be critical in distinguishing between theseacute and sustained effects is knowledge of the constellation ofmacromolecular events that transpire on both timescales e hoursto days for acute effects versus several months up to a year forsustained AHR activation.

The genomic changes induced by DLCs can be organized intotwo categories. The first is the rapid induction of a core batterygenes consisting of a number of drug metabolizing enzymes andthe second consists of changes in the expression of other genes thatare temporally concordant with the critical histopathological KEsleading to promotion of liver tumors (Ovando et al., 2006; Vezinaet al., 2004).

3.1. Core battery response

These genomic changes are adaptive responses and provideseveral reliable biomarkers of AHR activation (Boutros et al., 2004,2009, 2011; Boverhof et al., 2005; Boverhof et al., 2006).

The induction of CYP1A1 expression measured byethoxyresorufin-O,O-deethylase is a common marker of AHR acti-vation and remains elevated for at least one year with sustainedadministration of DLCs (NTP, 2006a; NTP, 2006b; NTP, 2006c; NTP,2006d; NTP, 2006e; NTP, 2006f; NTP, 2010). The induction ofCYP1A1, part of the core battery response, is used here as anassociative event or biomarker for AHR activation.

The one consistent and well-accepted transcriptional responseto AHR activation involves the core battery response that is rep-resented by the induction of a number of important xenobiotic

metabolic enzymes (CYP1A1, CYP1A2, Cyp1B1, UGT1A isoforms,Nqo1, and GST) (Rowlands et al., 1996). In-vivo and in-vitro genomicdata on both short-term and chronic time scales reflect theresponse of parenchymal cells to AHR activation to a greater extentthan other liver cell types. AHR activation results in the up- anddown-regulation of hundreds of genes (Black et al., 2012; Boutroset al., 2011; Boverhof et al., 2006; Brauze and Rawłuszko, 2012;Carlson et al., 2009; Fletcher et al., 2005; Forgacs et al., 2010;Franc et al., 2008; Heise et al., 2012; Le Vee et al., 2010; Ma et al.,2001; Moffat et al., 2010; Ovando et al., 2006, 2010; Rowlandset al., 2011; Vezina et al., 2004; Yao et al., 2012). These datapotentially offer insights into pathways and signaling networksthat might be important contributors to this AOP. For example, Nrf2induction is part of an antioxidant response pathway and is inducedby AHR activation (Wang et al., 2013). Induction of Nrf2 appears toprovide protection against TCDD-induced steatosis (Lu et al., 2011).Induction of Nrf2 protects against the effects of other liver carcin-ogens (Ikeda et al., 2004; Jaramillo and Zhang, 2013; Kwak et al.,2001; Liby and Sporn, 2012; Ma and He, 2012; Osburn et al.,2008; Shelton and Jaiswal, 2013; Yates and Kensler, 2007; Yateset al., 2007). Since these core battery responses may not bedirectly linked to toxicity they may best serve as biomarkers andhence, associative events, applicable for quantitative modeling ofAHR activation (Andersen et al., 2014; Budinsky et al., 2010, 2014;Julien et al., 2009; Simon et al., 2014).

3.2. Other gene expression changes

AHR activation results in transcription changes in many genesbeyond the core battery response. TiPARP is induced by TCDD andmay act to enhance ubiquitinization of proteins (Ma et al., 2001).Stearoyl coenzyme A desaturase 1 (Scd-1) is involved in fatty acidmetabolism andmay be involved in dioxin-induced steatosis; Scd-1is downregulated after 52 weeks of TCDD treatment in SpragueDawley Female rats but upregulated in immature mice followingacute TCDD exposure (Angrish et al., 2011; Ovando et al., 2010).Different strains of rats possess varying degrees of dioxin sensi-tivity; the dioxin-resistant Hans Wistar kupio strain has a majordeletion of the transactivation domain of the AHR and fewer geneexpression changes occur in this strain in response to high doses ofTCDD (Boutros et al., 2011).

At present, a clear and consistent transcriptional signalfollowing acute or short-term AHR activation across strains andspecies is not apparent. Early transcriptional changes so farmeasured in rats vary across strains and are not conserved acrossspecies, e.g., mice vs. rats, humans vs. rats (Angrish et al., 2011;Black et al., 2012; Ovando et al., 2010; Yao et al., 2012). Relevantpathways or gene networks cannot be identified due to the lack of aconsistent transcriptional response (Zhang and Andersen, 2007). Inaddition, the predominant transcriptional signal observed in vivorepresents the response of hepatocytes because these cellscomprise the bulk of the liver. Other cell types such as oval/stemcells, stellate cells, and bile duct cells may be central to the bio-logical response to sustained AHR activation that leads to tumorformation (Bendall and Nolan, 2012; Chia and Ng, 2012; Ma, 2011;Tanaka et al., 2011). However, the transcriptional response of theseother cell types is difficult to measure or observe.

3.3. Gene expression temporally concordant with the criticalhistopathological findings

Ovando et al. (2010) evaluated the 13 and 52 week liver tissuesfrom the 100 ng/kg/day 2006 NTP TCDD cancer bioassay treatmentgroup that showed clear evidence of hepatocellular and chol-angiolar carcinoma. Genes with altered expression at 24 h, 13

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weeks and 52 weeks of TCDD administration as well as a high leveldescription of the function of each gene (Ovando et al., 2010) arepresented in ST2 Supplemental Material. Histopathology that couldbe linked to the MIE was not observed in the livers of rats exposedfor 13 weeks; however, by 52 weeks, the histopathologic observa-tions were robust and clearly linked to the MIE (Hailey et al., 2005).Hence, the genomic signals observed at 52 weeks are thosetemporally concordant with the occurrence of histopathology.

