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Review
. 2018 Mar 6;7(3):347-357.
doi: 10.1039/c7tx00340d. eCollection 2018 May 8.

Paracetamol metabolism, hepatotoxicity, biomarkers and therapeutic interventions: a perspective

Toby J Athersuch  1 , Daniel J Antoine  2 , Alan R Boobis  3 , Muireann Coen  1 , Ann K Daly  4 , Lucia Possamai  5 , Jeremy K Nicholson  1 , Ian D Wilson  1
Affiliations
Review

Paracetamol metabolism, hepatotoxicity, biomarkers and therapeutic interventions: a perspective

Toby J Athersuch et al. Toxicol Res (Camb). .

Abstract

After over 60 years of therapeutic use in the UK, paracetamol (acetaminophen, N-acetyl-p-aminophenol, APAP) remains the subject of considerable research into both its mode of action and toxicity. The pharmacological properties of APAP are the focus of some activity, with the role of the metabolite N-arachidonoylaminophenol (AM404) still a topic of debate. However, that the hepatotoxicity of APAP results from the production of the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI/NABQI) that can deplete glutathione, react with cellular macromolecules, and initiate cell death, is now beyond dispute. The disruption of cellular pathways that results from the production of NAPQI provides a source of potential biomarkers of the severity of the damage. Research in this area has provided new diagnostic markers such as the microRNA miR-122 as well as mechanistic biomarkers associated with apoptosis, mitochondrial dysfunction, inflammation and tissue regeneration. Additionally, biomarkers of, and systems biology models for, glutathione depletion have been developed. Furthermore, there have been significant advances in determining the role of both the innate immune system and genetic factors that might predispose individuals to APAP-mediated toxicity. This perspective highlights some of the progress in current APAP-related research.

