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F3 Chemical Safety and Alternative Methods Unit incorporating EURL ECVAM
Directorate F – Health, Consumers and Reference Materials
Joint Research Centre, European Commission
Brigitte.LANDESMANN (at) ec.europa.eu
Point of Contact
Brigitte Landesmann (email point of contact)
- Brigitte Landesmann
|Author status||OECD status||OECD project||SAAOP status|
|Open for citation & comment||TFHA/WNT Endorsed||1.14||Included in OECD Work Plan|
This AOP was last modified on August 02, 2018 03:58
|N/A, Liver fibrosis||September 16, 2017 10:14|
|Alkylation, Protein||September 16, 2017 10:14|
|N/A, Cell injury/death||September 16, 2017 10:14|
|Activation, Stellate cells||September 16, 2017 10:14|
|Accumulation, Collagen||September 16, 2017 10:14|
|Tissue resident cell activation||October 08, 2018 05:22|
|Increased Pro-inflammatory mediators||August 02, 2018 02:47|
|Alkylation, Protein leads to N/A, Cell injury/death||November 29, 2016 20:02|
|N/A, Cell injury/death leads to Tissue resident cell activation||August 02, 2018 03:02|
|Tissue resident cell activation leads to Increased pro-inflammatory mediators||August 02, 2018 03:37|
|Increased pro-inflammatory mediators leads to Activation, Stellate cells||August 02, 2018 03:43|
|Activation, Stellate cells leads to Accumulation, Collagen||November 29, 2016 20:03|
|Accumulation, Collagen leads to N/A, Liver fibrosis||November 29, 2016 19:55|
|N/A, Cell injury/death leads to Activation, Stellate cells||November 29, 2016 19:54|
|Allyl Alcohol||November 29, 2016 21:18|
|Carbon tetrachloride||November 29, 2016 21:18|
|Retinol||November 29, 2016 21:20|
|Dimethyl nitrosamine||November 29, 2016 21:19|
|Thioacetamide||November 29, 2016 21:20|
Hepatotoxicity in general is of special interest for human health risk assessment. Liver fibrosis in particular is an important human health issue associated with chemical exposure and predictive assays are lacking; it is a typical result of chronic or repeated-dose toxic injury and one of the considered endpoints for regulatory purposes. It is a long-term process in which inflammation, tissue destruction, and repair occur simultaneously, together with sustained production of growth factors and fibrogenic cytokines due to a complex interplay between various hepatic cell types, various receptors and signalling pathways which lead to an imbalance between the deposition and degradation of extracellular matrix (ECM) and a change of ECM composition. Due to this complex situation an adequate cell model is not available and an in vitro evaluation of fibrogenic potential is therefore not feasible. A sufficiently detailed description of the AOP to liver fibrosis might support chemical risk assessment by indicating early (upstream) markers for downstream events and facilitate a testing strategy without the need for a sophisticated cell model. This systematic and coherent display of currently available mechanistic-toxicological information can serve as a knowledge-based repository for identification/selection/development of in vitro methods suitable for measuring key events (KEs) and their relationships along the AOP and to facilitate the use of alternative data for regulatory purposes. Identified uncertainties and knowledge gaps can direct future research by priority setting and targeted testing. The KE descriptions can be used for hazard identification and read-across to assess the toxic potential of an untested substance.
This AOP describes the linkage between hepatic injury caused by protein alkylation and the formation of liver fibrosis. The molecular initiating event (MIE) is protein alkylation, leading to structural and functional cell injury and further to cell death, the first KE. Apoptotic hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies. Upon engulfment of apoptotic bodies Kupffer cells (KCs) are activated, the next KE along the pathway. Activated KCs are the main source of TGF-β1, the most potent profibrogenic cytokine. TGF-β1 expression therefore is considered a KE that causes the next KE, hepatic stellate cell (HSCs) activation, meaning the transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast, the central effector in hepatic fibrosis. Activated HSCs cause progressive collagen accumulation, which together with changes in ECM composition signifies the KE on tissue level. The excessive accumulation of extracellular matrix proteins progressively affects the whole organ and alters its normal functioning, which corresponds to liver fibrosis, the adverse outcome (AO).