How do these genomic changes correspond to the genomicsignatures of other non-genotoxic liver carcinogens? A number ofstudies have attempted to separate genotoxic from non-genotoxichepatocarcinogens based on their genomic signature (Ellinger-Ziegelbauer et al., 2005, 2008; Fielden et al., 2007; Fielden et al.,2011; Nie et al., 2006; van Delft et al., 2005; Watanabe et al.,2012). Nie et al. (2006) reported six gene expression changes aspredictive of non-genotoxic carcinogens after 24 treatment in malerats; however, no overlap exists between these six and the genomicchanges observed after 52 weeks of DLC exposure. Fielden et al.(2011) proposed a signature consisting of expression changes in22 genes for non-genotoxic rat liver carcinogens, and the trainingset included TCDD; only a few of these genes were reported ashaving altered expression in the livers of the rats in the NTP bio-assays at 13 or 52 weeks (Ovando et al., 2010). Whether consistentpatterns of gene expression changes and pathways can be linked toestablished KEs, associative events and modulating factors remainto be determined.

3.4. MIEdSustained AHR activation

Insight into sustained AHR activation is provided by examiningthe induction of ethoxyresorufin-O,O-deethylase in liver by threeDLCs at three different time points (NTP, 2006a; NTP, 2006b; NTP,2006c). Plots of the fractional or normalized ethoxyresorufin-O,O-deethylase response from three NTP cancer bioassays are shown inthe plots on the left of Fig. 2. Induction of ethoxyresorufin-O,O-deethylase is easily measured and serves as a marker of CYP1A1gene expression. The dose term on the x-axis is the area under thecurve (AUC) of liver concentration. The normalizedethoxyresorufin-O,O-deethylase response on the y-axis is similarat 14, 31 and 53 weeks (Left column of Fig. 2). The plots for 31weeks and 53 weeks are shifted to the right given the dose term onthe x-axis is the AUC of hepatic concentration of the three chem-icals, TCDD, 4-PeCDF and PCB-126. To obtain a measure of sus-tained AHR activation, the fractional ethoxyresorufin-O,O-deethylase response is multiplied by the number of weeks.Hence, a fractional response of 50% at 14 weeks would be a sus-tained AHR activation index of 7. The sustained AHR activationindex is plotted versus the AUC of hepatic toxic equivalents (TEQ)for all three chemicals calculated using TEF values of 1.0, 0.3 and0.1 for TCDD, 4-PeCDF and PCB-126 respectively (Van den Berget al., 2006) (Fig. 2, right column). The sustained AHR activationindex shows a strong relationship to the AUC of hepatic TEQ andthus the sustained AHR activation index reflects the dose, potencyand duration of DLCs in the liver.

Dose-response modeling can be performed using the sustainedAHR activation index as the dose term and the measures of thevarious KEs or biomarkers as the response. Fig. 4 shows an examplein which the well-known Hill doseeresponse model was used. Oneof the model parameters is the ED50 or EC50 value e in otherwords, the effective dose or concentration sufficient to produce a50% of the maximal response. This parameter is also called the half-maximal dose. When this measure of sustained AHR activation isused, the ESA50 denotes the level of sustained AHR activationrequired for a half-maximal response.

4. Cellular responses associated with KEs

AHR activation results in cellular responses that range fromtranscriptional changes to alterations in biochemical pathways thatultimately impact cellular function. These changes are dose- andtime-dependent and range from adaptation to toxicity.

4.1. KE #1: changes in cellular homeostasis and decreased apoptosisin hepatic foci and/or damaged hepatocytes

Tumor promotion requires a perturbation in the balance be-tween cell gain via mitosis and cell loss via apoptosis (Roberts et al.,1997). Indirectly, the inhibition of apoptosis in either damaged orinitiated cells favors their survival, and inhibition of apoptosis af-fords initiated cells an increased opportunity for clonal expansionand autonomous growth with the chance to acquire additionalmutations during the process of tumor progression. AHR activationinhibits apoptosis in altered hepatic foci (i.e., initiated hepatic cells),and this inhibition affords cells within altered hepatic foci a sur-vival advantage and increases the likelihood that these cells willacquire additional mutations.

TCDD inhibited apoptosis in altered hepatic foci (Luebeck et al.,2000; Paajarvi et al., 2005; Schrenk et al., 1994, 2004;Stinchcombe et al., 1995). For this KE, initiation-promotionstudies provide indirect evidence of inhibition of intrafocalapoptosis due to sustained AHR activation and direct evidence ofa threshold for the clonal expansion of altered hepatic foci(Dragan and Schrenk, 2000; Teeguarden et al., 1999). Althoughincreases of cell proliferation could contribute to the increase insize and volume fraction of altered hepatic foci, the greatermagnitude of inhibition of apoptosis suggests it is the primaryfactor contributing to clonal expansion of altered hepatic foci, atleast early on (Luebeck et al., 2000; Moolgavkar et al., 1996;Stinchcombe et al., 1995). TCDD inhibits apoptosis produced byUV light exposure of human cell lines and rat primary hepatocytes(Ambolet-Camoit et al., 2010; Chopra et al., 2009, 2010; Schwarzet al., 2000). Inhibition of apoptosis in primary rat hepatocyteswas mediated through phosphorylation and inactivation of p53and modulation of Mdm2, Tfgb1/4, and AGR2; in addition, inhi-bition of apoptosis required protein synthesis (Ambolet-Camoitet al., 2010; Chopra and Schrenk, 2011; Chopra et al., 2009,2010; Davis et al., 2001; Franc et al., 2008; Paajarvi et al., 2005;Worner and Schrenk, 1996, 1998). Cytotoxicity appears to occurafter intrafocal apoptosis inhibition is measured andinflammation-driven cell proliferation is a somewhat later event.What remains unknown is whether a proliferative response instem and stellate cells occurs earlier or at the same time asintrafocal apoptosis inhibition.

The growth of altered hepatic foci is highly related to tumorformation. The relationship of the MIE to this KE is illustratedschematically in Fig. 3. When the increase in volume fraction inaltered hepatic foci is plotted versus the MIE measured by thesustained AHR activation index, a threshold is apparent at a sus-tained AHR activation index in the range of 5e10 (Fig. 3B).