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Figures

Fig. 1
Fig. 1. Major metabolic biotransformations of paracetamol related to therapeutic efficacy and observed hepatotoxicity. Metabolites: APAP: acetyl-p-aminophenol, acetaminophen, paracetamol; PAP: p-aminophenol; AM404: n-arachidonoylaminophenol; NABQI, NAPQI: n-acetyl-p-benzoquinoneimine; APAP-X: macromolecular conjugate of acetaminophenol; APAP-SG: glutathione conjugate of acetaminophen; APAP-Cys: cysteinyl acetaminophen; APAP-NAC: mercapturate of acetaminophen; APAP-S: acetaminophen sulfate; APAP-G: acetaminophen sulfate; PAP-S: p-aminophenol sulfate; PAP-G: p-aminophenol sulfate. Biotransformation enzymes and cofactors: 1: sulfotransferase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS); 2: uridine 5′-diphospho-glucuronosyltransferase (UGT), uridine 5′-diphospho-glucuronic acid (UDPGA); 3: cytochrome P450, O2, NADPH; 4: non-enzymatic electrophilic addition; 5: glutathione-s-transferase (GST), gluthathione (GSH); 6: gamma-glutamyl transpeptidase; 7: cysteinyl glycinase; 8: n-acetyltransferase (NAT); 9: n-deacetylase; 10: fatty acid amide hydrolase (FAAH).
Fig. 2
Fig. 2. Toxicity of paracetamol and NABQI to freshly isolated hepatocytes from mouse, hamster, rat and human (means ± SEM, n ≥ 4). Produced from data published in Tee et al. (1987).
Fig. 3
Fig. 3. Single pulse 1H NMR spectra of pooled urine from (a) control rats, (b) rats fed APAP in the diet (1%, 10 weeks) and (c) diet containing both APAP and methionine (both 1%). Key P, PS and PG = n-acetyl signals for APAP, APAP sulphate and APAP glucuronide respectively. PNC is the resolved n-acetyl on the side chain of the mercapturate. Reproduced from Ghauri et al. (1993).
Fig. 4
Fig. 4. Schematic of computational model of glutathione homeostasis. The network embraces pathways of methionine catabolism, glutathione metabolism, 5-oxoproline and ophthalmic acid synthesis and glutathione mediated detoxification. Variable metabolites are indicated by blue circles, fixed metabolites are indicated by green circles, enzymes are shown in white rectangles and rate numbers are shown in yellow squares. The shaded area refers to the intracellular space, while the prefixes ‘c’ and ‘b’ denote substances in the cell and the blood, respectively. The enzymes which catalyze the numbered reactions are: v[9]-mati-methionine adenosyl transferase i-2516; v[10]-matiii-methionine adenosyl transferase iii-2516; v[11]-meth-glycine n-methyltransferase-21120; v[12]-gnmt-dnamethyltransferase-21172; v[13]-ah-s-adenosyl-homocysteine hydrolase-3311; v[14]-bhmt-betaine-homocysteine methyltransferase-2115, v[15]-ms-methionine synthase-21113; v[16]-cbs-cystathionine gamma-synthase-42122; v[17]-ctgl-cystathionase-4411; v[18]-gcs-glutamylcysteine synthetase-6322; both v[19] and v[27]-gs-glutathione synthetase-6323; v[20]-gpx-glutathione peroxidase-11119; v[21]-gr-glutathione reductase-1817; v[26]-ggct-gamma-glutamylcyclotransferase-2324; v[25]-op-5-oxoprolinase-3529, v[27]-gcsglutamylcysteine synthetase-6322; v[31]-ap-aminopeptidase-34112; v[33]-ggct-gamma-glutamylcyclotransferase-2324; v[34]-gpx-glutathione s-transferase-25118; v[35]-gpx-glutathione s-transferase-25118. Metabolites assumed to be present at variable concentrations are: met—methionine; SAM—s-adenosyl-methionine; SAH—s-adenosylhomocysteine; hcy—homocysteine; cyt—cystathionine, ccys—cytosolic cysteine; bcys—blood cysteine; glc—glutamyl-cysteine; cGSH—cytosolic glutathione; bGSH—blood glutathione; cGSSG—cytosolic glutathione disulfide; bgssg—blood glutathione disulfide; cgly—cytosolic glycine; cglut—cytosolic glutamate; opa—ophthalmic acid; n—[n-(γ-glutamyl)-α-aminobutyryl]glycine; oxo—oxoproline = pyroglutamic acid; asg—acetaminophen glutathione adduct; gluab—glutamyl aminobutyrate; bgluaa—blood glutamyl amino acid; gln—glutamine; prot—protein. Metabolites assumed to be present at fixed concentrations are: ab—2-aminobutyrate; bet—betaine; bgly—blood glycine; bglut—blood glutamate; bmet—blood methionine; CNADPH—nicotinamide adenine dinucleotide phosphate; cser —cytosolic serine; H2O2—cellular hydrogen peroxide; bopa—blood ophthalmic acid; n-[n-(γ-glutamyl)-α-aminobutyryl]glycine; boxo—blood oxoproline, pyroglutamic acid; basg—blood acetaminophen glutathione adduct; para—paracetamol (acetaminophen). Reproduced from Geenen et al. (2013).
Fig. 5
Fig. 5. Paracetamol mechanistic complexity offers new opportunities for biomarker and therapeutic development. Based on decades of preclinical and clinical evidence, the mechanism of paracetamol induced hepatotoxicity has been largely defined. Key tipping points have been identified that modulate the overall response and may serve as crucial targets for therapeutic intervention. This include bioactivation of paracetamol to naqpi (1), mitochondrial dysfunction (2), cell death mode dynamics (3) and the balance between inflammation resulting in either enhanced injury or hepatic regeneration (4). Recently, it has been shown that these key events can also be reported by circulating biomarkers that offer improved diagnosis, prediction and prognostication during paracetamol toxicity. For example, monitoring APAP and GSH adducts may serve to report bioactivation, GLDH can be considered a marker of mitochondrial injury, cell death mode dynamics can be reported with kertin-18/caspase cleaved keratin-18 release and the balance between pro-inflammatory and pro-regenerative events can be assessed by HMGB1-acetyl and CSF-1 respectively.
Fig. 6
Fig. 6. Left: Still from a video showing neutrophil migration into the centrilobular region 8 hours after APAP dosing in a wild type mouse. Rhodamine 123 (cyan) is sequestered in mitochondria with an intact membrane potential and thus, stains viable hepatocytes. Black areas show an absence of rhodamine 123 staining in non-viable hepatocytes. Anti-Ly6G antibody (red) shows migration of neutrophils towards and their accumulation within the area of necrosis. Right: Still from a video showing mature inflammatory infiltrate around the centrilobular area 72 hours after APAP in a transgenic Cx3cr1gfp/+ Ccr2rfp/+ reporter mouse. Kupffer cells (blue, anti-f4/80) line the sinusoids throughout the lobule. Large monocyte derived macrophages with mixed CCR2 (red) Cx3cr1 (green) f4/80 (blue) signal are seen in the centrilobular region. CCR2 positive monocytes scan the sinusoids.
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