There are two further events that play an important role in driving fibrogenesis, namely oxidative stress and chronic inflammation. Both are on-going processes being present throughout the pathway and interconnected with most of the KEs. Hence, they are not classified as KEs themselves and described in the individual KE and key event relationship (KER) descriptions. The inflammatory response plays an important role in driving fibrogenesis, since persistent inflammation precedes fibrosis. Inflammatory signalling stems from injured hepatocytes, activated KCs and HSCs. Inflammatory and fibrogenic cells stimulate each other in amplifying fibrosis. Chemokines and their receptors provoke further fibrogenesis, as well as interacting with inflammatory cells to modify the immune response during injury. Oxidative stress, as well, plays a crucial role in liver fibrogenesis by inducing hepatocyte apoptosis, activation of KCs and HSCs and fuelling inflammation. ROS contributing to oxidative stress are generated by hepatocytes, KCs, HSCs and inflammatory cells.
This purely qualitative AOP description is plausible, the scientific data supporting the AOP are logic, coherent and consistent and there is temporal agreement between the individual KEs. Quantitative data on dose-response-relationships and temporal sequences between KEs are still lacking; the provision of quantitative data will further strengthen the weight of evidence and make the AOP applicable for a wide range of purposes.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||244||Alkylation, Protein||Alkylation, Protein|
|2||KE||55||N/A, Cell injury/death||N/A, Cell injury/death|
|3||KE||1492||Tissue resident cell activation||Tissue resident cell activation|
|4||KE||1493||Increased Pro-inflammatory mediators||Increased pro-inflammatory mediators|
|5||KE||265||Activation, Stellate cells||Activation, Stellate cells|
|6||KE||68||Accumulation, Collagen||Accumulation, Collagen|
|7||AO||344||N/A, Liver fibrosis||N/A, Liver fibrosis|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Alkylation, Protein leads to N/A, Cell injury/death||adjacent||Moderate|
|N/A, Cell injury/death leads to Tissue resident cell activation||adjacent||High|
|Tissue resident cell activation leads to Increased pro-inflammatory mediators||adjacent||High|
|Increased pro-inflammatory mediators leads to Activation, Stellate cells||adjacent||High|
|Activation, Stellate cells leads to Accumulation, Collagen||adjacent||High|
|Accumulation, Collagen leads to N/A, Liver fibrosis||adjacent||High|
|N/A, Cell injury/death leads to Activation, Stellate cells||non-adjacent||High|
Life Stage Applicability
|Not Otherwise Specified|
Overall Assessment of the AOP
Assessment of the Weight-of-Evidence supporting the AOP
Concordance of dose-response relationships
This is a qualitative description of the pathway; the currently available literature does not provide quantitative information on dose-response relationships. But there is empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown.
Temporal concordance among the key events and adverse outcome
Empirical evidence shows temporal concordance between the individual KEs leading to the AO.
Strength, consistency, and specificity of association of adverse outcome and initiating event
The scientific evidence on the linkage between MIE and AO has been described. The ample literature is consistent in describing this association between AO and MIE
Biological plausibility, coherence, and consistency of the experimental evidence
The available data supporting the AOP are logic, coherent and consistent with established biological knowledge.
Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP
There are some other important fibrogenic signalling pathways that influence HSC activation and fibrogenesis without constituting another AOP:
Adipokines are secreted mainly by adipose tissue, but also by resident and infiltrating macrophages and are increasingly recognised as mediators of fibrogenesis.
