The Acute Toxicity And Molecular Mechanism Of Mitoxantrone To Zebrafish Embryo

There is growing concern about ecotoxicity of non-target aquatic vertebrates exposed to cytotoxic drugs.Antineoplastic mitoxantrone,a typical cytotoxic drug,suppress tumor cell growth via inhibition of DNA replication and RNA transcription.In present research,acute developmental toxicity and mechanism of antitumor drug mitoxantrone hydrochloride(MA)were analyzed on non-target aquatic vertebrates with embryonic zebrafish as animal model.The results could provide a basis for the evaluation of ecotoxicity and management of environmental hazards of cytotoxic drugs.Our results indicated that MA was lethal to embryonic zebrafish during acute exposure,especially in the time before 24 hpf(hours postfertilization).The value of 96 h medium lethal concentration was880 μg/L,meaning MA high toxic to zebrafish.The death peak occurred in early development stages(24 hpf).Early developmental embryo zebrafish exposure to MA exhibited severe teratogenic effect such as post vent decreased length,tail defect,no tail and spinal curvature.Acridine orange staining confirmed the speculation.In additon,the expression levels of genes related with early tail development and P53-mediated apoptosis pathway were upregulated,suggesting MA-induced cell apoptosis which contributed to tail malformation,but tail development related genes overexpression as compensatory effect.The results of target organ toxicity showed that MA induced embryo hepatotoxicity.Pathology appearance incuding irregularly and loosely arranged liver tissue,vacuolization and hepatocyte swelling were clearly visible with HE staining.The activity of antioxidant enzymes(SOD,GST,CAT)and content of MDA were significantly changed in 72 and 96 hpf embryo exposed to MA,which were important symbols of oxidative stress and lipid peroxidation.The lipid peroxidation might be contributing to hepatotoxicity induced by MA.Neurotoxicity was also observed with decreased spontaneous movement.The expression of nervous system development related genes bdnf-v2、gap43、hand2、nkx2.2a-v1、neurog、shha and syn2 a were down regulated,indicating MA participated in inhibiting the embryonic neural development,especially growth of neurons and synapses formation.Immediately early genes fos 、 jun and myc were found participating in cell apoptosis and neurotoxicity mechanism.Extracellular MA was transported into cytoplasm via ABC transporter.MA inserted into double strands DNA and caused DNA damage,activating protein Fos and Jun.Fos and Jun binded as dimer protein AP-1.With binding to target genes,AP-1 induced cell proliferation and differentiation in nervous system.Jun protein also activated cell apptosis pathway erther by interacting with DNA or by activating P53 pathway.RNA sequencing techniques was further analyzed for the expression profile of 24 hpf and 48 hpf zebrafish embryo exposure to MA.We identified 6 456 lncRNA.The target genes of different expressed lncRNA were enriched in the pathway of apoptosis and cell cycle,suggesting that lnc RNA might paticipate in apoptosis and cell cycle induced by MA.32 849 circRNA were indentified for the first timein embryonic zebrfish.KEGG enrichment found that the origin genes of differentially expressed circ RNA might participate in neurotoxicity signaling pathway and cell prolifation.