The pathogenesis of diclofenac induced immunoallergic hepatitis in a canine model of liver injury

Abstract

Hypersensitivity to non-steroidal anti-inflammatory drugs is a common adverse drug reaction and may result in serious inflammatory reactions of the liver. To investigate mechanism of immunoallergic hepatitis beagle dogs were given 1 or 3 mg/kg/day (HD) oral diclofenac for 28 days. HD diclofenac treatment caused liver function test abnormalities, reduced haematocrit and haemoglobin but induced reticulocyte, WBC, platelet, neutrophil and eosinophil counts. Histopathology evidenced hepatic steatosis and glycogen depletion, apoptosis, acute lobular hepatitis, granulomas and mastocytosis. Whole genome scans revealed 663 significantly regulated genes of which 82, 47 and 25 code for stress, immune response and inflammation. Immunopathology confirmed strong induction of IgM, the complement factors C3&B, SAA, SERPING1 and others of the classical and alternate pathway. Alike, marked expression of CD205 and CD74 in Kupffer cells and lymphocytes facilitate antigen presentation and B-cell differentiation. The highly induced HIF1A and KLF6 protein expression in mast cells and macrophages sustain inflammation. Furthermore, immunogenomics discovered 24, 17, 6 and 11 significantly regulated marker genes to hallmark M1/M2 polarized macrophages, lymphocytic and granulocytic infiltrates; note, the latter was confirmed by CAE staining. Other highly regulated genes included alpha-2-macroglobulin, CRP, hepcidin, IL1R1, S100A8 and CCL20. Diclofenac treatment caused unprecedented induction of myeloperoxidase in macrophages and oxidative stress as shown by SOD1/SOD2 immunohistochemistry. Lastly, bioinformatics defined molecular circuits of inflammation and consisted of 161 regulated genes.Altogether, the mechanism of diclofenac induced liver hypersensitivity reactions involved oxidative stress, macrophage polarization, mastocytosis, complement activation and an erroneous programming of the innate and adaptive immune system.

An adverse outcome pathway for immune-mediated and allergic hepatitis: a case study with the NSAID diclofenac

“…In addition, reactive metabolites of diclofenac cause cellular stress and increased the level of ROS which leads to mitochondrial damage and subsequent apoptosis in hepatocytes (Boelsterli 2003;Gomez-Lechon et al 2003a). Consistent with the findings observed with mice, the genomic analysis of dog liver discovered genes related to oxidative stress, mitochondrial biogenesis and membrane transport and apoptosis as significantly regulated (Selvaraj et al 2017).…”

“…Previously reported genomic data of diclofenac-induced liver injury in mice and dog models were interrogated to construct this AOP framework (male C57BL/6 mice with daily intraperitoneal injection of 30 mg/kg/day and 150 mg/kg/days for 14 days; male beagle dogs with daily oral dosing of 1 mg/kg/day and 3 mg/kg/day for 28 days) (Lee et al 2016 ; Selvaraj et al 2017 ). Given the complex inference resulting from on-target but exaggerated pharmacological responses and toxicity related to the physicochemical characteristics of diclofenac and its effects on cells, organelles, membranes and/or metabolic pathways, a combined approach was taken to define MIE.…”

“…KE5: Kupffer cell activation and/polarization (M1/ M2) Diclofenac treatment caused an activation of Kupffer cells and strong expression of M1/M2 marker genes (Selvaraj et al 2017). Activated Kupffer cells release a range of inflammatory mediators, growth factors and acute-phase proteins to perpetuate liver inflammation (Kolios et al 2006;Roberts et al 2007).…”

“…This non-steroidal anti-inflammatory drug (NSAID) exerts anti-inflammatory, analgesic and anti-pyretic effects through various mechanisms; however, its use can lead to adverse drug reactions including DILI (Banks et al 1995 ). We entrained the AOP on our previous genomic studies as well as serum biochemistry, histopathology and immunohistochemistry and Western immunoblotting data (Lee et al 2016 ; Selvaraj et al 2017 ) and show that diclofenac reactive metabolism causes divergent immune responses among the two animal species commonly used in toxicity studies. We define iminoquinone and quinone reactive metabolites as MIE and confirm the relevance of these structural alerts for a larger group of drugs and chemicals undergoing iminoquinone and quinone reactive metabolite formation.…”

Glycosylation profiling of dog serum reveals differences compared to human serum

“…The domestic dog ( Canis familiaris ) is a widely used animal model for a variety of human diseases like Alzheimer’s, cancer, Duchenne muscular dystrophy ( Shearin and Ostrander 2010 ; Barth é lémy et al 2012 ; Head 2013 ; Villarnovo et al 2017 ; Gustafson et al 2018 ) as well as for the experimental infection of human pathogens ( Binhazim et al 1993 ; Cerri et al 1994 ). Dogs are commonly utilized in toxicology studies ( Selvaraj et al 2017 ; Sun et al 2018 ) and, furthermore, there is a large veterinary interest surrounding the diagnosis and treatment of canine diseases.…”