Leptin promotes HSC fibrogenesis and enhances TIMP-1 expression and further acts as a pro-fibrotic through suppression of peroxisome proliferator-activated receptor-gamma (PPARg), an anti-fibrogenic nuclear receptor that can reverse HSC activation. The expression of leptin receptor is up-regulated during HSC activation and leptin activity is therefore increased through enhanced signaling. Downstream effects include increased release of TGF-b1 from KCs. The counter-regulatory hormone adiponectin is reduced in hepatic fibrosis. 
The fibrogenic function of HSCs is also influenced by neurochemical and neurotrophic factors. Upon chronic liver injury, the local neuroendocrine system is up-regulated, and activated HSCs express specific receptors, most prominently those regulating cannabinoid signaling. Activated HSCs are additionally a key source of the endogenous cannabinoid,2-Arachidonylglycerol (2-AG), which drives increased (cannabinoid-receptor) CB 1 signalling. Stimulation of the CB1 receptor is profibrogenic, whereas the CB2 receptor is anti-fibrotic and hepatoprotective. Opioid signaling increases proliferation and collagen production in HSCs. Serotonin has a pro-fibrotic effect that synergizes with PDGF signaling. Also thyroid hormones enhance activation of HSCs (through increased p75 neurotrophin receptor (p75NTR) and activation of Rho), thereby accelerating the development of liver fibrosis. 
Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine that stimulates DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These fibrogenic actions are mediated by NOX.    
Uncertainties, inconsistencies and data gaps
Ths AOP description is plausible, though purely qualitative; the addition of quantitative data on dose response-relationships and temporal sequences is needed and would substantially improve its applicability.
Protein alkylation is a broad, non-specific MIE. Covalent protein alkylation is a feature of many hepatotoxic drugs but the overall extent of binding does not adequately distinguish toxic from non-toxic binding. For this AOP it is unclear whether protein alkylation per se is sufficient to start the pathway or whether alkylation to specific proteins or families of proteins needs to be affected and whether various binding sites influence the further downstream process. The identification and specification of the targeted biomolecules is needed for the structural definition of chemical initiators and consecutively for profiling and categorising of chemicals related to the initiation of this AOP. Likewise it is necessary for the establishment of a distinct relationship with the next downstream event. Further it is unknown whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins. Future studies could provide a better mechanistic basis for interpreting protein alkylation in chemical safety evaluation.
By definition, an AOP has only one MIE and one final AO, the two anchor points of the AOP that have to be clearly defined. Any other MIE that leads to cell injury and further to liver fibrosis via the same downstream KEs would constitute another AOP. There are various types of liver injury that are caused by different agents, initiated by various MIEs and finally lead to fibrosis via the same described pathway; therefore, the question arises whether hepatocyte injury itself, independently from the cause of injury, might be the initiating event for this pathway to fibrosis. Obviously hepatocyte injury does not inevitably lead to fibrosis in all cases and there is a wide range of hepatotoxic chemicals (like Acetaminophen, Aflatoxin or Chlorpromazine) for which liver fibrosis cannot be observed. Apoptosis, necrosis, transdifferentiation/transition and repair/regeneration, all these might occur in response to cellular stressors and the difference in progression to liver fibrosis might lie in these various cellular responses. There is increasing evidence for apoptosis being the main fibrogenic trigger. Yet, both necrosis and apoptosis are often present simultaneously and necrosis may only represent the more severe cellular response to stronger damaging stimuli. It also might well be that hepatocyte insult/injury, rather than death is sufficient to trigger fibrosis and the key question would then be whether there are fibrosis-specific features of cell injury. It could be rather the amount (quantitative difference) than the kind (qualitative difference) of cell injury that matters. The rate of cell injury/death, i.e. the amount of injury within a certain time frame could be another plausible initiating parameter, as fibrosis is resulting from chronic injury. Assuming hepatocyte injury being the crucial KE without which fibrosis could not occur via this AOP, then simple investigation of in vitro hepatotoxicity could provide relevant information for potential fibrosis prediction without the need of highly elaborated cell models.
