API

Aop: 38

AOP Title

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Protein Alkylation leading to Liver Fibrosis

Short name:

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Protein Alkylation to Liver Fibrosis

Authors

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Brigitte Landesmann

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

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Brigitte Landesmann

Contributors

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  • Brigitte Landesmann

Status

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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 December 02, 2016 12:55

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Revision dates for related pages

Page Revision Date/Time
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
Increased, Activation and Recruitment of Hepatic macrophages (Kupffer Cells) September 16, 2017 10:14
Up Regulation, TGFbeta1 expression September 16, 2017 10:14
Activation, Stellate cells September 16, 2017 10:14
Accumulation, Collagen September 16, 2017 10:14
Alkylation, Protein leads to N/A, Cell injury/death November 29, 2016 20:02
N/A, Cell injury/death leads to Increased, Activation and Recruitment of Hepatic macrophages (Kupffer Cells) November 29, 2016 19:54
N/A, Cell injury/death leads to Activation, Stellate cells November 29, 2016 19:54
Increased, Activation and Recruitment of Hepatic macrophages (Kupffer Cells) leads to Up Regulation, TGFbeta1 expression November 29, 2016 19:57
Up Regulation, TGFbeta1 expression leads to Activation, Stellate cells November 29, 2016 20:04
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
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

Abstract

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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.


Background (optional)

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Summary of the AOP

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Stressors

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Name Evidence Term
Allyl Alcohol Strong
Carbon tetrachloride Strong
Retinol Strong
Dimethyl nitrosamine Strong
Thioacetamide Strong

Molecular Initiating Event

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Title Short name
Alkylation, Protein Alkylation, Protein

Key Events

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Title Short name
N/A, Cell injury/death N/A, Cell injury/death
Increased, Activation and Recruitment of Hepatic macrophages (Kupffer Cells) Increased, Activation and Recruitment of Hepatic macrophages (Kupffer Cells)
Up Regulation, TGFbeta1 expression Up Regulation, TGFbeta1 expression
Activation, Stellate cells Activation, Stellate cells
Accumulation, Collagen Accumulation, Collagen

Adverse Outcome

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Title Short name
N/A, Liver fibrosis N/A, Liver fibrosis

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
Not Otherwise Specified

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI

Sex Applicability

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Sex Evidence
Unspecific

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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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:

Adipokine pathways

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. [1][2]

Neuroendocrine pathways

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. [3][2][1]

Renin–angiotensin pathway

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. [4] [2] [1] [5]

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)[6][7][8][9][10][11][12][13][14][15][16][17] [18][19] [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] [40][41][42] and Allyl Alcohol [43][44][45][46][47][48]. Further knowledge was gathered by looking for mechanistic data of other chemicals which are known inducers of liver fibrosis, namely Thioacetamide [49][50][51][52][53][54][55][56][57][58][59][60] [32][61][62][63][64][65], Amiodarone [66][67][68] [69][70][71][72], Methotrexate [73][74][75][76][77][78][79], Isoniazid [80][81][82][83], Dimethyl Nitrosamine [84][85][86][87], Ethanol [88][89][31][90][91][92][93], Retinol[94][95][96], Ethinyl Estradiol [97][98][99], and Chlopromazine [100][101] [102]. 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.

Mechanistic data matrix.jpg

Assessment of the quantitative understanding of the AOP

See above

Domain of Applicability

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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

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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


Weight of Evidence Summary

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Support for Essentiality of KEs

 

MIE

Protein Alkylation

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. [103][104]

KE 1

cell injury/death

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. [105][106] [107][108][109][110][111][112][113]

KE 2

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. [114][4][115][55][113][116][117][118]

KE 3

TGF-β1 expression

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). [119][120][121][122][123]

KE 4

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.

Experimental inhibition of HSC activation prevents fibrosis. [4][124][125][126][127][128]

KE 5

collagen accumulation

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. [1]

Adverse Outcome

liver fibrosis

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 [129][130][131][132][133]

 

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.

 

Associated Event

chronic inflammation

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. [134][135][136][137][138][1][139][140][141]

 

Associated Event

oxidative stress

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. [142][143] [139][144][145][146][147][148]

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.

There is a mechanistic relationship between MIE and KE 1 consistent with established biological knowledge. [103][46][149][28]

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.

There is a functional relationship between KE 1 and KE 2 consistent with established biological knowledge. [150][23] [115][105][106] [107][114][4] [151][152][143]

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. [153][120][105][106] [107][114][4] [152][3][1]

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.

The functional relationship between KE 2 and KE 3 is consistent with biological knowledge. [154][152][114][140][1][155][156][157][148] [158]

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.

[147][159][123][120][114][140][155][156] [160] [134][152][161][4][143] [142][3][119]

 

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.

The functional relationship between KE 4 and KE 5 is consistent with biological knowledge and generally accepted. [162][163][164][114][140][1][155][156][152][134][165]

KE 5 => AO

Excessive accumulation of ECM proteins leads to disruption of normal hepatic architecture.

