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Jung-Hwa Oh, Korea Institute of Toxicology
Juergen Borlak, Hannover Medical School
Seokjoo Yoon, Korea Insitute of Toxicology
Point of Contact
Jung-Hwa Oh (email point of contact)
- Jung-Hwa Oh
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite|
This AOP was last modified on November 02, 2020 07:34
|benzoquinone imine and acylglucuronide metabolites||November 02, 2020 07:03|
|Mitochondrial dysfunction||November 02, 2020 07:11|
|Apoptotic cell death||November 02, 2020 07:11|
|Immune cell activation||November 02, 2020 07:12|
|IFNγ signaling||November 02, 2020 07:13|
|Increase in inflammation||May 03, 2019 14:27|
|Immune mediated hepatitis||November 02, 2020 07:14|
|Activation of ER stress||November 02, 2020 07:09|
|Reactive Metabolite leads to Mitochondrial dysfunction||November 02, 2020 07:14|
|Mitochondrial dysfunction leads to Apoptotic cell death||November 02, 2020 07:15|
|Reactive Metabolite leads to Immune cell activation||November 02, 2020 07:16|
|Mitochondrial dysfunction leads to ER stress||November 02, 2020 07:18|
|Immune cell activation leads to IFNγ signaling||November 02, 2020 07:18|
|IFNγ signaling leads to Increase in inflammation||November 02, 2020 07:19|
|Apoptotic cell death leads to Increase in inflammation||November 02, 2020 07:19|
|Increase in inflammation leads to hepatitis||November 02, 2020 07:20|
|ER stress leads to Apoptotic cell death||November 02, 2020 07:20|
Many drugs have the potential to cause drug induced liver injury (DILI); however underlying mechanisms are diverse. We report an AOP for immune mediated and allergic hepatitis as a case study with diclofenac and the findings of this NSAID can be extended to other ones. By considering genomic, histo- and clinical pathology data from mice and dogs we developed AOPs specific for immune mediated and drug hypersensitivity/allergic hepatitis. We define reactive metabolites including quinone imine catalysed by CYP monooxygenase and myeloperoxidases of Kupffer cells as well as acylglucuronide as molecular initiating events (MIE). The reactive metabolites bind to proteins and act as neo-antigen and involve antigen presenting cells to elicit B-and T-cell responses. Given the different immune system between mice and dogs 6 different key events (KE) at the cellular and up to 4 KEs at the organ level are defined with mechanistic plausibility for the onset and progression of liver inflammation. With mice, cellular stress response, interferon gamma-, adipocytokine- and chemokine signaling provide a rational for the AOP of immune-mediated hepatitis. With dogs, an erroneous programming of the innate and adaptive immune response results in mast cell activation; their infiltration into liver parenchyma and the shift to M2 polarized Kupffer cells signifies allergic hepatitis and the occurrence of granulomas of the liver. Taken together, diclofenac induces divergent immune response among two important preclinical animal species and the injury pattern seen among clinical cases confirms the relevance of the developed AOP for immune-mediated hepatitis.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||1814||benzoquinone imine and acylglucuronide metabolites||Reactive Metabolite|
|2||KE||1816||Mitochondrial dysfunction||Mitochondrial dysfunction|
|7||KE||1815||Activation of ER stress||ER stress|
|3||KE||1817||Apoptotic cell death||Apoptotic cell death|
|4||KE||1818||Immune cell activation||Immune cell activation|
|5||KE||1819||IFNγ signaling||IFNγ signaling|
|6||KE||1633||Increase in inflammation||Increase in inflammation|
|8||AO||1820||Immune mediated hepatitis||hepatitis|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Reactive Metabolite leads to Mitochondrial dysfunction||adjacent||High||High|
|Mitochondrial dysfunction leads to Apoptotic cell death||adjacent||High||High|
|Reactive Metabolite leads to Immune cell activation||adjacent||High||High|
|Mitochondrial dysfunction leads to ER stress||adjacent||High||High|
|Immune cell activation leads to IFNγ signaling||adjacent||High||Low|
|IFNγ signaling leads to Increase in inflammation||adjacent||High||Low|
|Apoptotic cell death leads to Increase in inflammation||adjacent||High||Moderate|
|Increase in inflammation leads to hepatitis||adjacent||High||High|
|ER stress leads to Apoptotic cell death||adjacent||High||High|
Life Stage Applicability
|Homo sapiens||Homo sapiens||High||NCBI|
Overall Assessment of the AOP
Domain of Applicability
Essentiality of the Key Events