The initial AOP case study was based on data of two prototypic fibrogenic chemicals, namely Carbon Tetrachloride (CCl4)    and Allyl Alcohol . Further knowledge was gathered by looking for mechanistic data of other chemicals which are known inducers of liver fibrosis, namely Thioacetamide  , Amiodarone  , Methotrexate , Isoniazid , Dimethyl Nitrosamine , Ethanol , Retinol, Ethinyl Estradiol , and Chlopromazine  . Mechanistic data related to these additional chemicals are rather scarce, because classical in vivo studies were mainly looking at the AO than at intermediate (key) events and in vitro studies investigating liver fibrosis tend to use always the same reference chemicals. The overall gathered information was summarised in a data matrix that displays how many (if any) individual studies have observed the same findings at the MIE, KE and AO levels. Blue boxes refer to the KEs described in this AOP to liver fibrosis and green boxes indicate the observation of the event (the number within the box showing how many individual publications reported this specific event). It must be noted that these studies have not intended to investigate KEs on various levels of biological information; therefore, absence of a KE description does not necessarily mean that this KE did not occur, but rather that it has not been investigated or described. This matrix shows that protein binding was indicated as MIE only for three more chemicals Thioacetamide, Retinol and Dimethylnitrosamine) and therefore only these were added to the list of chemical initiators of this AOP. This matrix also demonstrates that hepatocyte injury/death is an early convergent KE that is valid for all described fibrogenic chemicals.
Assessment of the quantitative understanding of the AOP
Domain of Applicability
The described AOP is valid for both sexes and any life stage. This pathway description is also based on studies of formation and progression of fibrosis in human patients. Findings suggest common conserved pathways across different species which initiate and promote liver fibrosis. Animal models are used to study fibrogenesis and CCl4 intoxication in rats and mice is probably the most widely studied and therefore best characterised model with respect to histological, biochemical, cell, and molecular changes associated with the development of fibrosis
Essentiality of the Key Events
The essentiality of each of the KEs for this AOP was rated high as there is much experimental evidence that the blocking of one KE prevents (or attenuates where complete blocking is not possible) the next downstream KE and therefore the whole AOP. Much evidence arises from preclinical research for antifibrotic agents, which is mainly based on the interference with or blockade of a key event. For details see the table above
Support for Essentiality of KEs
Alkylating agents are highly reactive chemicals that introduce alkyl groups into biologically active molecules and thereby prevent their proper functioning.
Essentiality of the MIE is high.
Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity over 40 years ago. These reactions remain a major cause of chemical-induced toxicity. 
Covalent binding to liver proteins and oxidative stress can directly affect cells or influence signalling pathways, finally leading to necrotic or apoptotic cell death.
Essentiality of KE 1 is high.
Up-regulated apoptosis of hepatocytes is increasingly viewed as a nexus between liver injury and fibrosis. Pharmacological inhibition of liver cell apoptosis attenuates liver injury and fibrosis, suggesting a critical role for hepatocyte apoptosis in the initiation of HSC activation and hepatic fibrogenesis.  
Kupffer cell (KC) activation and macrophage recruitment
Activated KCs are a major source of inflammatory mediators, including cytokines, chemokines, lysosomal and proteolytic enzymes and a main source of TGF-β, as well as a major source of reactive oxygen species (ROS).
Essentiality of KE 2 is high.
Probably there is a threshold of KC activation and release above which liver damage is induced. Pre-treatment with gadolinium chloride (GdCl), which inhibits KC function, reduced both hepatocyte and sinusoidal epithelial cell injury, as well as decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-b1 mRNA expression in macrophages. Experimental inhibition of KC function or depletion of KCs appeared to protect against liver injury from the alkylating agent melphalan, the chemical thioacetamide and the immunostimulants concanavalin A and Pseudomonas exotoxin. 
TGF-β1 is the most potent profibrogenic cytokine and plays a central role in fibrogenesis, mediating a cross-talk between parenchymal, inflammatory and collagen expressing cells.
Essentiality of KE 3 is high.
