Paracetamol-induced liver injury modelled in Xenopus laevis embryos

Katy Saide, Victoria Sherwood, Grant N. Wheeler (Lead / Corresponding author)

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Abstract

Introduction: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC).

Method: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention.

Results: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively).

Conclusion: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research. 

Original languageEnglish
Pages (from-to)83-91
Number of pages9
JournalToxicology Letters
Volume302
Early online date30 Sep 2018
DOIs
Publication statusPublished - 1 Mar 2019

Fingerprint

Xenopus laevis
Acetaminophen
Liver
Embryonic Structures
Wounds and Injuries
Acetylcysteine
Xenopus
Cysteine
Lead compounds
Chemical and Drug Induced Liver Injury
Pharmaceutical Preparations
MicroRNAs
Mammals
Oxidative stress
Imines
Metabolites
Glutathione
Animals
Tail
Antioxidants

Keywords

  • Drug-induced liver injury (DILI)
  • Paracetamol
  • Pre-clinical studies
  • Xenopus laevis

Cite this

Saide, Katy ; Sherwood, Victoria ; Wheeler, Grant N. / Paracetamol-induced liver injury modelled in Xenopus laevis embryos. In: Toxicology Letters. 2019 ; Vol. 302. pp. 83-91.
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title = "Paracetamol-induced liver injury modelled in Xenopus laevis embryos",
abstract = "Introduction: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC).Method: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention.Results: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively).Conclusion: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research. ",
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Paracetamol-induced liver injury modelled in Xenopus laevis embryos. / Saide, Katy; Sherwood, Victoria; Wheeler, Grant N. (Lead / Corresponding author).

In: Toxicology Letters, Vol. 302, 01.03.2019, p. 83-91.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Paracetamol-induced liver injury modelled in Xenopus laevis embryos

AU - Saide, Katy

AU - Sherwood, Victoria

AU - Wheeler, Grant N.

N1 - Article processing charges were paid from the Medical Research Council contribution to the RCUK block grant to the University of East Anglia.

PY - 2019/3/1

Y1 - 2019/3/1

N2 - Introduction: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC).Method: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention.Results: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively).Conclusion: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research. 

AB - Introduction: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC).Method: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention.Results: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively).Conclusion: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research. 

KW - Drug-induced liver injury (DILI)

KW - Paracetamol

KW - Pre-clinical studies

KW - Xenopus laevis

U2 - 10.1016/j.toxlet.2018.09.016

DO - 10.1016/j.toxlet.2018.09.016

M3 - Article

C2 - 30282005

VL - 302

SP - 83

EP - 91

JO - Toxicology Letters

JF - Toxicology Letters

SN - 0378-4274

ER -