Biological Plausibility of KE5 => AO is high.

By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between KE 5 and the AO is undisputed. [1][140][166][167][168][169]

 

 

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.


Empirical Support of the MIE => KE 1 is moderate.

There is exposure-dependent change in both events following exposure with temporal concordance. [170][171][172][173][103]

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.

There are limited experimental studies which could show that there is a direct relationship between these two events with temporal concordance. [174][175][176][110]

 

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.

There is experimental evidence for this KER. [109][113][177][178]

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.

Cytokine release is one of the features that define KC activation and there is sound empirical evidence for this KER. [161][179]

 

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.

Qualitative empirical evidence with temporal and incidence concordance for this KER exists. [180][181][182][183]

 

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.

It is general accepted knowledge that activated HSCs (=myofibroblasts) are collagen-producing cells. [184][185]

 

 

 

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 [140]

 


Quantitative Considerations

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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)

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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.

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 [166][124][2]. For example the reference compound CCl4 equally affects lymphoid organs, lungs and kidneys [4]. 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 [186][137][187][188]. 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?

Findings also suggest common conserved pathways across different species which initiate and significantly modulate the progression of liver fibrosis [189] [190][191][192][193].

Acknowledgements

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.


References

?


  1. 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. 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. 3.0 3.1 3.2 Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.
  4. 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.
  5. 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.
  6. 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).
  7. 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.
  8. Brattin, W. et al. (1985), Pathological mechanisms in carbon tetrachloride hepatotoxicity, J Free Radic Biol Med, vol. 1, no. 1, pp. 27-38.
  9. Calabrese, E.J. and H.M. Mehendale (1996), A review of the role of tissue repair as an adaptive strategy: why low doses are often non-toxic and why high doses can be fatal, Food Chem Toxicol, vol. 34, no. 3, pp. 301-311.
  10. Calabrese EJ et al., 1993,G2 subpopulation in rat liver induced into mitosis by low-level exposure to carbon tetrachloride: an adaptive response, Toxicol Appl Pharmacol;121(1):1-7.
  11. Clawson, G.A. (1989), Mechanisms of carbon tetrachloride hepatotoxicity, Pathol Immunopathol Res, vol. 8, no. 2, pp. 104-112.
  12. Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.
  13. Dalu, A. and H.M. Mehendale (1996), Efficient tissue repair underlies the resiliency of postnatally developing rats to chlordecone + CCl4 hepatotoxicity, Toxicology,vol. 111, no. 1-3, pp. 29-42.
  14. Feng, Y. et al. (2011), Hepatoprotective effect and its possible mechanism of Coptidis rhizoma aqueous extract on carbon tetrachloride-induced chronic liver hepatotoxicity in rats, J Ethnopharmacol, vol.138, no. 33, pp. 683-690.
  15. Hamdy, N. and E. El-Demerdash. (2012), New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol Appl Pharmacol, vol. 261, no. 3, pp. 292-299.
  16. Jaeschke, H. et al. (2013), Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products, Food and Chemical Toxicology,vol. 55, pp. 279–289.
  17. Jang, J.H. et al. (2008), Reevaluation of experimental model of hepatic fibrosis induced by hepatotoxic drugs: an easy, applicable, and reproducible model, Transplantation Proceedings;, vol. 40, no. 8, pp. 2700-2703.
  18. Knockaert, L. et al. (2012), Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver, Lab Invest, vol. 92, no. 3, pp. 396-410.
  19. Lee Kwang-Jong et al. (2004), Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage, Cell Stress Chaperones, vol. 9, no. 1, pp. 58-68.