TGF-β1 is considered the most potent pro-fibrogenic cytokine and several reviews assign this cytokine a central role in fibrogenesis, especially in HSC activation. Strategies aimed at disrupting TGF-β1 expression or signalling pathways are extensively being investigated because blocking this cytokine may not only inhibit matrix production, but also accelerate its degradation. Animal experiments using different strategies to block TGF-β1 have demonstrated significant anti-fibrotic effect for liver fibrosis. Experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TbRs (TGF-β receptors). 
hepatic stellate cell (HSC) activation
HSC activation (in response to TGF-β1) means a transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast and is the dominant event in liver fibrosis. Activated HSCs (myofibroblasts) are the primary collagen producing cells, the key cellular mediators of fibrosis and a nexus for converging inflammatory pathways leading to fibrosis.
Essentiality of KE 4 is high.
Excess ECM (extracellular matrix) deposition and changes in ECM composition.
Essentiality of KE 5 is high.
Continuing imbalance between the deposition and degradation of ECM is a pre-requisite of liver fibrosis; therefore this KE is essential for the AO. 
Excessive deposition of ECM proteins occurs as a result of repeated cycles of hepatocytes injury and repair and results in liver fibrosis.
It is generally accepted that any chronic form of liver damage, including any chemical causing sub-massive hepatocellular injury, can result in myofibroblast activation, leading to hepatic fibrosis and cirrhosis in humans 
There are two further events that play an important role in driving fibrogenesis, namely chronic inflammation and oxidative stress. Both are on-going processes being present throughout the pathway and interconnected with most of the KEs. Therefore they are not classified as KEs themselves and described in the individual KE and KER descriptions. Nevertheless a short overview is given below.
Hepatic fibrosis is commonly preceded by chronic inflammation and persistent inflammation has been associated with progressive hepatic fibrosis. Hepatic inflammation is a driver of hepatic fibrosis as the whole fibrinogenic cascade is initiated and maintained by inflammatory mediators and inflammatory and fibrogenic cells stimulate each other in amplifying fibrosis. Damaged hepatocytes release inflammatory cytokines that activate KCs and stimulate the recruitment of inflammatory cells, which produce profibrotic cytokines and chemokines that further activate fibroblastic cells. Activated HSCs secrete various cytokines (like macrophage colony-stimulating factor (M-CSF), MCP-1 and IL-6) and inflammatory chemokines, they interact directly with immune cells through expression of adhesion molecules (mediated by TNF-α and facilitating the recruitment of inflammatory cells), and they modulate the immune system through antigen presentation. Signaling of HSCs in response to either lipopolysaccharides (LPS) or endogenous TLR4 ligands down-regulates the protein activin membrane-bound inhibitor (BAMBI), a transmembrane suppressor of TGF-β1. Other inflammatory cells regulating progression and resolution of fibrosis include T-cells, dendritic cells, liver sinusoidal endothelial cells (LSECs) and natural killer cells (NKs), which exert an anti-fibrotic activity by inducing HSC apoptosis through production of IFN γ. Chronic inflammatory response is often accompanied simultaneously by tissue destruction and repair. Activated inflammatory cells represent a major source of oxidative stress-related molecules.
Essentiality of inflammation is high.
Suppression of inflammatory activity by eliminating the etiological agent (e.g. a virus) or dampening the immune response (lymphocytic proliferation and infiltration) can halt and even reverse the fibrotic process. 
Oxidative stress corresponds to an imbalance between the rate of oxidant production and that of degradation and plays a crucial role in liver fibrogenesis by inducing hepatocyte apoptosis and activation of KCs and HSCs. Oxidative stress-related molecules act as mediators to modulate tissue and cellular events responsible for the progression of liver fibrosis. ROS, including superoxide, hydrogen peroxide, hydroxyl radicals and aldehydic end products, may be derived from hepatocytes (generated through cytochrome P450, lipid peroxidation), as well as from activated KCs, other inflammatory cells and HSCs (by NOX). Excessive levels of ROS can lead to hepatocellular injury and death. Under conditions of oxidative stress macrophages are activated, which leads to a more enhanced inflammatory response. Oxidative stress can activate a variety of transcription factors like NF-κB, PPAR-γ which may further lead to increased gene expression for the production of growth factors, inflammatory cytokines and chemokines.