5. Organ-level response

Pre-neoplastic histopathological changes represent a significantorgan-level KE following sustained AHR activation and necessaryfor tumor promotion (Goodman and Sauer, 1992; Hailey et al.,2005). Quantitative doseeresponse modeling of the eventsincluded in toxic hepatopathy provides a path to understanding theAOP (Fig. 4).

Fig. 2. Measuring sustained AHR activation (SAA). Left: EROD (ethoxyresorufin-O-deethylase) responses of three DLCs at 14, 31 and 53 weeks in the NTP cancer bioassays inresponse the hepatic AUC (2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD); 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF); 3,30 ,4,40 ,5-Pentachlorobiphenyl (PCB 126)). Right: Combinedresponses of all chemicals expressed as AUC of hepatic TEQ. All curves were fit with Hill model implemented in Graphpad Prism.

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5.1. KE#2: toxic hepatopathy

As defined in NTP (2006a), toxic hepatopathy consists of aconstellation of different histological observations including, butnot limited to, hepatocyte hypertrophy, multinucleated hepato-cytes, inflammation, pigmentation, steatosis, portal fibrosis, bileduct cysts, cholangiofibrosis, mitochondrial injury, necrosis,fibrosis, and porphyria, as well as bile duct and oval cell hyperplasia(Boverhof et al., 2005; Chang et al., 2005; Hailey et al., 2005; Jonesand Greig, 1975; NTP, 2006; NTP, 2006; NTP, 2006; NTP, 2006; NTP,2006; NTP, 2006; NTP, 2006; Walker et al., 2006; Simon et al.,2009).

For the purpose of distinguishing KE#2 from KE#3, KE#2 isconsidered to represent the effects included in hepatopathywithout bile duct hyperplasia and oval cell hyperplasia. Transitional

dose values as a measure of possible thresholds for toxic hepat-opathy, bile duct hyperplasia and oval cell hyperplasia in units ofthe sustained AHR activation index are shown in Table ST3(Supplemental Materials). This value for hepatopathy is consider-able lower than those for bile duct hyperplasia or oval cell hyper-plasia and thus, support the idea that toxicity and proliferation aredistinct KEs.

The relationship of this constellation of organ-level effects tohepatocellular cancer is well established in mammalian species; forexample, steatosis and nonalcoholic steatohepatitis increase therisk of liver cancer in humans and rodents (Ip and Wang, 2014;Nakamura and Terauchi, 2013). Sustained AHR activation inducessteatosis and could act in a similar fashion.

Necrosis and inflammation are not observed after 52 weeks ofDLC administration but occur during the second year of treatment

Fig. 3. Rat liver tumor promotion by persistent AHR ligands. A) Simplified diagram showing changes in both cell proliferation and apoptosis appear to promote tumors. B) Plot of theincrease in volume fraction of ATPase-deficient hepatic foci versus sustained AHR activation (SAA) index. Data from Teeguarden et al. (1999). The Hill model was used to fit the dataand implemented in Graphpad Prism.

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and are observed at the 2-year termination of the NTP cancerbioassays. Necrosis and inflammation are observed at lower dosesthan those at which tumors were observed (Hailey et al., 2005).Damage to hepatocytes and cytotoxicity are common features ofthese histopathological changes. This provides empirical supportfor the hypothesis that regenerative repair may be a contributor tothe proliferative response and the adverse effects observed at thehigher TCDD dosages.

The quantitative KER linking the MIE to KE#2 is shown in thetop panel of Fig. 4A. Usingmethods described in Simon et al. (2014),the transitional dose values of the sustained AHR activation indexbased on a 21% response level are shown in ST3 SupplementalMaterial. Here, the transitional dose value is the projection fromthe 21% response level to the background response level using theslope of the doseeresponse at this same 21% response level (Sandet al., 2006; Simon et al., 2014). While not definitive thresholds,transitional dose values based on the 21% response level can beused as an approximation of a threshold. As noted, toxic hepatop-athy includes a constellation of effects and the transitional dosevalue has the lowest value of the three effects representing thetoxicity of KE#2 and the proliferation/hyperplasia of KE#3.

ESA50 (Fig. 4) is similar to an EC50 e it is the effective level ofsustained AHR activation necessary to achieve a 50% response. Asdescribed, the level of sustained AHR activation can be calculatedby multiplying the fractional level of AHR activation measured byCYP1A1 induction by the number of weeks of dosing (Fig. 2B). Bileduct hyperplasia occurs at a higher level of sustained AHR activa-tion and oval cell hyperplasia at an even higher level (Fig. 4A,middle and bottom plots). The doseeresponse for oval cell hyper-plasia is much steeper than the other two histopathological effectsthat comprise KE#3. Although speculative, this higher level ofsustained AHR activation needed for bile duct hyperplasia may bethe reason why Kociba et al. (1978) failed to observe bile duct tu-mors whereas they were observed in NTP (2006a). Possibly, thedistinction between the two studies may be that dosage regimen(diet vs. gavage) or changes in the SpragueeDawley strain overtime. In the rats used in Kociba et al. (1978), the degree of sustainedAHR activation needed for promotion of bile duct tumors may nothave been achieved.

5.2. KE#3: altered proliferation and hyperplasia of hepatocytes,stem cells and other cell types in the liver

Over the time period of high levels of sustained AHR activation,DLCs produce a complex pattern of cell proliferative responses.Teeguarden et al. (1999) observed that rats initiated with

diethylnitrosamine (DEN) and then dosed with either 0.1 or 1 ng/kg-d TCDD for one month exhibited a reduced labeling indexrelative to controls, and reduced BrdU-labeling was also observedfollowing the 0.1 ng/kg-d TCDD dose after three months. Maronpotet al. (1993) observed a reduction in BrdU labeling index in hepa-tocytes at a low dose of 3.5 ng/kg-d TCDD in DEN-initiated rats after30 weeks of TCDD administration but, at a dose of 125 ng/kg/d thelabeling index was increased.