  20. Leung, T.M. et al. (2008), Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis, Int J Exp Pathol, vol. 89, no. 4, pp. 241-250.
  21. Li, Li et al. (2012), Establishment of a standardized liver fibrosis model with different pathological stages in rats, Gastroenterol Res Pract; vol. 2012, Article ID 560345.
  22. Li Xiaowei et al. (2014), NMR-based metabonomic and quantitative real-time PCR in the profiling of metabolic changes in carbon tetrachloride-induced rat liver injury, J Pharm Biomed Anal, vol. 89, pp. 42-49.
  23. 23.0 23.1 Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.
  24. Luster, M.I. et al. (2001), Role of inflammation in chemical-induced hepatotoxicity, Toxicol Lett, vol. 120, no. 1-3, pp. 317-321.
  25. Luster, M.I. et al. (2000), Immunotoxicology: role of inflammation in chemical-induced hepatotoxicity, Int J Immunopharmacol, vol. 22, no. 12, pp. 1143-1147.
  26. Luster MI et al., 2001, Role of inflammation in chemical-induced hepatotoxicity, Toxicol Lett; 120(1-3):317-21.
  27. Lv, L. et al. (2012), Protective effects of lotus (Nelumbo nucifera Gaertn) germ oil against carbon tetrachloride-induced injury in mice and cultured PC-12 cells, Food Chem Toxicol, vol. 50, no. 5, pp. 1447-1453.
  28. 28.0 28.1 Manibusan, M.K., M. Odin and D.A. Eastmond (2007), Postulated carbon tetrachloride mode of action: a review, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, vol. 25, no. 3, pp. 185-209.
  29. Masuda, Y. (2006), [Learning toxicology from carbon tetrachloride-induced hepatotoxicity], Yakugaku Zasshi, vol. 126, no. 10, pp. 885-899.
  30. Morio, L.A. et al. (2001), Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice, Toxicol Appl Pharmacol, vol. 172, no. 1, pp. 44-51.
  31. 31.0 31.1 Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids Health Dis, vol. 12, p.11.
  32. 32.0 32.1 Natajaran, S.K. et al. (2006), Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J Gastroenterol Hepatol, vol. 21, no. 6, pp. 947-957.
  33. Natsume, M et a.l, (1999), Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice, J Leukoc Biol, vol. 66, no. 4, pp. 601-608.
  34. Neubauer K. et al. (1998), Sinusoidal intercellular adhesion molecule-1 up-regulation precedes the accumulation of leukocyte function antigen-1-positive cells and tissue necrosis in a model of carbontetrachloride-induced acute rat liver injury, Lab Invest, vol.78, no. 2, pp. 185-194.
  35. Nissar, A.U. et al. (2013), Effect of N-acetyl cysteine (NAC), an organosulfur compound from Allium plants, on experimentally induced hepatic prefibrogenic events in Wistar rat, Phytomedicine, vol. 20, no. 10, pp. 828-833.
  36. Nagano, K. et al. (2007), Inhalation carcinogenicity and chronic toxicity of carbon tetrachloride in rats and mice, Inhal Toxicol, vol 19, no. 13, pp. 1089-1103.
  37. Park, D.H. et al. (2004), Chronic hepatotoxicity of carbon tetrachloride in hsp-70 knock-out mice, Exp Anim, vol. 53, no. 1, pp. 27-30.
  38. Recknagel, R.O. (1976), Carbon tetrachloride hepatotoxicity, Pharmacol Rev, vol. 19, no. 2,pp. 145-208.
  39. Simeonova, P.P. et al. (2001), The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride, Toxicol Appl Pharmacol, vol. 177, no. 2, pp. 112-120.
  40. Tipoe, G.L. et al. (2006), Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury, Histol Histopathol, vol. 21, no. 11, pp. 1157-1165.
  41. Weber, L.W., M. Boll and A. Stampfl (2003), Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model, Crit Rev Toxicol, vol. 33, no. 2, pp. 105-136.
  42. Zhu, W. and P.C. Fung (2000), The roles played by crucial free radicals like lipid free radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)-induced acute liver injury of mice, Free Radic Biol Med, vol. 29, no. 9, pp. 870-880.
  43. Auerbach, S. et al. (2008), A comparative 90 day toxicity study of allyl acetate, allyl alcohol and acrolein, Toxicology; vol. 253, no. 1-3, pp. 79–88.
  44. Huang, L. et al. (2008), Genes related to apoptosis predict necrosis of the liver as a phenotype observed in rats exposed to a compendium of hepatotoxicants, BMC Genomics, vol. 9: 288.
  45. Jung, S.A. et al. (2000), Experimental model of hepatic fibrosis following repeated periportal necrosis induced by allylalcohol, Scand J Gastroenterol, vol. 35, no. 9, pp. 969-975.
  46. 46.0 46.1 Kehrer, J.P. and S. Biswal (2000), The Molecular Effects of Acrolein, Toxicol. Sciences, vol. 57, no. 1, pp. 6-15.
  47. Mohammad, M.K. et al. (2012), Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress, Toxicol Appl Pharmacol, vol. 265, no. 1, pp. 73-82.