Drug induced hepatitis is a multistep process and frequently involves reactive metabolites which subsequently form drug-protein adducts. These are sensed by antigen presenting cells to elicit innate and adaptive immune responses (Adams et al. 2010). Diclofenac is extensively metabolized by CYP monooxygenases and myeloperoxidases (MPO) of neutrophils and Kupffer cells into reactive metabolites notably quinoneimine intermediates. The metabolism of diclofenac to reactive metabolites causes organ toxicity and this defines the MIE. A further example for a benzoquinone imine reactive metabolite causing liver injury is the metabolism of paracetamol to NAPQI. Correspondingly, NAPQI is the initial cause of hepatotoxicity and once again defines the MIE. Indeed, for a wide range of drugs quinoneimines are implicated in liver injury. Diclofenac caused significant regulation of CYP monooxygenases after single and repeated treatment of mice (Lee et al. 2016). Independent investigations also support the key role of the acyl glucuronide produced by uridine diphosphoglucuronosyl transferase as a molecular initiation event (Oda et al. 2017; Seitz and Boelsterli 1998) and among NSAIDs the salicyl acyl glucuronide derived from aspirin is a further example. Thus, reactive metabolites are formed by hepatocytes, neutrophils and Kupffer cells with benzoquinone imine intermediates and acyl glucuronides being particularly harmful. If not sufficiently detoxified the reactive metabolites damage organelles, proteins and membrane lipids and eventually trigger programmed cell death. Specifically, the accumulation of the reactive metabolite triggers oxidative stress and mitochondrial permeability transitions, i.e. mitochondrial toxicity by inhibiting ATP synthesis that leads to hepatocellular damage (Syed et al. 2016). Next to direct effects the reactive metabolites can covalently bind to proteins to form adducts. These function as
neoantigens and are sensed and phagocytosed by APCs. Through interaction with the major histocompatibility complex APCs elicit B and T cell responses (Aithal 2011; Boelsterli 2003). Additionally, diclofenac acyl glucuronide inhibit the Mrp2 transport. This results in intrahepatic cholestasis and damage of the biliary epithelium (Boelsterli 2003; Lagas et al. 2010; Seitz and Boelsterli 1998). Similar to diclofenac the NSAIDs lumiracoxib and indomethacin produce quinoneimine reactive intermediates and next to ibuprofen and naproxen a wider range of carboxylic acid containing drugs are associated with allergic reactions (Stepan et al. 2011). The reactivity of acyl glucuronides derived from carboxylic acid containing drugs and the evidence for its toxicological concerns was recently summarized (Darnell et al. 2015; Van Vleet et al. 2017). Note, the covalent binding of acyl glucuronides to proteins constitutes a mechanism of toxicity and the safety assessment of acyl glucuronides was the subject of a recent commentary with zomepirac being a prominent example for NSAID toxicity (Smith et al. 2018). Importantly, inhibition of MPO ameliorate adverse effects of MPO-derived oxidants (Malle et al. 2007) and MPO ko mice are an excellent system to study the importance of MPO in systemic inflammatory reactions. Alike, amelioration of diclofenac induced toxicity was observed with cytochrome P450 reductase (CPR) null mice (Zhu and Zhang 2012) and multidrug resistance-associated protein 3 plays an important role in protection against acute toxicity of diclofenac acyl glucuronide as evidenced in Mrp3-null (KO) mice (Scialis et al. 2015). Moreover, NSAIDs are known to produce reactive oxygen species that result in cardiovascular disease (Ghosh et al. 2015) and a structural alert/reactive metabolite concept of 200 common drugs producing a wide range of different reactive metabolites was reported (Stepan et al. 2011). Collectively, the structural alerts quinoneimine and acyl glucuronides function as MIEs in immune-mediated and allergic
hepatitis. Notwithstanding, defining an unequivocal MIE in the AOP framework can be complex as discussed in the seminal paper of Allen and coworkers (Allen et al. 2014).
KEs related to immune mediated hepatitis
The biochemical and genomic data revealed diclofenac treatment to induce complex immune mediated inflammatory signalling particularly from resident and migratory cells of the sinusoidal and space of Disse. Within hepatocytes, the reactive metabolites elicit cellular stress responses including oxidative stress, mitochondrial dysfunction, apoptosis and ER stress/unfolded protein responses. In repeated dose studies with mice, diclofenac treatment induced expression of the plasma membrane cysteine carrier (Slc3a1) to imply adaptive responses to oxidative stress. Furthermore, cysteine is an essential building block for the hepatic synthesis of reduced GSH and therefore of fundamental importance in alleviating oxidative stresses.
