Table 5.

Details of relevant processes and mechanism involved in DILI of the training compounds

Training compound	Phase I	Phase II	Phase III and BSEP inhibition	Cell death	Mitochondrial dysfunction	Lysosomal dysfunction and phospholipidosis	Immune response	Association studies
Acetaminophen	NAPQI, CRM responsible for APAP-hepatotoxicity CYP2E1 is primary enzyme, other CYPs (including CYP1A2 and CYP3A4) also involved in bioactivation At therapeutic dose NAPQI is efficiently deactivated through spontaneous or enzymatic (GST) GSH conjugation Overdose of APAP results in GSH depletion and protein binding	Mainly glucuronidation and sulfation APAP-glucuronide (UGT1A1 and 1A6): 50–70 % of the dose APAP-sulphate (SULT1A1, 1A3/4 and 1E1) accounts for 25–35 % of the dose At a therapeutic dose 5—15 % excreted in urine as mercapturic acid or cysteine conjugate	Unlike APAP, elimination of APAP metabolites require transporters Biliary excretion: APAP-glucuronide—MRP2 and BCRP, APAP-sulphate: MRP2 and BCRP, APAP-GSH: MRP2 Basolateral excretion: APAP-glucuronide: MRP3, APAP-sulphate: MRP3 and MRP4	Majority of animal studies suggest necrosis Increasing number of reports suggest apoptosis also plays a key role	Associated with binding of NAPQI to mitochondrial proteins
Amiodarone	Primarily metabolized to its active metabolite, mono-N-desethylamiodarone (DEA) by several hepatic CYPs (primarily by CYP3A4 but also CYP1A1, 2D6 and 2C8) Both, Amiodarone and its major active metabolite contribute to observed overall hepatotoxicity	Glucuronide conjugation is an important elimination pathway of amiodarone and its phase 1 metabolites (conjugates found in human bile)	Amiodarone and DEA are excreted through the biliary system Inhibitory effects of amiodarone towards Phase 3 transporters observed (primarily uptake, OATP2) MDR1 possibly plays a role in elimination of amiodarone (induced in rat) Mdr2 expression progressively decreases upon amiodarone treatment	Micro- and macrovesicular steatosis, phospholipid laden lysosomes (phospholipidosis, impairment of lysosomal function), intense ballooning of hepatocytes, presence of abundant mallory bodies, and fibrosis Pseudoalcoholic hepatitis End-stage liver disease occurs rarely in human probably due to repair	Inhibition of mitochondrial beta-oxidation → triglyceride accumulation and microvesicular steatosis Interference with mitochondrial energy production, disruption of electron transport chain enzyme complexes and uncoupling of oxidative phosphorylation subsequent to amiodarone accumulation in mitochondria Severe mitochondrial impairment → ATP depletion and subsequent cell death	Accumulation in lysosomes and trapping due to protonation in acidic environment Amiodarone complexes with phospholipids and inhibits intralysosomal phospholipase A resulting in accumulation of phospholipids
Bosentan	Metabolites are reported to inhibit rat BSEP Reactive metabolites +are formed, but not thought to play a role through the formation of ROS CYP3A4 and CYP2C9 dependent metabolism	UGT1A1 dependent metabolism and transport	Prototypical inhibitor of BSEP dependent transport Substrate (MRP2) and stimulates MRP2 activity, uncoupling bile dependent (BSEP) and independent (MRP2) flow inducing cholestasis Trans inhibition of BSEP ruled out
Diclofenac	Oxidation of DF at 4′ position by CYP2C9 major oxidative route Minor route is 5-OH-DF formation by CYP3A4 Both 4′OH-DF and 5OH-DF form reactive quinone imines, inactivated by GSH Quinone imines are protein reactive and implicated in redox cycling/causing oxidative stress Minor bioactivation pathways result in reactive arene oxide (CYP2C9) and o-imine methide (CYP3A4), inactivated by GSH Multiple minor oxidative routes result in stable metabolites NQO1 and NQO2 reduce QIs back to the OH– metabolites	Glucuronidation is major route → diclofenac acyl-glucuronide (DF–AC), mainly by UGT2B7 but also by UGT1A9 and 1A6 Subsequent hydroxylation of DF–AC by CYP2C8 results in 4-hydroxy acyl-glucuronide formation DF–AC is protein and GSH reactive GSH conjugation results in a reactive DF-S-acyl-glutathione thioester Quinone imines inactivated by GSH S-transferase (GST) catalysed GSH conjugation	DF is efficiently transported by human Bcrp1 and to a lesser extent by BCRP DF–AC, hydroxy acyl-glucuronides as well as 4′hydroxy and 5-hydroxy diclofenac are excreted into bile by ABCC2 In addition Bcrp1 and Mrp3 are also involved in the biliary excretion and sinusoidal efflux of DF–AC, respectively	At low concentrations apoptosis by mechanisms related to MPT induction When a large proportion of the mitochondria are affected, necrotic cell death can occur Metabolism related cell death in rat hepatocytes → inhibition of CYP activity reduced cell toxicity while inhibition of UGT aggravated the toxicity	DF induces mitochondrial permeability transition (MPT) 4′hydroxy DF, DF–AC and DF glutathione thioester are stronger inhibitors of ATP synthesis compared to DF No mitochondrial toxicity observed in HepG2 gal-glu assay at low concentrations		Immune cell activation by 5-hydroxy-DF and DF-2,5-quinoneimine in mice; DF and DF-AC no immune cell activation Hepatic adducts and circulating antibodies in patients with hepatotoxicity	Association with UGT2B7*2, CYP2C8, and ABCC2 polymorphisms: increased formation of reactive DF metabolites GWAS study: association UGT2B7; no HLA association Association GSTM1 and T1-null genotype