  48. Yamada, T. et al. (2013), A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Regulatory Toxicology and Pharmacology, vol. 65, no. 2, pp. 189–195.
  49. Akhtar, T. and N. Sheikh (2013), An overview of thioacetamide-induced hepatotoxicity, Toxin Reviews, vol. 32, no. 3, pp. 43-46.
  50. Altomonte, J. et al. (2013), Antifibrotic properties of transarterial oncolytic VSV therapy for hepatocellular carcinoma in rats with thioacetamide-induced liver fibrosis, Mol Ther, vol. 21, no. 11, pp. 2032-2042.
  51. Amin, Z.A. et al. (2013), Gene expression profiling reveals underlying molecular mechanism of hepatoprotective effect of Phyllanthus niruri on thioacetamide-induced hepatotoxicity in Sprague Dawley rats, BMC Complement Altern Med, vol. 5, no. 13, p. 160.
  52. Chilakapati, J. et al. (2005), Saturation toxicokinetics of thioacetamide: role in initiation of liver injury, Drug Metab Dispos, vol. 33, no.12, pp. 1877-1885.
  53. Hajovsky, H. et al. (2012), Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes, Chem Res Toxicol, vol. 25, no. 9, pp. 1955-1963.
  54. Ide, M. et al. (2003), Emergence of different macrophage populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats, J Comp Patho, vol. 128, no. 1, pp. 41-51.
  55. 55.0 55.1 Ide, M. et al. (2005), Effects of gadolinium chloride (GdCl(3)) on the appearance of macrophage populations and fibrogenesis in thioacetamide-induced rat hepatic lesions, J Comp Pathol, vol. 133, no. 2-3, pp. 92-102.
  56. Kang, J.S. (2008), Role of CYP2E1 in thioacetamide-induced mouse hepatotoxicity, Toxicol Appl Pharmacol, vol. 228, no. 3, pp. 295-300.
  57. Kim, J.H. et al. (2013), Chronic vitamin C insufficiency aggravated thioacetamide-induced liver fibrosis in gulo-knockout mice, Free Radic Biol Med, vol. 67, pp. 81-90.
  58. Ledda-Columbano, G.M. et al. (1991), Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide, Am J Pathol, vol. 139, no. 5, pp. 1099-1109.
  59. Low, T.Y. et al. (2004), A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers, Proteomics, vol. 4, no. 12, pp. 3960-3974.
  60. Nafees ,S. et al. (2013), Carvacrol ameliorates thioacetamide-induced hepatotoxicity by abrogation of oxidative stress, inflammation, and apoptosis in liver of Wistar rats, Hum Exp Toxicol, vol. 32, no.12, pp. 1292-1304.
  61. Sarma, D. et al. (2012), Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites, Chem Res Toxicol, vol. 25, no. 9, pp. 1868-1877.
  62. Shirai, M. et al. (2013), Thioacetamide-induced hepatocellular necrosis is attenuated in diet-induced obese mice, J Toxicol Pathol, vol. 26, no. 2, pp. 175-186.
  63. Staňková, P. et al. (2010), The toxic effect of thioacetamide on rat liver in vitro, Toxicol In Vitro, vol. 24, no. 8, pp. 2097-2103.
  64. Sun, F. et al. (2000), Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver, Biochim Biophys Acta, vol. 1500, no. 2, pp. 181-185.
  65. Waters, N.J. et al. (2005), Metabonomic deconvolution of embedded toxicity: application to thioacetamide hepato- and nephrotoxicity, Chem Res Toxicol, vol.18, no. 4, pp. 639-654.
  66. Cimic, A. and J. Sirintrapun (2013), Amiodarone hepatotoxicity with absent phospholipidosis and steatosis: a case report and review of amiodarone toxicity in various organs, Case Reports in Pathology, Article ID 201095. doi:10.1155/2013/201095.
  67. Felser, A. et al. (2013), Mechanisms of hepatocellular toxicity associated with dronedarone-a comparison to amiodarone, Toxicological Sciences, vol. 131, no. 2, pp. 480-490.
  68. Golli-Bennour, E.E. et al. (2012), Cytotoxicity effects of amiodarone on cultured cells, Exp Toxicol Pathol, vol. 64, no. 5, pp. 425-430.
  69. Isomoto, S. et al. (2004), Antiarrhythmic amiodarone mediates apoptotic cell death of hepg2 hepatoblastoma cells through the mitochondrial pathway, Acta medica Nagasakiensia, vol. 49, no. 1-2, pp. 13-17.
  70. Kicker, J.S. et al. (2012), Hepatotoxicity after continuous amiodarone infusion in a postoperative cardiac infant, J Pediatr Pharmacol Ther, vol. 17, no. 2, pp. 189-195.
  71. Lu, J. et al. (2013), Tumor necrosis factor-alpha potentiates the cytotoxicity of amiodarone in Hepa1c1c7 cells: roles of caspase activation and oxidative stress, Toxicological Sciences, vol. 131, no. 1, pp.164-178.
  72. Nasser .M. et al. (2013), Hyperacute drug-induced hepatitis with intravenous amiodarone: case report and review of the literature, Drug Healthc Patient Saf, vol. 5, pp. 191-198.
  73. Al-Ali, S.Y., I.M. Hassan and S. Sadek (2005), Ultrastructural changes in rat livers perfused in vitro and in vivo with a high dose of methotrexate, Histol Histopathol, vol. 20, no. 4, pp. 1131-1145.
