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...
Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.
1617 The intrinsic signaling pathways regulating hematopoietic stem cells (HSC) are increasingly well recognized. However, less is known about how in utero exposure to common environmental xenobiotic compounds may alter HSC development and increase the risk of carcinogenesis. RUNX1 (AML1), required for definitive HSC induction in all vertebrates, is the target of frequent chromosomal alterations associated with leukemia. Through a chemical genetic screen for modifiers of runx1 expression in the zebrafish, estrogen-related compounds were identified. Here, we found that exposure to 17β-estradiol (E2) throughout the initial waves of hematopoietic development (5 somites (som) to 36 hours post fertilization (hpf)) significantly altered the number of runx1+ HSCs in the zebrafish Aorta-Gonad-Mesonephros Region (AGM) compared to controls (n≥25-50 embryos /condition). Other physiological estrogens, such as estrone and estriol, elicited a similar hematopoietic response. However, treatment with either the isomer 17α-estradiol, or the related steroid hormones testosterone or progesterone, could not mimic the effect of E2 on HSCs. Use of the aromatase inhibitor anastrozole and the pan-estrogen receptor inhibitor fulvestrant confirmed that estrogen was both required for nascent HSC regulation and functioned through classical estrogen receptor (esr) signaling. Microarray analysis of FACS-sorted cell populations during zebrafish development demonstrated differential spatio-temporal regulation of esr1 (esrα) and esr2a/b (esrβ) in vascular and hematopoietic cell types. During the primitive wave of hematopoiesis, exposure to E2 and the esr1-agonist PPT significantly enhanced red blood cell number as seen by in situ hybridization for embryonic globin (hbbe3) and quantified by fluorescent microscopy and FACS analysis of the Tg(globin:GFP) line. Conversely, the esr2-specific agonist DPN diminished definitive HSC formation after exposure from 5 som to 24 hpf; this phenotype was mediated by disruption of vessel formation, as indicated by flk1 (kdrl) expression, and alteration in the assignment of artery-vein identity. Interestingly, when exposure to E2 or DPN occurred from 24 – 36 hpf, after the establishment of ephb2+ arteries and the initiation of blood flow, estrogen treatment enhanced HSC formation; this was confirmed by FACS analysis and fluorescent microscopy using the Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP) HSC-reporter lines. E2 treatment was found to elicit both pro-apoptotic (TUNEL+) and pro-proliferative (BrdU+) effects on HSCs and the vascular niche depending on the timing of exposure, but independent of the concentration of E2 over the physiological range and above (10nM to 10mM). Morpholino-mediated gene knockdown of esr1 and the two esr2 alleles alone and in combination with E2 confirmed that esr2 was responsible for the effects on definitive hematopoiesis. Using the Tg(TOP:GFP)w25 line, alterations in estrogen signaling were shown to mediate effects on wnt activity. To determine whether exposure to environmental estrogens could mediate similar alterations in HSC specification and proliferation, we exposed embryos to the phytoestrogen genistein, the synthetic estrogen ethinylestradiol, and the xenoestrogen bisphenol A (BPA) and found results reminiscent of E2; using fulvestrant, we confirmed that the phenotype elicited by each was dependent on estrogen receptor stimulation. In an adult zebrafish marrow injury model, E2 significantly enhanced stem and progenitor cell regeneration in males and females by day 10 post irradiation (n≥10 /condition). Intriguingly, we found that females, with higher circulating estrogen levels, recovered better after injury than male siblings, both in the presence and absence of exogenous estrogen. Finally, murine bone marrow treated with E2 or DPN produced significantly (n=10 /condition, p<0.0001) higher numbers of spleen colonies at day 12 post-transplantation than vehicle-only controls, demonstrating functional conservation of estrogenic regulation of HSCs/progenitor cells. These data identify stage-specific, differential roles for estrogen during hematopoiesis, highlighting the potent impact of environmental exposure to estrogenic compounds on blood formation and revealing potential therapeutic options for the treatment of bone marrow failure and leukemia. (equal contribution: KJC, MCD; WG, TEN). Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North:Fate Therapeutics: Consultancy, Patents & Royalties.
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