Both parenchymal calls and liver stem cells are likely involved inthe organ-level response to sustained AHR activation. Early acti-vation of the AHR in zone 3 of the liver acinus causes decreasedhepatocyte replication and may act as an indirect proliferativestimulus for stem cells and hepatoblasts (Andersen et al., 1997;Conolly and Andersen, 1997; Tritscher et al., 1992). Oval cells inthe periportal region likely function as a source of replacement ofhepatocytes after inhibition of normal hepatocyte replication-replacement (Paku et al., 2001; Sahin et al., 2008; Tanaka et al.,2011; Wang et al., 2003). While hepatocyte replication is consid-ered the normal means for replacement of liver parenchyma, in-hibition of hepatocyte replication in centrilobular regions inducedby TCDD may induce normally quiescent liver stem cells toproliferate.

Following longer period of sustained AHR activation, organ-level increases in cell proliferation ensue, demonstrated by an in-crease in BrdU labeling and likely reflecting the regenerativeresponse to organ-wide toxicity (Hailey et al., 2005).

Non-parenchymal cells, including stem cells, hepatoblasts,biliary cells, stellate cells, endothelial cells, and Kuppfer cells, play arole in this AOP. In rodents, TCDD elicits a fibrogenic and bile ductproliferative response that requires pathological alteration of stel-late cell function and increased differentiation and growth ofhepatoblasts and bile duct cells before 33 weeks of exposure.Retinoid depletion induces stellate cell proliferation, production ofextracellular matrix components, and the transition to fibroblast;stellate cells maintain vitamin A homeostasis and respond to liverinjury with formation of proliferative cytokines such as TGF-a andEGF (Friedman, 2008; Pintilie et al., 2010; Senoo et al., 2010). TCDDinduces loss of retinoid content (presumably from stellate cells) andmay disrupt the extensive communication between various livercell types (Fletcher et al., 2001; Hoegberg et al., 2005; Pierre et al.,2014; Schmidt et al., 2003). Thus, TCDD-induced retinol loss fromhepatic stellate cells may contribute to cell proliferation, biliaryfibrosis, and cholangiolarcarcinoma (Fattore et al., 2000; Friedman,2008; Hakansson and Hanberg, 1989; Schmidt et al., 2003).

AHR activation also induces changes in stem/oval cells. All of therats receiving 100 ng/kg/day TCDD, the highest dose group animals

Fig. 4. Indirect key event relationships. Sustained AHR activation was identified as the MIE and determined as the product of fractional AHR activation measured by CYP1A1induction and time in weeks. The frequency of histopathology and tumors observed in rats in the NTP bioassays was plotted against sustained AHR activation. A) Relationshipsbetween the MIE and aspects of KE2 (hepatopathy and hyperplasia; all response shown as filled circles). B) Relationships between the MIE and the two tumor types representing theadverse outcome (AO) (responses shown as an X). The Hill coefficients, BMD21 and TDV21 values are shown in ST3 Supplemental Material. The values shown as open circles in thetwo plots in B show tumor responses in animals in the stop-exposure group that were exposed to the maximum DLC concentration for 31 weeks (see text for discussion). In eachplot in B, three values, one each for TCDD, 4-PeCDF and PCB-126, are shown for the stop-exposure experiments and only two are not obscured by the other responses. The numericalESA50 values are the half-maximal level of sustained AHR activation similar to an EC50 and described in the text.

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in the 2-year cancer bioassay, developed oval cell hyperplasia withclear statistical increases in this endpoint at 22 ng/kg/day or greater(Hailey et al., 2005).

Evidence points to the involvement of TNF-a regulation in theproliferative response of hepatic stem cells; this is likely mediatedthrough modulation of the levels of TNF-a, altered b-cateninsignaling, and inhibition of cell-to-cell contact (Knight et al., 2000;Umannov�a et al., 2007; Vondracek et al., 2009; Dietrich et al., 2002;Prochazkova et al., 2011; Proch�azkov�a et al., 2011; Weiss et al.,2008). TNF-a is an inflammatory cytokine with an important rolein liver tumor promotion. More research on how sustained AHRactivation dysregulates normal TNF-a activity could be very im-pactful on evolving the AOP.

5.3. Adverse outcome: hepatocellular adenoma/carcinoma andcholangiolar carcinoma in rodents

As well as toxic hepatopathy, the organ-level response includesthe adverse effects of hepatocellular adenomas/carcinomas, andcholangiocarcinomas as the apical adverse outcomes. Adenomasmay arise from altered hepatic foci that are derived from hepato-cytes or hepatoblasts whereas hepatocellular carcinomas andcholangiocarcinomas likely arise from initiated stem cells. How-ever, the actual cellular origin of the various liver tumor types is notknown with certainty and involvement of both liver stem cells andhepatocyte-like cells have been observed in hepatocellular ade-nomas (Libbrecht et al., 2001; Libbrecht, 2006).

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These tumor types (hepatocellular adenomas/carcinomas, andcholangiocarcinomas) occur at a transitional dose value for sus-tained AHR activation of about 100 (ST3 Supplemental Material).This would represent maximal or 100% activation of the AHR sus-tained for 100 weeks. The calculation is borne out by the extremelysteep doseeresponse curves (Fig. 4B). In each of two plots in Fig. 4B,three open circles (one each for TCDD, 4-PeCDF and PCB-126) showtumor responses in animals in the stop-exposure group that wereexposed to the maximum DLC concentration for 31 weeks. In thesestop-exposure groups maximal AHR activationwas sustained for 31weeks, about 30% of the lifespan. The tumor incidence for bothtypes of tumors in the animals in these stop-exposure groups was5% or less for both tumor types and not significantly increased.However, the occurrence of tumors indicates that a sustained AHRactivation value of 30 is sufficient for tumor formation, e.g., a pointof departure consideration for anchoring the threshold response.