  74. Al-Motabagani, M.A. (2006), Histological and histochemical studies on the effects of methotrexate on the liver of adult male albino rat, Int. J. Morphol;, vol. 24, no. 3, pp. 417-422.
  75. Belinsky, G.S. et al. (2007), The contribution of methotrexate exposure and host factors on transcriptional variance in human liver, Toxicological Sciences, vol. 97, no. 2, pp. 582–594.
  76. Fathi, N.H. et al. (2002), Longitudinal measurement of methotrexate liver concentrations does not correlate with liver damage, clinical efficacy, or toxicity during a 3.5 year double blind study in rheumatoid arthritis, J Rheumatol, vol. 29, no. 10, pp. 2092-2098.
  77. Hall, P.D. et al. (1991), Hepatotoxicity in a rat model caused by orally administered methotrexate, Hepatology, vol. 14, no. 5, pp. 906-910.
  78. Hytiroglou ,P. et al. (2004), The canals of hering might represent a target of methotrexate hepatic toxicity, Am J Clin Pathol, vol. 121, no. 3, pp. 324-329.
  79. Lindsay, K. et al. (2009), Liver fibrosis in patients with psoriasis and psoriatic arthritis on long-term, high cumulative dose methotrexate therapy, Rheumatology, vol. 48: no. 5, pp. 569–572.
  80. Bhadauria, S. et al. (2007), Isoniazid induces oxidative stress, mitochondrial dysfunction and apoptosis in Hep G2 cells, Cell Mol Biol (Noisy-le-grand), vol. 53, no. 1, pp. 102-114.
  81. Tostmann, A et. al. (2008), Isoniazid and its toxic metabolite hydrazine induce in vitro pyrazinamide toxicity, Int J Antimicrob Agents, vol. 3, no. 6, pp. 577-580.
  82. Schwab, C.E. and H. Tuschl (2003), In vitro studies on the toxicity of isoniazid in different cell lines, Hum Exp Toxicol, vol. 22, no. 11, pp. 607-615.
  83. Singh, M. et al. (2010), Studies on toxicity of antitubercular drugs namely isoniazid, rifampicin, and pyrazinamide in an in vitro model of HepG2 cell line, Med. Chem. Res, vol. 20, no. 9, pp. 1611-1615.
  84. George, J. et al. (2001), Dimethylnitrosamine-induced liver injury in rats: the early deposition of collagen, Toxicology , vol.156, n0. 2-3, pp. 129–138.
  85. George, J. (2003), Ascorbic acid concentrations in dimethylnitrosamine-induced hepatic fibrosis in rats, Clin Chim Acta, vol. 335, no. 1-2, pp. 39–47.
  86. Ju, H.K. et al. (2013), Investigation of metabolite alteration in dimethylnitrosamine-induced liver fibrosis by GC-MS, Bioanalysis, vol. 5, no. 1, pp. 41-51.
  87. Usunomena, U. et al. (2012),N-nitrosodimethylamine (NDMA), liver function enzymes, renal function parameters and oxidative stress parameters: a review, British Journal of Pharmacology and Toxicology, vol. 3, no. 4, pp. 165-176.
  88. Das, S.K. et al. (2010), Effects of long-term ethanol consumption on adhesion molecules in liver, Indian Journal of Experimental Biology, vol. 48, no. 4, pp. 394-401.
  89. Friedman, S.L. (1999), Stellate cell activation in alcoholic fibrosis - an overview, Alcohol Clin Exp Res, vol. 23, no. 5, pp. 904-910.
  90. Purohit, V. and D.A. Brenner (2006), Mechanisms of alcohol-induced hepatic fibrosis: A summary of the Ron Thurman symposium, Hepatology, vol. 43, no. 4, pp. 872-878.
  91. Siegmund, S.V. et al. (2005), Molecular mechanisms of alcohol-induced hepatic fibrosis, Dig Dis, vol. 23, no. 3-4, pp. 264-274.
  92. Thompson, K.J., I.H. McKillop and L.W. Schrum (2011), Targeting collagen expression in alcoholic liver disease, World J Gastroenterol, vol. 17, no. 20, pp. 2473-2481.
  93. Wang, J.H. et al. (2006), Role of ethanol in the regulation of hepatic stellate cell function, World J Gastroenterol, vol. 12, no. 43, pp. 6926-6932.
  94. Castano, G. et al. (2006), Vitamin A toxicity in a physical culturist patient: a case report and review of literature, Annals of Hepatology, vol 5, no. 4, pp. 293-295.
  95. Levine, P.H. et al. (2003), Stellate-cell lipidosis in liver biopsy specimens. Recognition and significance, Am J Clin Pathol, vol. 119, no. 2, pp. 254-258.
  96. Nollevaux, M.C. et al. (2006), Hypervitaminosis A-induced liver fibrosis: stellate cell activation and daily dose consumption, Liver Int, vol. 26, no. 2, pp. 182-186.
  97. Pandey, G. et al. (2010), Hepatic cell injury by ethinyl estradiol estrogen, IJPSR, vol. 1, no. 1, pp. 49-53.
  98. Pandey, G. et al. (2011), Experimental hepatotoxicity produced by ethinyl estradiol, Toxicol Int, vol. 18, no. 2, pp. 160-162.
  99. Radzikowska, E. et al.(2012), Estrogen-induced hepatotoxicity in rats, Journal of Pre-Clinical and Clinical Research, vol. 6, no. 1, pp. 10-13.
  100. Antherieu, S. et al, (2013), Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells, Hepatology, vol. 57, no. 4, pp. 1518-29.