Flucloxacillin	Hydroxylation of the 5-methyl group in the isoxazole ring to a 5-hydroxymethyl derivative by CYP 3A4		An inhibitor of BSEP				Activates CD4+ and CD8+ T cells in patients with DILI Drug-specific CD8+ T cell response HLA-B*57:01 restricted	GWAS study showed HLA-B*57:01 as major determinant of DILI
Fialuridine	FIAU to FMAU to FAU (via thymidylate synthase)—FMAU also has activity against mtDNA following phosphorylation	Unknown glucuronide conjugates reported Phosphorylation through mono-phosphate (thymidine kinase), di-phosphate (thymidylate kinase) to tri-phosphate (DPK) species	Localized to mitochondria via hENT1	Inhibition of mitochondrial DNA polymerase γ by the triphosphate metabolite → depletion of mtDNA Loss of mtDNA encoded proteins leads to loss of function of mitochondrial respiratory chain
Nefazodone	Metabolized to active hydroxynefazodone, m-chlorophenyl-piperazine and triazoledione Inhibitor of CYP3A4 in vitro and in vivo Extensively metabolized in the liver by n-dealkylation, aliphatic and aromatic hydroxylation less than 1 % of intact drug recovered in urine The clearance of mCPP is CYP2D6 dependent High first-pass metabolism and plasma protein binding	Metabolites are finally eliminated as glucuronide or sulphate conjugates	Strong inhibition of BSEP Inhibits the efflux activity of MDR1	Apoptotic cell death	Mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase Inhibition of mitochondrial respiratory complex I and IV, associated with accelerated glycolysis → mitochondrial membrane potential collapse, GSH depletion and oxidative stress
Perhexiline	Hydroxylation by CYP2D6 (highly polymorphic, 10–20-fold inter-individual variability)	Secondary metabolism to dihydroxy-metabolites and glucuronide conjugates	No reports of interaction		Protonated perhexiline accumulates in mitochondrial membrane, leading to multitude of effects, incl. uncoupling of mitochondrial oxidative phosphorylation, inhibition of complexes I and II, decreased ATP formation and reduced mitochondrial β-oxidation of long-chain fatty acids → triglyceride accumulation and microvesicular steatosis and/or cell death	Non-covalent complexes of protonated perhexiline with phospholipids are formed which inhibit the action of intralysosomal phospholipases resulting in accumulation of phospholipids along with lysosomal accumulation of the drug
Tolcapone	Several metabolites derived from reduced drug at 5-nitro Oxidation of amine and acetylamine by several P450 s (CYP2E1, −1A2) to reactive species that could be trapped with glutathione (GSH) Oxidative hydroxylation of the methyl group by CYP3A4 and to the carboxylic acid is a minor pathway CYP2C9 as major P450 in hydroxylation of tolcapone	The major early and most abundant metabolite is 3-O-β,d-glucuronide conjugate; UGT polymorphism The major late metabolite in plasma is 3-O-methyltolcapone (methylation by COMT) N-acetylamino glucuronide in urine and faeces	Modest inhibition of MRP3, MRP4 and BSEP		Uncoupling of oxidative phosphorylation
Troglitazone	Reactive metabolites formation, including quinones and quinone methides, catalysed by CYP3A potentially leading to toxicity	Mainly glucuronidation and sulfation leading to relatively stable metabolites	Inhibition of the BSEP transporter by troglitazone and its troglitazone sulfate Troglitazone sulfate inhibits OATP and is toxic at similar concentrations as the parent compound ₠	Induce apoptotic cell death in human hepatocytes at high concentrations by by ₠! effects on mitochondria resulting in depletion of ATP and release of cytochrome c	Induces mitochondrial membrane permeability in isolated mitochondria ₠	Lipid peroxidation and PPARγ‐dependent steatosis
Ximelagatran	Stepwise esterase-mediated hydrolysis and N-reduction (and vice versa) Outer mitochondrial membrane enzymes—mitochondrial reducing component 1 and 2 (mARC1 and mARC2) involved in melagatran formation	No phase II metabolism reported	Potential involvement of PgP transport metabolism Transport of intermediates thought to be greater than for parent	Cell viability: tolerated well up to at least 200 uM (ATP content) Apoptosis: after exposure of HepG2 cells at 100 uM for 24 h	Toxicity in vitro via mitochondrial mechanism: 1. higher mitochondrial concentrations of melagatran (mARC) 2. asymptomatic hepatic stress (e.g. virus/inflammation) leads to mitochondrial stress Mitochondrial functions: no effects