KE1: Mitochondrial dysfunction. Diclofenac caused mitochondrial dysfunction through an inhibition of ATP synthesis (Boelsterli 2003, Kang, et al. 2016, Syed, et al. 2016). The significant repression of mitochondrial membrane transport proteins and key members of the oxidative phosphorylation pathway is testimony of an impaired mitochondrial respiration and ATP synthesis (Lee, et al. 2016). Conversely, the plasma-membrane Mg2+ transporter is strongly induced to increase intracellular Mg2+ concentration. Note, increased Mg2+ uptake counteracts the detrimental effects of diclofenac treatment to alleviate mitochondrial stress and the opening of the Ca2+ dependent permeability transition pore to dampen apoptotic signaling.
KE2: Apoptotic cell death. Reactive metabolites of diclofenac can directly or indirectly induce apoptotic cell death by activating several pro- and anti-apoptotic factors notably toll like receptors, cytokine signaling inducible factors such as S100 calcium binding proteins and pro-inflammatory adipokines to augment ER stress-induced apoptosis (Lee, et al. 2016, Sawa, et al. 2009). Damaged hepatocytes send alarm signals like the damage-associated molecular patterns (DAMPs) that induce the immune and inflammatory response by activating immune cells. Although, several DAMP molecules including S100 proteins were upregulated after repeated dosing of mouse study, the major components of the inflammasome are not regulated at the transcript level. Meanwhile, the danger hypothesis proposed that DAMPs can also be influenced by immune or inflammatory response. Our previous study demonstrated that diclofenac treatment induced the expression of inflammatory proteins which are released from macrophages/Kupffer cells. The subsequent inflammatory responses can reinforce the cellular damage of hepatocyte and in a vicious cycle strengthen inflammation (Lee, et al. 2016).
KE3: ER stress/unfolded protein response. Genes involved in ER stress and unfolded protein response (UPR) were significantly regulated in the liver of diclofenac treated mice. Independent studies evidenced diclofenac to trigger ER stress and UPR by PERK and ATF6 pathways as well as eIF2α phosphorylation (Foufelle and Fromenty 2016, Franceschelli, et al. 2011, Fredriksson, et al. 2014). However, the prolonged activation of PERK/eIF2α pathway induces apoptosis by activating the pro-apoptotic factor CHOP (Franceschelli, et al. 2011, Fredriksson, et al. 2014). In addition, ER stress can alter the lipid metabolism by UPR and leads to dyslipidemia (Basseri and Austin 2012). As reported by us, diclofenac caused hepatic cholersterolosis in mice with significant alteration in the transcription of genes coding for fatty acid and cholesterol metabolism (Lee, et al. 2016). On the other hand, the repeated diclofenac treatment induced Cyp7a1 expression which catalyzes the hydroxylation of cholesterol into bile acids (Lee, et al. 2016). Alike, the induction of the apical sodium–bile acid transporter (Slc10a2) evidences changes in the transcellular transport of bile acids across the biliary epithelium to support the enterohepatic cycling of bile acids (Lee, et al. 2016). Moreover, the organic anion transporter Slc10a6 was induced and functions on taurolithocholic acid-3-sulfate (TCA-3S). Note TCA-3S excretion into urine is about 90-fold higher in patients diagnosed with intrahepatic cholestasis of pregnancy thus highlighting its potential as a biomarker of hepatic cholestasis (Lee, et al. 2016).
KE4: Immune cell activation. Diclofenac adducts are sensed by APC and other phagotysing cells and trigger immune responses. The immune-mediated hepatitis is the result of complex interplay of innate and adaptive immune responses and involves the regulation of various cytokines/chemokines and their receptors (Lee, et al. 2016). Specifically, the released chemokines recruit the neutrophils, leukocytes, and B lymphocytes to the sinusoidal space or to harmed hepatocytes, while cytokines endorse differentiation of myeloid and cytotoxic CD8+ T-cells (Saiman and Friedman 2012, Sawa, et al. 2009). Increased expressions of interleukins modulate the activation and proliferation of T and/or NK cell responses (Hammerich and Tacke 2014, Zwirner and Domaica 2010) while members of the interleukin-1 superfamily stimulate the production of type 2 cytokines by T-helper cells (Miller 2011). Thus, diclofenac treatment resulted in an activation of several cytokines to affect T cell differentiation. Collectively, diclofenac stimulated an activation of diverse immune cells including monocytes, Kupffer cells and APC (Lee, et al. 2016).