  101. Parmentier, C. et al. (2013), Transcriptomic hepatotoxicity signature of chlorpromazine after short- and long-term exposure in primary human sandwich cultures, Drug Metab Dispos, vol. 41, no. 10, pp. 1835-1842.
  102. Wen, B. and M. Zhou (2009), Metabolic activation of the phenothiazine antipsychotics chlorpromazine and thioridazine to electrophilic iminoquinone species in human liver microsomes and recombinant P450s, Chem Biol Interact, vol. 181, no. 2, pp. 220-226.
  103. 103.0 103.1 103.2 Codreanu, S.G. et al. (2014), Alkylation damage by lipid electrophiles targets functional protein systems, Molecular & Cellular Proteomics, vol. 13, no. 3, pp.849–859.
  104. Liebler, D.C. (2008), Protein Damage by Reactive Electrophiles: Targets and Consequences, Chem Res Toxicol, vol. 21, no. 1, pp. 117-128.
  105. 105.0 105.1 105.2 Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.
  106. 106.0 106.1 106.2 Canbay, A., S.L. Friedman and G.J. Gores (2004), Apoptosis: the nexus of liver injury and fibrosis, Hepatology, vol. 39, no. 2, pp. 273-278.
  107. 107.0 107.1 107.2 Orrenius, S., P. Nicotera and B. Zhivotovsky (2011), Cell death mechanisms and their implications in toxicology, Toxicol. Sci, vol. 119, no. 1, pp. 3-19.
  108. Jaeschke, H. (2002), Inflammation in response to hepatocellular apoptosis, Hepatology, vol. 35, no. 4, pp. 964–966.
  109. 109.0 109.1 Canbay, A. et al. (2002), Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis, Gastroenterology, vol. 123, no. 4, pp. 1323-1330.
  110. 110.0 110.1 Canbay, A. et al. (2003), Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis, J Clin Invest, vol. 112, no. 2, pp. 152-159.
  111. Takehara, T. et al. (2004), Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses, Gastroenterology, vol. 127, no. 4, pp. 1189-1197.
  112. Faubion, W.A. and G.J. Gores GJ (1999), Death receptors in liver biology and pathobiology, Hepatology, vol. 29, no. 1, pp. 1-4.
  113. 113.0 113.1 113.2 Canbay, A. et al. (2004), The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse, J Pharmacol Exp Ther, vol. 308, no. 3, pp. 1191-1196.
  114. 114.0 114.1 114.2 114.3 114.4 114.5 Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.
  115. 115.0 115.1 Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.
  116. Kresse, M. et al. (2005), Kupffer cell-expressed membrane-bound TNF mediates melphalan hepatotoxicity via activation of both TNF receptors, J. Immunol, vol. 175, no. 6, pp. 4076–4083.
  117. Andrés, D. et al. (2003), Depletion of Kupffer cell function by gadolinium chloride attenuates thioacetamide-induced hepatotoxicity. Expression of metallothionein and HSP70, Biochem. Pharmacol, vol. 66, no. 6, pp. 917–926.
  118. Schümann, J. et al. (2000), Importance of Kupffer cells for T-cell-dependent liver injury in mice, Am. J. Pathol, vol. 157, no, 5, pp. 1671–1683.
  119. 119.0 119.1 Liu, Xingjun et al. (2006), Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int, vol.26, no.1, pp. 8-22.
  120. 120.0 120.1 120.2 Gressner , A.M. et al.(2002), Roles of TGF-β in hepatic fibrosis. Front Biosci, vol. 7, pp. 793-807.
  121. Cheng, K., N.Yang and R.I. Mahato (2009), TGF-beta1 gene silencing for treating liver fibrosis, Mol Pharm, vol. 6, no. 3, pp. 772–779.
  122. Tang, L.X. et al. (2012), Asiatic acid inhibits liver fibrosis by blocking TGF-β /Smad signaling in vivo and in vitro, PLoS One, vol. 7, no. 2, e31350.
  123. 123.0 123.1 Qi Z et al. (1999), Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat, Proc Natl Acad Sci USA, vol. 96, no. 5, pp. 2345-2349.
  124. 124.0 124.1 Friedman, S.L. (2002), Hepatic Fibrosis-Role of Hepatic Stellate Cell Activation, MedGenMed, vol. 4, no. 3, pp. 27.
  125. Friedman, S.L. (2004), Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications, Nat Clin Pract Gastroenterol Hepatol, vol. 1, no. 2, pp. 98-105.
  126. Anan, A. et al. (2006), Proteasome inhibition induces hepatic stellate cell apoptosis, Hepatology, vol. 43, no. 2, pp. 335-344.
  127. Son G. et al. (2009), Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis, Hepatology, vol. 50, no. 5, pp. 1512–1523.
  128. Nakamura, I. et al. (2014), Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling, PLoS One, vol. 9, no. 4, e92273.
  129. Lee William M. (2003), Drug- induced hepatotoxicity, N Engl J Med, vol. 349, no. 5,pp. 474-485.
  130. Russmann, S., G.A. Kullak-Ublick and I. Grattagliano (2009), Current concepts of mechanisms in drug-induced hepatotoxicity, Curr Med Chem, vol. 16, no. 23, pp. 3041-3053.
