Abstract
Notwithstanding the widespread use and promising clinical value of chemotherapy, the pharmacokinetics, toxicology, and mechanism of mitoxantrone remains unclear. To promote the clinical value in the treatment of human diseases and the exploration of potential subtle effects of mitoxantrone, zebrafish embryos were employed to evaluate toxicity with validated reference genes based on independent stability evaluation programs. The most stable and recommended reference gene was gapdh, followed by tubα1b, for the 48 h post fertilization (hpf) zebrafish embryo mitoxantrone test, while both eef1a1l1 and rpl13α were recommended as reference genes for the 96 hpf zebrafish embryo mitoxantrone test. With gapdh as an internal control, we analyzed the mRNA levels of representative hepatotoxicity biomarkers, including fabp10a, gclc, gsr, nqo1, cardiotoxicity biomarker erg, and neurotoxicity biomarker gfap in the 48 hpf embryo mitoxantrone test. The mRNA levels of gclc, gsr, and gfap increased significantly in 10 and 50 μg/L mitoxantrone-treated 48 hpf embryos, while the transcript levels of fabp10a decreased in a dose-dependent manner, indicating that mitoxantrone induced hepatotoxicity and neurotoxicity. Liver hematoxylin–eosin staining and the spontaneous movement of embryos confirmed the results. Thus, the present research suggests that mitoxantrone induces toxicity during the development of the liver and nervous system in zebrafish embryos and that fabp10a is recommended as a potential biomarker for hepatotoxicity in zebrafish embryos. Additionally, gapdh is proposed as a reference gene for the 48 hpf zebrafish embryo mitoxantrone toxicity test, while eef1a1l1 and rpl13α are proposed as that for the 96 hpf test.
Highlights
Zebrafish is a recent addition to vertebrate models of human disease and drug screening, rapidly contributing major insights into these fields
More than 75% of human genes implicated in disease have counterparts in zebrafish, providing an opportunity to analyze their roles in this model system [3]
With diluted cDNA of embryonic zebrafish (24 and 48 hpf) from control and mitoxantrone-treated groups as templates, the Quantitative real-time PCR (qPCR) assay was carried out for transcript abundance based on quantification cycle (Cq) values
Summary
Zebrafish is a recent addition to vertebrate models of human disease and drug screening, rapidly contributing major insights into these fields. More than 75% of human genes implicated in disease have counterparts in zebrafish, providing an opportunity to analyze their roles in this model system [3]. Zebrafish tumors share conservation of expression profiles at levels different from tumors of humans [4]. The technology developments of transgenes and xenografts give rise to abundant zebrafish models of cancer, including lymphoblastic T-cell leukemia and pancreatic cancer [5]. There is some similarity between zebrafish and humans in terms of the nervous system, the cardiovascular system, and the digestive system. Zebrafish tissues and organs (brain, liver, heart, intestinal, etc.) fully develop within 72 hpf
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