Essentiality of oxidative stress is moderate.
Oxidative stress-related molecules act as mediators to modulate tissue and cellular events responsible for the progression of liver fibrosis. Hence ROS likely contribute to both onset and progression of fibrosis, being simultaneously cause and consequence of the observed condition.  
Support for Biological Plausibility of KERs
MIE => KE 1
Hepatocytes are damaged by alkylating agents via both covalent binding to liver proteins and lipid peroxidation accompanied by oxidative stress and collapse of mitochondrial membrane potential, which triggers apoptotic cell death.
Biological Plausibility of the MIE => KE1 is high.
KE 1 => KE 2
Damaged hepatocytes release ROS, cytokines and chemokines which lead to oxidative stress, inflammatory signalling and activation of KCs. Apoptotic hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies. Upon engulfment of apoptotic bodies KCs are activated. Liver cells trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs). Through toll-like receptors KCs are additionally activated.
Biological Plausibility of KE1 => KE2 is high.
KE 1 => KE 4
Like KCs, also HSCs are activated by damaged hepatocytes through the release of ROS, cytokines and chemokines and upon engulfment of apoptotic bodies from hepatocytes. DNA from apoptotic hepatocytes induces toll-like receptor 9 (TLR9)-dependent changes of HSCs that are consistent with late stages of HSC differentiation (activation), with up-regulation of collagen production and inhibition of platelet derived growth factor (PDGF)-mediated chemotaxis to retain HSCs at sites of cellular apoptosis. The release of latent TGF-β complex into the micro-environment by damaged hepatocytes is likely to be one of the first signals for adjacent HSCs leading to their activation.
Biological Plausibility of KE1 => KE4 is high.
HSCs activation by hepatocytes is only a contributing factor and not the main route; partly it is mediated by TGF-β1; therefore this relationship is classified as indirect. Nevertheless, there is a functional relationship between KE 1 and KE 4 consistent with established biological knowledge.   
KE 2 => KE 3
Following activation KCs become a main source of TGF-β1, the most potent profibrogenic cytokine, as well as a major source of inflammatory mediators, chemokines, and ROS.
Biological Plausibility of KE2 => KE3 is high.
KE 3 => KE 4
TGF-β1 activates HSCs, i.e. stimulates cell proliferation, matrix synthesis, and release of retinoids by HSCs and is the most potent fibrogenic factor for HSCs.
Biological Plausibility of KE3 => KE4 is high.
There is good understanding and broad acceptance of the KER between KE 3 and KE 4.
KE 4 => KE 5
In response to TGF-β1 activated HSCs up-regulate collagen synthesis. Together with decreased matrix degradation ECM composition changes and further stimulates HSC activation and production of TGF-β1, which further promotes activation of neighbouring quiescent HSCs.
Biological Plausibility of KE4 => KE5 is high.
KE 5 => AO
Excessive accumulation of ECM proteins leads to disruption of normal hepatic architecture.
Biological Plausibility of KE5 => AO is high.
Empirical Support for KERs
There is a need for more advanced in vitro models systems for chemical-induced hepatotoxicity to study intercellular signalling and dose-response data on KERs. Nevertheless, some empirical evidence exists to support that a change in KEup leads to an appropriate change in the respective KEdown.
MIE => KE 1
It is general accepted knowledge that alkylating chemicals damage cells. Although covalent protein alkylation is a feature of many hepatotoxic drugs the overall extent of binding does not adequately distinguish toxic from non-toxic binding. It is not known whether protein alkylation to certain proteins is required and whether particular proteins and various binding sites influence the further downstream process. Further, we do not know whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins.