6. Species specificity and relevance to humans

Overall, empirical evidence supporting the applicability of thisAOP to humans is absent. Only a single KE has been observed inhumansdbinding and activation of the AHR by DLCs with accom-panying hepatic CYP1A induction (Abraham et al., 2002; Budinskyet al., 2010; Coenraads et al., 1999; Lambert et al., 2006; Tang et al.,2008). For perspective purposes regarding an unequivocal dioxineffect occurring in highly exposed human subjects, high levels ofAHR activation in humans alter the growth and differentiation ofkeratinocytes and produce chloracne (Forrester et al., 2014; Geusauet al., 2001; Ju et al., 2011; Moses and Prioleau, 1985; Saurat andSorg, 2010; Saurat et al., 2012; Sorg, 2014; Sutter et al., 2009,2010, 2011). In contrast, the epidemiological evidence for TCDD-associated liver cancer in humans is negative or equivocal(Akintobi et al., 2007; Bertazzi et al., 1989; Du et al., 2006; Geusauet al., 2005; Hankinson, 2009; Loertscher et al., 2001). Tri-chlorophenol workers exposed toTCDD show no increase in liver orbiliary cancer (Collins et al., 2009; McBride et al., 2009). Theoccurrence of chloracne indicates high levels of exposure to DLCsand significant AHR activation; even in such cases, no evidence ofliver injury or cancer has been reported (Ghezzi et al., 1982;Mocarelli et al., 1986, 1991; Pocchiari et al., 1979; Reggiani, 1980).In other trichlorophenol workers, transient changes in liver enzymelevels were reported in alcohol consumers only (Calvert et al.,1992).

The unique Yusho and Yucheng rice oil poisonings are con-foundeddthe exposures were to a mixture of complex PCBs, pol-ychlorinated dibenzofurans, and mixtures of quarterphenyl, andterphenyl compounds. Clearly, these compounds possess dioxin-like properties and the mixture was sufficiently potent to inducea chloracne-like condition in some individuals (Lambert et al.,2006). An increase in mortality from cirrhosis and chronic liverdisease has been observed among the victims of the Yushopoisoning incident, whereas liver cancer was not elevated(Onozuka et al., 2009). The rate of mortality from chronic liverdisease was increased in men only among the victims of the similarYucheng poisoning incident without excess liver cancer in eithersex (Tsai et al., 2007).

In contrast to humans, rodents are highly susceptible to thehepatotoxic, proliferative, and carcinogenic effects of TCDD (Haileyet al., 2005; Goodman and Sauer, 1992; Kociba et al., 1978). Tosummarize, the sustained AHR activation rodent liver tumor pro-motion AOP appears to be a pathway that very likely requires ex-ceedance of a threshold for sustained AHR activation for livercancers to occur in rodents (e.g. Fig. 4). In humans, increases in livercancer have not been observed even in highly exposed populations,and no population level data in humans are available showing an

increased liver cancer response, even in individuals with chloracneand obvious high exposure to DLCs. However, as is often the case forevaluations of chemicals which lead to tumor formation in labo-ratory animals, for regulatory purposes, assumptions are made thatpotential risks to human health can be estimated from animalstudies. In many cases where there is sparse data, a default linearno-threshold extrapolation method is used. However, in applyingthis AOP for such an assessment, the extensive body of scientificevidence clearly indicates that liver tumor promotion by DLCs onlyoccurs after a threshold level of sustained AHR activation isexceeded. These thresholds also become apparent in Figs. 3B and 4.Therefore, a quantitative application to derive an exposure guid-ance value for humans to address the potential for tumor promo-tion by DLCs should be based on a threshold mode of action (e.g.,Simon et al., 2009).

7. Scientific confidence in the AOP

The Bradford Hill (BH) considerations (doseeresponse, tempo-rality, strength, consistency, specificity and biological plausibility ofthe proposed association) form the basis for evaluating weight ofevidence within the U.S. Environmental Protection Agency'sGuidelines for Carcinogen Risk Assessment, the WHO/IPCS humanrelevance-MOA framework, the key events/doseeresponse 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 beenupdated and additionally tailored for AOPs by the OECD to facilitateevaluations of KEs and KERs as well as the overall AOP (Meek et al.,2013, 2014a, 2014b; OECD, 2014; Becker et al., 2015).

Below, we summarize the weight of evidence evaluation con-ducted using these AOP-tailored BH considerations of biologicalplausibility, essentiality and empirical evidence for the sustainedAHR activation rodent liver tumor promotion AOP.

7.1. Support for biological plausibility of the KERs

The OECD AOP guidance (OECD, 2014) for evaluation of biolog-ical plausibility of an AOP provides this defining question forevaluating biological plausibility: “is there a mechanistic (i.e.,structural or functional) relationship between KEup and KEdownconsistent with established biological knowledge?” Under theOECD guidance, a high degree of confidence is afforded when thereis an establishedmechanistic basis and “extensive understanding ofthe KER based on extensive previous documentation and broadacceptance.” For biological plausibility, for this AOP, the WoE foreach KER is judged to be strong, as is the WoE for the overall AOP.

All the elements in this AOP are strongly associated with thebiological steps and elements of carcinogenesis (Hanahan andWeinberg, 2011). First, there is extensive body of mechanistic evi-dence in support the biological plausibility of this MOA (see recentreview by Budinsky et al., 2014). Further, the relationships betweensustained 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 asurfeit of published studies (e.g., Fig. 4). Moreover, overall consis-tency with knowledge of the pathogenesis of liver tumor promo-tion is supported by replication of events related to tumorpromotion across different laboratories and the multiple lines ofevidence for sustained AHR activation acting as a mechanism ofliver tumor promotion. Thus, the AOP is well supported by the KEs,consistent with the biology of carcinogenesis and the events oftumor promotion (Dietrich and Kaina, 2010; Gasiewicz et al., 2008;Roberts et al., 1997).

The unique sensitivity of the female rat response suggests a

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possible role for estrogen as a modulating factor in the tumorigenicMOA. Estrogen is an established co-promoter of tumorigenesis andthus 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 re-ceptor pathway may also be a contributing factor (Matthews andGustafsson, 2006). Such receptor mediated cross talk is consistentwith the sustained AHR MOA.

