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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: (1) microRNA-122 (miR-122) expression (a hepatic marker), (2) free GSH concentration (a marker of oxidative stress) and (3) 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 tailtissue, 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 a significantly higher amount of 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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30282005
???displayArticle.link???Toxicol Lett ???displayArticle.grants???[+]
NC/L001659/1 National Centre for the Replacement, Refinement and Reduction of Animals in Research
Fig. 1. Paracetamol exposure dose-response phenotype
Xenopus laevis embryos were exposed to a paracetamol concentration in the range 0-5 mM (n = 4). The embryos were exposed to paracetamol from stage 38 and harvested at stage 45. Phenotypes include wildtype (A), damaged tail (B), oedema (C), abnormal gut (D) and bent tail (E). The surviving embryos at stage 45 were photographed and the different phenotypes found for each paracetamol concentration were calculated as a percentage of total embryos tested (F). Where possible, the embryos were photographed to show the lateral view and the ventral view. Scale bar represents 1 mm.
Fig. 2. The amount of free GSH inside Xenopus embryos treated with paracetamol.
Xenopus laevis embryos were exposed to 0-5 mM paracetamol from the age of stage 38, and harvested at stage 45 (A, n = 10) or at stage 41 (B, n = 3). The amount of free GSH (nmol/mL) was measured inside the embryos and normalised to the amount of protein (mg/mL). An ordinary one-way ANOVA compared each exposure group to the group exposed to Xenopus media alone (0 mM). For the embryos exposed until stage 45 (A), the amount of free GSH for 3, 3.5, 4 and 4.5 mM paracetamol exposure groups was statistically significant compared to the group exposed to Xenopus media only (P < 0.05). The 5 mM paracetamol exposure group was also statistically significantly different (P < 0.01). There was no significant difference between treated and untreated embryos that were harvested at stage 41 (B).
Fig. 3. Wholemount in situ hybridisation (WISH) for liver markers.
WISH assay for Alpha-1-Microgobulin/Bikunin Precursor (AMBP) (A) and miR-122 (B) at stage 38 (Ai and Bi) and stage 45 (Aii and Bii). The stage 38 embryos are shown in lateral view and the stage 45 embryos are shown ventrally. The specific purple WISH stain is indicated with an arrow. These embryos are representatives of the typical expression patterns seen (n = 10). Scale bar represents 1 mm.
Fig. 4. Expression of miR-122 in paracetamol-treated embryos.
Stage 38 embryos were exposed to paracetamol (0-5 mM) and harvested at stage 45. The expression of miR-122 in the gut (purple) and tail (red) was measured using qRT-PCR (log (fold change ± SEM)) and normalised to embryos exposed to Xenopus media only (0 mM). The statistically significant difference of miR-122 expression between tissues from embryos treated with the same paracetamol concentration was measured using the Mann-Whitney test (P < 0.05) (n = 5).
Fig. 5. The amount of free GSH inside Xenopus embryos treated with paracetamol (APAP) or N-acetyl-meta-aminophenol (AMAP).
Xenopus laevis embryos were exposed to up to 5 mM paracetamol (APAP) or up to 5 mM N-acetyl-meta-aminophenol (AMAP) from the age of stage 38 and harvested at stage 45 (n = 3). The amount of free GSH (nmol/mL) was measured inside the embryos and normalised to the amount of protein (mg/mL). There was no significant difference between embryos treated with the same concentration of APAP and AMAP.
Fig. 6. Free GSH in embryos treated with paracetamol and N-acetyl cysteine (NAC).
Stage 38 embryos were exposed to paracetamol (up to 5 mM) for 72 h with or without the addition of 0.5 mM NAC, they were harvested at stage 45. For the initial experiments (A) there were 3 conditions: exposure to paracetamol alone (blue), 2 h incubation with NAC prior to the addition of paracetamol (pink) and concurrent treatment with NAC and paracetamol (yellow) (n = 10). The embryos were also exposed to APAP from stage 38 and then with NAC for 24 h prior to harvest (green) at stage 45, a 72 h incubation period, or with paracetamol alone (blue) (B) (n = 5). The amount of free GSH inside the embryos was measured and normalised to the amount of protein. This value was then normalised to the embryos exposed to Xenopus media alone (0 mM). The amount of free GSH measured was calculated as a percentage of the 0 mM paracetamol exposure group (±SEM). A multiple comparison 2 way ANOVA was performed to measure the statistical significance between groups of them same paracetamol exposure, with and without additional NAC exposure (P < 0.05).