KE 1 => KE 2
Specific markers for activated KCs have not been identified yet. KC activation cannot be detected by staining techniques since cell morphology does not change, but cytokines release can be measured (with the caveat that KCs activate spontaneously in vitro). Tukov et al. examined the effects of KCs cultured in contact with rat hepatocytes. They found that by adding KCs to the cultures they could mimic in vivo drug-induced inflammatory responses. Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated cytokine expression by KCs.
Empirical Support of the KE 1 => KE 2 is moderate.
KE 1 => KE 4
Canbay et al. could show that Fas-mediated hepatocyte injury is mechanistically linked to liver fibrogenesis. Markers of HSC activation were significantly reduced when apoptosis was prevented in Fas-deficient bile duct ligated mice. These findings (reduction of inflammation, markers of HSC activation, and collagen I expression) could be repeated by pharmacological inhibition of liver cell apoptosis using a pan-caspase inhibitor. Watanabe et al. could demonstrate in vitro that DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and inducing a stationary phenotype- associated up-regulation of collagen production. Coulouarn et al found in a co-culture model that hepatocyte - HSC crosstalk engenders a permissive inflammatory micro-environment.
Empirical Support of the KE 1 => KE 4 is moderate.
KE 2 => KE 3
Experiments by Matsuoka and Tsukamoto already 1990 showed that KCs isolated from rat liver with alcoholic fibrosis express and release TGF-β1 and that this cytokine is largely responsible for the KC-conditioned medium-induced stimulation of collagen formation by HSCs. Accumulated CD11b1 macrophages are critical for activating HSCs (via expression of TGF-β1) (Chu et al, 2013)
Empirical Support of the KE 2 => KE 3 is moderate.
KE 3 => KE 4
Czaja et al could prove that treatment of cultured hepatic cells with TGF-β1 increased type I pro-collagen mRNA levels 13-fold due to post-transcriptional gene regulation. Tan et al. discovered that short TGF-β1 pulses can exert long-lasting effects on fibroblasts. Difficulties are that HSCs cultured on plastic, undergo spontaneous activation and HSCs activated in culture do not fully reproduce the changes in gene expression observed in vivo. De Minicis et al investigated gene expression changes in 3 different models of HSC activation and compared gene expression profiles in culture (mice HSCs in co-culture with KCs) and in vivo and did not find a proper correlation.
Empirical Support of the KE 3 => KE 4 is moderate.
KE 4 => KE 5
It is difficult to stimulate sufficient production of collagen and its subsequent incorporation into a pericellular matrix in vitro; therefore analytical methods have focused on measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver began to express α-SMA coincident with culture-induced activation.
Empirical Support of the KE 4 => KE 5 is moderate.
KE 5 => AO
Liver fibrosis results from chronic damage in conjunction with the accumulation of ECM proteins, which distorts the hepatic architecture by forming a fibrous scar. The onset of liver fibrosis is usually insidious and progression to cirrhosis occurs after an interval of 15–20 years.
Empirical Support of the KE 5 => AO is high.
There is a smooth transition from ECM accumulation to liver fibrosis without a definite threshold and plenty in vivo evidence exists that ECM accumulation is a pre-stage of liver fibrosis 
More advanced in vitro models systems are needed to study chemical-induced hepatotoxicity. Modulations of hepatotoxicity by intercellular signalling cannot be addressed in primary cultures of hepatocytes alone but require co-cultures of different liver cell types. Various co-cultures systems with two or more different liver cell types are currently being developed, but quantitative data on KERs are not available yet.