7.2. Support for essentiality of KEs

The defining question contained in the OECD AOP guidance(OECD, 2014) for evaluation of essentiality is “are downstream KEsand/or the AO prevented if an upstream KE is blocked?” Overall, theevidence in support of essentiality for sustained AHR activation, theMIE, is strong. There is direct evidence of essentiality from the stop-exposure group in the cancer bioassay; the 100 ng/kg/d dose ofTCDDwas stopped after 30 weeks and at the 2-year termination, nostatistically significant increase in tumor frequency was observed(NTP, 2006a; NTP, 2006b; NTP, 2006c). This observation also in-dicates that theMIE of sustained AHR activation requiresmore than30weeks of continuous exposure and is consistent with the generalonset of hepatopathy around the same time (Hailey et al., 2005).Additional support for essentiality comes from studies that showthat 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 beenconfirmed 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 theincidence of tumors and hepatotoxicity (Andersson et al., 2002;Brunnberg et al., 2006; Chopra and Schrenk, 2011; Moennikeset al., 2004).

7.3. Empirical evidence

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

When the KEs and associative events are placed in sequentialorder based on dose and temporality, the doseeresponse slopesfrom Hill doseeresponse model fits of the data increase in value.Thus, the later KEs occur with a steeper slope than early KEs and thenumber 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 earliestand 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 thatare more aligned with the concept of a pre-MIE and thus providesan associative event for AHR activation. Since measurable enzymeinduction persists for at least one year, we used the AUC of abiomarker for this enzyme induction as ameasure of sustained AHRactivation. Both Hill equation coefficients and half maximal con-centrations increase with increasing values of sustained AHR acti-vation and reflect dose-dependent transitions as KEs occur at thevarious levels of biological organization (Simon et al., 2009;

Budinsky et al., 2014).Table 2, the doseeresponse temporality table, 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 rele-vance MOA framework and is recommended in the OECD AOPguidance (OECD, 2014). Here, the value of the sustained AHR acti-vation index has been provided for each doseetime combination.One can easily see the increase in sustained AHR activation due tothe increase in dose going down the table and the increase induration 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 inthe TCDD cancer bioassay, which showed that when the adminis-tration of 100 ng/kg/d TCDD was stopped after 30 weeks, a statis-tically significant increase in tumor frequency was not observed(NTP, 2006a). This observation also indicates that the AHR activa-tion needs to be sustained for more than 30 weeks for KE#2 tooccur (Hailey et al., 2005).

7.4. Discussion of likelihood of alternative modes of action

Alternative MOA(s) or KEs and KE elements can be examined tohelp 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; Seedet al., 2005; Sonich-Mullin et al., 2001; USEPA, 2005).

One such alternative MOA would be that DLCs act to produceliver tumors in rodents by a mutagenic mechanism. However, thereis substantial evidence that DLCs are neither mutagenic nor geno-toxic compounds and thus do not act by a mutagenic MOA (Bockand 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 andWilliams, 1996).

Effects on gap junctions or induction of oxidative stress are twoother potential mechanisms. TCDD disrupts normal gap junctionactivity and intercellular communication in rat primary hepato-cytes and WB-344 cells (Andrysík et al., 2013; Bager et al., 1997;Herrmann et al., 2002; Weiss et al., 2008). Further research isneeded to understand the contribution of this mechanism to DLC-induced rodent liver tumor formation. Oxidative stress appears lesslikely as an alternativeMOA andmay be a late-occurring associativeevent due to continued high activity of phase 1 mixed functionoxidases and accompanying cytotoxicity.

In summary, the WoE in support of sustained AHR activationleading to changes in cellular growth homeostasis and eventuallypromotion of liver tumors in rodents is strong. TheWoE supportingthe alternative MOAs is much weaker.

8. Discussion: applications of this AOP

The OECD guidance for AOP development (OECD, 2013) suggestsa number of potential uses for AOPs. These include 1) categoryformation for read-across, 2) integrated approaches for testing andassessment 3) development or refinement of test methods such asOECD test guidelines and 4) hazard identification (classification/labeling) and risk assessment. The use of a specific AOP for any oneor more of these applications depends on scientific confidence inthe AOP for each specific use. An AOP can offer practical utility incertain applications even if confidence is not sufficient to quanti-tatively predict the AO from the MIE. Application of the sustainedAHR activation AOP for several applications was described in briefin Patlewicz et al. (2015); herein we discuss the confidence of thisAOP in more detail.

Table 2Empirical 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) andMaronpot 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 the TCDD bioassay. The number in parentheses is the administered gavage dose inng/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 in the text and is used as the dose term in Figs. 3 and 4.

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8.1. Which KEs can Be used to predict the adverse outcome?

To date, no quantitative models have been developed to predictthe adverse outcome from AHR activation by ligand binding. Withthe exception of DLCs, PCDDs, PCDFs and co-planar PCBs, thispredictive capability is highly uncertain for the plethora of AHRligands. For example, indole 3-carbinol is an AHR ligand occurringin cruciferous vegetables and acts as a cancer chemopreventiveagent. In the stomach, indole 3-carbinol forms 3,3-diinolylmethane, a potent AHR ligand that has shown promise forpreventing tumor reoccurrence in humans (Banerjee et al., 2011).Dietary administration of indole-3-carbinol for 23 weeks inhibitedtumor formation in rats initiated with diethylnitrosamine (Tanakaet al., 1990). However, also in diethylnitrosamine-initiated rats,prolonged indole 3-carbinol administration (>26 weeks) increasedthe progression of altered hepatic foci to hepatocellular adenomas(Yamamoto et al., 2013). A recent NTP 2-year cancer bioassay failedto demonstrate indole 3-carbinol as a liver tumor promoter in fe-male rats (NTP, 2014). Furthermore, endogenous AHR ligands andnaturally occurring exogenous ligands occurring in foods havecancer-preventive properties and likely contribute to a relativelyhigh level of AHR activation activity in human blood (Connor et al.,2008; Navarro et al., 2009, 2011; Peterson et al., 2009; Wincentet al., 2009). These naturally occurring and endogenous ligandsinduce both their own metabolism and that of other AHR ligandsthrough 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 toKEs related to liver tumor promotion.