  131. Jaeschke, H. et al. (2002), Mechanisms of hepatotoxicity, Toxicological Sciences, vol. 65, no. 2, pp. 166–176.
  132. Mehta N and Ozick LA, (2014), Drug-Induced Hepatotoxicity. Medscape E-Medicine http://emedicine.medscape.com/article/169814-overview (accessed on 12 December 2014).
  133. Ramachandran, R. and S. Kakar (2009), Histological patterns in drug-induced liver disease, J Clin Pathol, vol. 62, no. 6, pp. 481-492.
  134. 134.0 134.1 134.2 Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.
  135. Henderson, N.C. and J.P. Iredale (2007), Liver fibrosis: cellular mechanisms of progression and resolution, Clin Sci (Lond), vol. 112, no. 5, pp. 265-280.
  136. Czaja, A.J. (2014), Hepatic inflammation and progressive liver fibrosis in chronic liver disease, World J Gastroenterol, vol. 20, no. 10, 2515-2532.
  137. 137.0 137.1 Sivakumar, P. and A.M. Das (2008), Fibrosis, chronic inflammation and new pathways for drug discovery, Inflamm Res, vol. 57, no. 9, pp. 410-418.
  138. Marra, F. (2002), Chemokines in liver inflammation and fibrosis, Front Biosci, vol. 7, pp. 1899-1914.
  139. 139.0 139.1 Parola, M. and G. Robino (2001), Oxidative stress-related molecules and liver fibrosis. J Hepatol, vol. 35, no. 2, pp. 297-306.
  140. 140.0 140.1 140.2 140.3 140.4 140.5 Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.
  141. Guo, J. and S. L. Friedman (2010),Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis, Fibrogenesis Tissue Repair, vol. 3, p. 21.
  142. 142.0 142.1 Parsons, C.J., M.Takashima and R.A. Rippe (2007), Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, vol. 22, Suppl.1, pp. S79-S84.
  143. 143.0 143.1 143.2 Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98.
  144. El-Rigal, N.S. et al. (2013), Oxidative Stress in Liver Diseases, JPCR, vol. 5, pp. 155-164.
  145. Paik, Y.H. et al. (2011), The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice, Hepatology, vol. 53, no. 5, pp. 1730-1741.
  146. Sánchez-Valle, V. et al. (2012), Role of oxidative stress and molecular changes in liver fibrosis: a review, Curr Med Chem, vol. 19, no. 28, pp. 4850-4860.
  147. 147.0 147.1 Kisseleva, T. and Brenner, D.A. (2007), Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis, Journal of Gastroenterology and Hepatology, vol. 22, Suppl. 1; pp. S73–S78.
  148. 148.0 148.1 Kirkham, P. (2007), Oxidative stress and macrophage function: a failure to resolve the inflammatory response, Biochem Soc Trans, vol. 35, no. 2, pp. 284-287.
  149. Tanel, A. et al. (2007), Activation of the death receptor pathway of apoptosis by the aldehyde Acrolein, Free Radic Biol Med, vol. 42, no. 6, pp. 798-810.
  150. Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.
  151. Jaeschke, H. (2011), Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts, J Gastroenterol Hepatol. vol. 26, suppl. 1, pp. 173-179.
  152. 152.0 152.1 152.2 152.3 152.4 Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.
  153. Roth, S., K. Michel and A.M. Gressner (1998), (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat HSCs, Hepatology, vol. 27, no. 4, pp.1003-1012.
  154. Kamimura, S. and H. Tsukamoto (1995), Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease, Hepatology, vol. 22, no. 4, pp. 1304-1309.
  155. 155.0 155.1 155.2 Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.
  156. 156.0 156.1 156.2 Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.
  157. Fujiwara, N. and K. Kobayashi (2005), Macrophages in inflammation, Curr Drug Targets Inflamm Allergy, vol. 4, no. 3, pp. 281-286.
  158. Reuter, S. et al. (2010), Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med, vol. 49, no. 11, pp. 1603-1616.
  159. Williams, E.J. et al. (2000), Increased expression of connective tissue growth factor infibrotic human liver and in activated hepatic stellate cells, J Hepatol, vol. 32, no. 5, pp. 754-761.
  160. Kaimori, A. et al. (2007), Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro, J Biol Chem, vol. 282, no. 30, pp. 22089-22101.
  161. 161.0 161.1 Matsuoka, M. and H. Tsukamoto, (1990), Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis, Hepatology, vol. 11, no. 4, pp. 599-605.
  162. Benyon, R.C. and M.J. Arthur (2001), Extracellular matrix degradation and the role of stellate cells, Semin Liver Dis, vol. 21, no. 3, pp. 373-384.
  163. Milani, S. et al. (1994), Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver, Am J Pathol, vol. 144, no. 3, pp. 528-537.
  164. Safadi, R. and S.L. Friedman (2002), Hepatic fibrosis--role of hepatic stellate cell activation, MedGenMed, vol 4, no. 3, p. 27.