Considerations for Potential Applications of the AOP (optional)
This systematic and coherent display of currently available mechanistic-toxicological information can serve as a knowledge-based repository for identification/selection/development of in vitro methods suitable for measuring KEs and their relationships along the AOP and to facilitate the use of alternative data for regulatory purposes. Identified uncertainties and knowledge gaps can direct future research by priority setting and targeted testing. The KE descriptions can be used for hazard identification and read-across to assess the toxic potential of an untested substance. A sufficiently detailed description of the AOP to liver fibrosis might support chemical risk assessment by indicating early (upstream) markers for downstream events and facilitate a testing strategy without the need for an elaborated cell model.
Confidence in the AOP
Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.
The biological plausibility, i.e the mechanistic relationship between of each of the KERs in this AOP was rated high, because there is good scientific understanding of these relationships and they are consistent with established biological knowledge. The empirical support for the KERs is considered moderate because there are only limited (nevertheless consistent) data available. For the KER between collagen accumulation and liver fibrosis exists a lot of empirical and clinical evidence and therefore empirical support is rated high. Modulations of hepatotoxicity by intercellular signalling cannot be addressed in primary cultures of hepatocytes alone but require co-cultures of different liver cell types. Due to the limited availability of adequate cell models dose-response data on KERs are not available yet. But there is some empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown; some experimental studies could demonstrate a dependent relationship between two consecutive KEs with temporal concordance following exposure to a toxicant.
How well characterised is the AOP?
The adverse outcome is well understood qualitatively, but quantitative data are lacking.
How well are the initiating and other key events causally linked to the outcome?
The relationships between each key event and adverse outcome are well established.
What are the limitations in the evidence in support of the AOP?
This AOP description is plausible and consistent with existing literature in describing the association between AO and MIE across different levels of biological organisation. Animal studies are mainly focused on the AO and do not describe mechanistic sequences in detail. Due to the pathogenic complexity of liver fibrosis involving many different cells there is currently no suitable cell model available to mimic and further explore the sequence of events, especially in quantitative terms.
Prestigiacomo et al. generated a cell system containing the three key players of liver fibrosis (hepatocytes, Kupffer cells and hepatic stellate cells) to assess the response to fibrogenic compounds and they could recapitulate in vitro the KEs leading to liver fibrosis, as described in this AOP.
Is the AOP specific to certain tissues, life stages / age classes?
The complex mechanism of fibrogenesis does not only affect a single organ, but causes a systemic response which equally damages other organs and tissues. The described findings in liver fibrosis parallel those in studies of fibrogenesis in other organs; everywhere are the same kind of cells and soluble factors involved . For example the reference compound CCl4 equally affects lymphoid organs, lungs and kidneys . Fibrosis may affect lung, kidney, heart and blood vessels, eye, skin, pancreas, intestine, brain and bone marrow. Multi-organ fibrosis occurs due to mechanical injury or can be drug- or radiation-induced . As many fibrogenic pathways are conserved across tissues, recent findings in the liver could be extended to studies of fibrosis in the lungs, the kidneys, the heart and other organs.
Are the initiating and key events expected to be conserved across taxa?
I want to thank Clemens Wittwehr for his repeated and patient editing assistance, as well as Steve Edwards for his prompt availability whenever a technical problem occurred.
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract ResClin Gastroenterol, vol. 25, no. 2, pp. 195-206.
- ↑ 2.0 2.1 2.2 2.3 Friedman, S.L (2010), Evolving challenges in hepatic fibrosis, Nat. Rev. Gastroenterol. Hepatol, vol. 7, no. 8, pp. 425–436.
- ↑ 3.0 3.1 3.2 Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.
- ↑ 4.0 4.1 4.2 4.3 4.4 4.5 4.6 Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233,no. 2, pp. 109-122.
- ↑ Bataller, R. et al. (2003), NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis, J Clin Invest, vol. 112, no.9, pp. 1383-1894.
- ↑ EPA, (2010),Toxicological review of Carbon Tetrachloride (CAS No. 56-23-5). EPA/635/R-08/005F available at:http://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0020tr.pdf (accessed on 24 October 2015).
- ↑ Basu, S. (2003), Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients, Toxicology, vol. 189, no. 1-2, pp. 113-127.
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