Neither binding of ligand to the AHR nor short-term transcrip-tional changes and cellular responses are sufficient to produce livertumors in rats. Strains of rats that show resistance towards the toxicand carcinogenic effects of DLCs express different genomic profilesoutside 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 occurwithin hours of ligand activation; yet the subsequent KEs and AOrequire months of sustained AHR activation for tumors to occur.Hence, the distinction between short-term and sustained activationof the AHR is an important one and AHR activation must be sus-tained for more than 30% of the rodent lifespan to result in tumorpromotion.

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

8.2. Using this AOP for grouping chemicals into chemical categoriesfor read-across

Without some measure of sustained activation, the use of pre-MIEs for any purpose other than preliminary screening is prob-lematic as a predictive criteria for liver tumor promotion. Evidenceclearly shows it is the combination of sustained AHR activation andthe subsequent biological changes involving complex parenchymaland non-parenchymal cell interactions that underlie the hepato-toxicity, 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 theAHR and the species- and strain-specificity of the initial genomicresponses suggest that category development may prove a chal-lenge (Denison, 2011; Dere, 2011).

8.3. Using this AOP for integrated approaches to testing andassessment (IATA)

The most straightforward use of this AOP within an integratedtesting and assessment approach for hazard evaluationwould be todetermine the potential for a substance to activate the AHR in asustained manner with long-term changes in gene transcriptioninvolving multiple cell types, which leads to increased liver cellproliferation. An IATA decision-tree approach, for illustrative pur-poses has been already presented by Patlewicz et al. (2015).

The initial steps in the IATA focus on evaluating molecular andcellular events related to AHR binding and transcriptional activa-tion using rapid and cost effective in silico or in vitro assays. Com-pounds found to be inactive in such assays would not proceedforward into further testing.

At the present time, there is insufficient understanding topermit the use transcription profiling as a metric of sustained AHRactivation to quantitatively predict development of rat liver foci andliver tumors. Therefore, the IATA proposes that substances found tobe active in the AHR mechanistic assays be subjected to a decisionframework for further evaluating the potential to act as rodent livertumor promoter. For example, a subchronic study, utilizing anappropriate dosing regimen, may be able to rule-in or rule-out thesubstance's ability to trigger the critical histological components ofhepatopathy (Hailey et al., 2005). Or a rodent liver initiation-promotion assay could be considered, though interpretation canbe challenging, given that indole-3-carbinol has been reported toinhibit, and also to enhance, development of liver tumors in initi-ated animals, whereas in the recent, more definitive 2-year rodentstudy indole-3-carbinol was negative for liver tumors (Tanaka et al.,1990; Yamamoto et al., 2013; NTP, 2014). Patlewicz et al. (2015) alsoillustrate how exposure information can be used in conjunctionwith the AOP to inform IATA decisions.

8.4. Using this AOP to inform test method development orrefinement

An IATA consisting of a suite of in vitro and in vivo (e.g., sub-chronic) assays to predict hepatopathy, a complex histologicalresponse, may be needed to differentiate AHR ligands with andwithout liver tumor promotion potential. Theoretically, it may beplausible to consider using a combination of AHR-binding, AHR-transcriptional activation and rat liver initiation-promotion assaysto develop a prediction model for sustained AHR activation-induced rat liver tumors. Therefore, within the OECD test guide-lines program, it may be worthwhile to consider developing per-formance criteria that could be applied to judge the scientificquality and reliability of in vitro AHR-binding and transactivationassays, liver stem cell assays, as well as a validated test guideline fora rat liver tumor (hepatic foci) initiation-promotion assay.

8.5. Taxonomic applicability: using this AOP for screening-levelevaluations of hazard and risks to humans

Over the last 15e20 years, screening-level hazard and riskevaluations have commonly used the World Health OrganizationInternational Programme on Chemical Safety (WHO/IPCS) TEFs forDLCs (Van den Berg et al., 2006). To be assigned a TEF, a compoundmust 1) show a structural relationship to the PCDDs and PCDFs; 2)

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bind to the AHR; 3) elicit AHR-mediated biochemical and toxicresponses; and 4) be persistent and accumulate in the food chain.The advantage of the TEF approach is that the TEQ can be defined bythe sum of the products of the concentration of each compoundmultiplied by its TEF. However, a wide variety of structurallydiverse, non-dioxin-like naturally occurring chemicals in vegeta-bles, fruits, teas, etc. can also bind to, and activate, the AHR (Connoret al., 2008; Navarro et al., 2009, 2011; Peterson et al., 2009). Thus,what is not clear is whether substances with low TEFs would beexpected to act in vivo in a manner similar to these naturallyoccurring substances or in a manner more akin to TCDD. Non-dioxinelike AHR ligands can increase or decrease the toxicity of2,3,7,8-TCDD and related compounds (Van den Berg et al., 2006).Therefore, hazard or risk assessments relying on theWHO-IPCS TEFapproach need to be appropriately caveated. Similar challenges arefaced in using this AOP for conducting screening level hazard or riskevaluations for human health.