KE5: IFNγ signaling. Increased expression of IFNγ hallmarks innate and adaptive immune responses. IFNγ plays a pivotal role in host defence in response to infections and mediating the inflammation by producing the pro-inflammatory cytokines (Muhl and Pfeilschifter 2003). Genomic analysis revealed Ptpn2 (Protein tyrosine phosphatase non-receptor type 2) to be significantly regulated in mice after diclofenac treatment and Ptpn2 plays a critical role in modulating IFNγ signaling (Lee, et al. 2016, Scharl, et al. 2010). Together, diclofenac treatment induced expression of inflammatory cytokines including IFNγ, interleukins and TNFα (Dutta, et al. 2008, Yano, et al. 2012).
KE6: Inflammation. Our computational studies defined key master regulatory molecules and their associated networks. Based on independent RT-qPCR studies induced expression of suppressor of cytokine signaling (Socs), leptin, growth hormone receptor (Ghr), and Ptpn proteins could be confirmed and these function in IFNγ, Jak/Stat, pro- and anti-inflammatory signaling pathways. Stat3 is one of the major transcription factors activated by cytokines and growth factors and influences pro-inflammatory (Mapk, p38, Jnk, and IκB kinase) and anti-inflammatory signaling (Pi3k-Akt) events Among individual animals marked induction of this protein was observed which controls transcription of Socs and inhibits Jak/Stat3 signaling (Kong, et al. 2002). In addition, leptin and other adipokine signaling molecules play a major role in energy intake to influence monocyte and macrophage activity during inflammation (Fantuzzi and Faggioni 2000). Next to its role in the control of energy intake leptin and its receptor modulates Jak/Stat, Erk 1/2 and Pi3k signaling (Bjorbaek and Kahn 2004, Cottrell and Mercer 2012, Paz-Filho, et al. 2012) and therefore play decisive role in inflammation. Similarly, the growth hormone receptor is influenced by multiple intracellular signaling cascades (Jak-Stat and chemokine signaling) and functions in liver regeneration. Diclofenac treatment reinforced Ghr degradation as evidenced by immunoblotting to suppress hepatic Ghr signaling (Takahashi 2017). Diclofenac also induced expression of the endothelial-leukocyte adhesion molecule selectin which recruits leukocytes to the inflammatory site and the computational analysis defined selectin as a master regulator (Lee, et al. 2016, Ley 2003). Furthermore, the highly significant induction of lipocalin-2 (LCN2) signifies sterile inflammation and neutralization of LCN2 controls neutrophilic inflammation as had been summarized (Moschen, et al. 2017). Taken together, the IFNγ, Jak/Stat, adipocytokine and chemokine signaling pathways provide a rational for the AOP of immune-mediated hepatitis observed in mice in response to diclofenac treatment.
Considerations for Potential Applications of the AOP (optional)
A long-term prospective clinical trial involving 17,289 arthritis patients who were randomly assigned to
diclofenac (150 mg daily) or etoricoxib (60 or 90 mg daily) evidenced diclofenac to cause common
aminotransferase elevations (Laine et al. 2009). Alike, a systematic review of randomized clinical trial data of 3
NSAIDs revealed diclofenac to be top ranking for hepatotoxic events (Sriuttha et al. 2018). Earlier studies
already reported cases of acute hepatitis induced by diclofenac (Helfgott et al. 1990; Iveson et al. 1990; Purcell
et al. 1991; Sallie 1990) and liver biopsy findings defined a histological injury pattern of granulomatous
hepatitis. Note our studies with dogs also demonstrated granulomatous hepatitis as a key finding (see above
KE4) and the granulomas are composed of inflammatory cells and histocytes (Ramachandran and Kakar 2009).
Moreover, a current study compared 30 DILI cases among 8 NSAIDs and found diclofenac to be the most
frequently implicated NSAID. The cases are characterized by hepatocellular injury, prolonged hospitalization
and included a patient with fatal Stevens-Johnson syndrome (Schmeltzer et al. 2015). In their study 38% of
diclofenac DILI cases presented fever, rash, and eosinophilia and the findings are consistent with the clinical
features of drug hypersensitivity reactions (Schmeltzer et al. 2015). Altogether, the proposed AOP of immunemediated
and allergic hepatitis is relevant for human DILI induced by NSAIDs.