  165. López-Novoa, J.M. and M.A. Nieto (2009), Inflammation and EMT: an alliance towards organ fibrosis and cancer progression, EMBO Mol Med, vol. 1. no. 6-7, pp. 303–314.
  166. 166.0 166.1 Pellicoro, A. et al. (2014), Liver fibrosis and repair: immune regulation of wound healing in a solid organ, Nat Rev Immunol, vol. 14, no. 3, pp. 181-194.
  167. Brancatelli, G. et al. (2009), Focal confluent fibrosis in cirrhotic liver: natural history studied with serial CT, AJR Am J Roentgenol, vol. 192, no. 5, pp. 1341-1347.
  168. Rockey, D.C. and S.L. Friedman (2006), Hepatic fibrosis and cirrhosis, Zakim and Boyer's Hepatology, 5th edition, section 1, chapter 6, pp. 87-109.
  169. Poynard, T., P. Bedossa and P. Opolon (1997), Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups, Lancet, vol. 349, no. 9055, pp. 825-832.
  170. Schwend, T. et al. (2008), Alkylation of adenosine deaminase and thioredoxin by acrylamide in human cell cultures, Z Naturforsch, vol. 64, no. 5-6, pp. 447-453.
  171. Thompson, C.A. and P.C. Burcham (2008), Protein alkylation, transcriptional responses and cytochrome c release during Acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents, Toxicol In Vitro, vol. 22, no. 4, pp. 844-853.
  172. Bauman, J.N. et al. (2009), Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction, Chem Res Toxicol, vol. 22, no. 2, pp. 332-340.
  173. Bauman, J.N. et al. (2008), Comparison of the bioactivation potential of the antidepressant and hepatotoxin nefazodone with aripiprazole, a structural analog and marketed drug, Drug Metab. Dispos, vol. 36, no. 6, pp. 1016–1029.
  174. LeCluyse, E.L. et al. (2012), Organotypic liver culture models: meeting current challenges in toxicity testing, Crit Rev Toxicol, vol. 42, no. 6, 501-548.
  175. Soldatow, V.Y. et al. (2013), In vitro models for liver toxicity testing, Toxicol Res, vol. 2, no.1, pp. 23–39.
  176. Tukov, F.F. et al. (2006), Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte co-culture system, Toxicol In Vitro, vol. 20, no. 8, pp. 1488-1499.
  177. Coulouarn, C. et al. (2012), Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma, Cancer Res, vol. 72, no. 10, pp. 2533–2542.
  178. Watanabe, A. et al. (2007), Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9, Hepatology, vol. 46, no. 6, pp. 1509-1518.
  179. Chu, P.S. et al. (2013), C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice, Hepatology, vol. 58, no. 1, pp. 337-350.
  180. Czaja, M.J. et al. (1989), In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis, J Cell Biol, vol. 108, no. 6, pp. 2477-2482.
  181. Tan, A.B. et al. (2013), Cellular re- and de-programming by microenvironmental memory: why short TGF-β1 pulses can have long effects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 12.
  182. Yin, C. et al. (2013), Hepatic stellate cells in liver development, regeneration, and cancer, J Clin Invest, vol. 123, no. 5, pp. 1902–1910.
  183. De Minicis, S. et al. (2007), Gene expression profiles during hepatic stellate cell activation in culture and in vivo, Gastroenterology, vol. 132, no. 5, pp. 1937-1946.
  184. Chen, C. and M. Raghunath (2009), Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art, Fibrogenesis Tissue Repair, vol. 15, no. 2, p. 7.
  185. Rockey, D.C. et al. (1992), Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture, J Submicrosc Cytol Pathol, vol. 24, no. 2, pp. 193-203.
  186. Liu Youhua (2011), Cellular and molecular mechanisms of renal fibrosis, Nat. Rev.nephrol,vol 7, no. 12, pp. 684-696.
  187. Wynn, T.A. (2008), cellular and molecular mechanisms of fibrosis, J Pathol, vol. 214, no. 2, pp. 199-210.
  188. Chatziantoniou, Ch. and J.C. Dussaule (2005), Insights into the mechanisms of renal fibrosis: is it possible to achieve regression, Am J Physiol Renal Physiol, vol. 289, no. 2, pp. F227–F234.
  189. Weber, S. et al. (2010), Liver fibrosis: from animal models to mapping of human risk variants, Best Practice & Research Clinical Gastroenterology, vol. 24, no. 5, pp. 635–646.
  190. Iredale, J.P. (2007), Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ, J Clin Invest, vol. 117, no. 3, pp. 539-548.
  191. Constandinou, C., N.C. Henderson and J.P. Iredale (2005), Modelling liver fibrosis in rodents, Methods Mol Med, vol. 117, pp. 237-250.
  192. Tsukamoto, H., M. Matsuoka and S.W. French (1990), Experimental models of hepatic fibrosis: a review, Semin Liver Dis, vol. 10, no. 1, pp. 56-65.
  193. Crespo Yanguas, S. et al. (2015), Experimental models of liver fibrosis, Arch Toxicol.[Epub ahead of print] DOI 10.1007/s00204-015-1543-4.