At a number of levels of biological organization, differences existbetween the human and rodent AHR. Considering toxicodynamics,the human AHR binding affinity is an order of magnitude or morelower than that in rodents that is generally correlated with reducedsensitivity in human hepatocytes relative to rats (Black et al., 2012;Budinsky et al., 2010; Connor and Aylward, 2006). In addition, thesespecies differences include AHR binding affinity, different recruit-ment of co-regulatory proteins, and different patterns of generegulation (Black et al., 2012; Budinsky et al., 2010; Carlson et al.,2009; Connor and Aylward, 2006; Dere et al., 2011; Flaveny et al.,2010). Considering toxicokinetics, TCDD tissue concentrationsrequired to induce hepatic enzyme induction in humans are higherthan those that elicit similar responses in rodents or non-humanprimates (Abraham et al., 2002; Coenraads et al., 1999; Kim et al.,2009; Lambert et al., 2006; Schrenk et al., 1995; Silkworth et al.,2005; Sutter et al., 2010; Tang et al., 2008; Xu et al., 2000). Interms of the whole organism, a minimal increase in CYP1A activitycan be observed at lipid-normalized blood concentrations less than1000 ppt in humans (Abraham et al., 2002; Guzelian et al., 2006).The corresponding adipose concentration in rats corresponding to aminimal increase in CYP1A activity would be about 100 ppt in lipid,consistent with earlier work showing rats are about an order ofmagnitude more sensitive than humans (Budinsky et al., 2014).Mice transfected with the human AHR are less responsive to dioxinthan mice carrying the wild-type AHR (Flaveny et al., 2009). Inhuman primary cell lines, dioxin-mediated changes in gene tran-scription also require higher concentrations than those in rodents(Budinsky et al., 2010; Haarmann-Stemmann et al., 2007; Kim et al.,2009; Schrenk et al., 1995; Uno et al., 2009; Westerink et al., 2008;Silkworth et al., 2005; Xu et al., 2000).

Table 3 summarizes the qualitative concordance of KEs in theAOP between rats and humans. To the extent humans have been

Table 3Concordance of KEs in the AOP between rats and humans for the purpose of assessing h

Key Event Pre-MIE and MIE KE#1 KE#2AHR Binding and Activation(Pre-MIE and MIE)

Altered Cellular GrowthHomeostasis & Inhibitionof Apoptosis

Hepatopa

QualitativeConcordance

Yes; occurs in both species Biologically plausible(no direct evidence)

Biological(no direct

QuantitativeConcordance

Yes; occurs in vivo andin vitro in both species;observed as increases inxenobiotic metabolizingenzymes (e.g., CYP1A)

Yes but limited; occursin vitro in cells of humanorigin, but in vivo datafrom humans is lacking

Has not bin highly ehumans

inadvertently, accidentally, or intentionally exposed to TCDD, noevidence of increased liver cancer or even liver injury have beenobserved, consistent with rats being more sensitive than humans.Given that tumorigenic responses in rodents only occur when AHRactivation is sustained for a period approximating 30% of the life-span, and the steep slopes corresponding to responses elicitedwhen this apparent threshold of AHR activation is exceeded, riskassessments for humans using this AOP should employ a thresholdmodel.

As noted above, binding to the AHR is insufficient to infer activityleading to the adverse outcome of liver tumors. Moreover, there isconsiderable scientific debate as to whether the rat liver tumori-genic responses induced by TCDD are relevant endpoints for humanhealth. WHO indicates that cancer may not be the most sensitiveresponse in either humans or animals and EPA's latest assessment isbased on non-cancer effects in humans (sperm deficits amongyoung males exposed between the ages of 1e9 and increased TSHlevels in 72-hour neonates born of Seveso mothers with elevatedserum TCDD concentrations). Nonetheless, the utility of the AOP isthe identification and ordering of effects, demonstration of dos-eeresponse concordance and illustrating that rodent liver tumorpromotion by sustained AHR is a threshold phenomenon.

9. Conclusions

Many excellent reviews and an extensive database of publishedresearch provide the foundation for this AOP (e.g., Budinsky et al.,2014). This case study provides a systematic construction of theAOP according to the OECD template, drawing from this extensivebody of scientific studies. When one considers the amount of in-formation on DLCs and AHR, one of the most significant challengesis gathering, distilling and organizing all of the relevant informa-tion into a concise AOP that adequately reflects the currentknowledge and hypotheses on how sustained AHR activation leadsto tumor promotion and hepatic carcinogenesis in rodents. We fullyexpect that as experience is gained in developing and applyingAOPs, this AOP and its applications will evolve and improve. Moredata regarding doseeresponse, species sensitivity, and differentAHR ligands, that provide additional insight into KEs, associativeevents and/or modulating factors will certainly strengthen the AOP.In addition, employment of a consistent, structured scientific con-fidence framework to evaluate AOPs (OECD, 2014; Becker et al,2014; Patlewicz et al., 2013, 2015) will help to document andcommunicate the evidence in support of KEs, methods to measureKEs, KERs, and prediction models. Users of AOPs can then makemore informed decisions regarding which applications of a givenAOP can be confidently undertaken (Patlewicz et al., 2015).

uman relevance (adapted from Budinsky et al., 2014).

KE#3 AOthy Alterations in Cellular

Proliferation/HyperplasiaAdverse Outcome: HepatocellularTumors and Cholangiolar Carcinomas

ly plausibleevidence)

Biologically plausible(no direct evidence)

Plausible but weak (e.g., highestrelative risk in highest exposurehuman cohorts for all cancerscombined is 1.4) (WHO IPCS, 1998))

een observedxposed

No evidence in humans Evidence in humans largely negative(e.g., no evidence of increased hepatictumors among former trichlorophenolworkers who were exposed to TCDD(Collins et al., 2009))

R.A. Becker et al. / Regulatory Toxicology and Pharmacology 73 (2015) 172e190 185

Acknowledgments

The authors had complete control over the design, conduct,interpretation, and reporting of the analyses included in thismanuscript. The contents of this manuscript are solely the re-sponsibility of the authors and do not necessarily reflect the viewsor policies of their employers. Ted Simon received funding tosupport this research from the American Chemistry Council (ACC).Craig Rowlands and Robert Budinsky are employed by a companyengaged in chemical product manufacturing and remediation ofsoils and sediments containing dioxins. Richard Becker is employedby ACC, a trade association of U. S. chemical manufacturers. Note:during development of this manuscript Grace Patlewicz wasemployed by a company engaged in chemical productmanufacturing; subsequently she became an employee of the Na-tional Center for Computational Toxicology, U.S. EnvironmentalProtection Agency.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.yrtph.2015.06.015.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.yrtph.2015.06.015.

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