PGE2-regulated wnt signaling and N -acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury

Abstract

Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage.acetaminophen liver toxicity | chemical screen A cetaminophen (N-acetyl-p-aminophenol; APAP) is a commonly used analgesic and antipyretic. Although safe at therapeutic doses, accidental or suicidal drug overdose can cause dosedependent liver damage. APAP is the most common cause for liver transplantation for toxin-induced fulminant hepatic failure and results in more than 300 deaths annually in the United States (1). The only antidote in clinical use is N-acetylcysteine (NAC), which reduces mortality by ∼20-28% (2); however, the time interval of effective intervention after ingestion is typically <12 h, and delayed or prolonged treatment can negatively impact clinical outcome and survival (3). Therapeutic options for hepatic failure are limited to best supportive care and liver transplantation.APAP toxicity results from a hepatotoxic metabolite, N-acetylp-benzoquinone imine (NAPQI), produced by the cytochrome P450 enzymes CYP1A2, 2E1, and 3A4 (4). At therapeutic doses, NAPQI is efficiently inactivated in the liver by glutathione (GSH) conjugation (5). At toxic doses, excess production of NAPQI depletes hepatic GSH. Unconjugated NAPQI causes dysfunction of critical liver proteins, oxidative stress, and mitochondrial damage (6-8). NAC is a true antidote and limits damage by repleting GSH. Clinical efficacy of NAC treatment was shown for patients presenting after APAP ingestion; however, no conclusive clinical trials were conducted to elucidate its efficacy and optimal treatment window. Therapeutic benefits have been shown in mammalian models for other antioxidants (9, 10) that function to restore GSH levels. Compounds that support liver recovery from AP...

Critical function for the Ras-GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis

“…We exposed zebrafish control and rasa3 morphant embryos from the Tg(globin LCR:eGFP) transgenic line to N-acetyl cysteine (NAC), a potent antioxidant, reactive oxygen scavenger (25,26). There was no increase in the number of GFP + erythrocytes after NAC treatment compared with the −NAC treatment cohorts among rasa3 morphants (Fig.…”

In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

“…For example, zebrafish are proving useful for alcohol research (Jang et al, 2012;Monk et al, 2013;North et al, 2010;Passeri et al, 2009;Tsedensodnom et al, 2013;Yin et al, 2012). By using transgenic zebrafish expressing a GFP-tagged secreted glycoprotein in hepatocytes Tsedensodnom et al, 2013;Xie et al, 2010), and zebrafish that express fluorescent protein markers of the hepatocyte secretory organelles and of other cells in the liver (Yin et al, 2012), the mechanisms by which alcohol and other drugs cause organ-specific and organelle-mediated toxicity can be uniquely addressed.…”

Using zebrafish as the model organism to understand organ regeneration

“…Additionally, the topoisomerase 2a, Wnt, Fgf and Bmp signaling pathways [3,84] play roles. Recently, through the chemical screen, PGE2 was identified as a hepatoprotective compound that limited liver damage [84,86], and this function is mediated by the interaction between PGE2 and Wnt signaling [84,87]. Treatment of a novel GSNOR inhibitor (GSNORi) after PHx in adult fish enhanced cellular proliferation and regeneration [3, 84,88].…”