Toxicity Evaluation and Biomarker Selection with Validated Reference Gene in Embryonic Zebrafish Exposed to Mitoxantrone

• Main Authors: Lili Liu, Hua Zhu, Yanchun Yan, Peng Lv, Wei Wu
• Format: Article
• Language: English
• Published: MDPI AG 2018-11-01
• Series: International Journal of Molecular Sciences
• Subjects: reference genes; stability evaluation; zebrafish embryos; mitoxantrone; toxicity evaluation; biomarker selection
• Online Access: https://www.mdpi.com/1422-0067/19/11/3516

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 <i>gapdh</i>, followed by <i>tub&#945;1b</i>, for the 48 h post fertilization (hpf) zebrafish embryo mitoxantrone test, while both <i>eef1a1l1</i> and <i>rpl13&#945;</i> were recommended as reference genes for the 96 hpf zebrafish embryo mitoxantrone test. With <i>gapdh</i> as an internal control, we analyzed the mRNA levels of representative hepatotoxicity biomarkers, including <i>fabp10a</i>, <i>gclc</i>, <i>gsr</i>, <i>nqo1</i>, cardiotoxicity biomarker <i>erg</i>, and neurotoxicity biomarker <i>gfap</i> in the 48 hpf embryo mitoxantrone test. The mRNA levels of <i>gclc</i>, <i>gsr</i>, and <i>gfap</i> increased significantly in 10 and 50 &#956;g/L mitoxantrone-treated 48 hpf embryos, while the transcript levels of <i>fabp10a</i> decreased in a dose-dependent manner, indicating that mitoxantrone induced hepatotoxicity and neurotoxicity. Liver hematoxylin&#8315;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 <i>fabp10a</i> is recommended as a potential biomarker for hepatotoxicity in zebrafish embryos. Additionally, <i>gapdh</i> is proposed as a reference gene for the 48 hpf zebrafish embryo mitoxantrone toxicity test, while <i>eef1a1l1</i> and <i>rpl13&#945;</i> are proposed as that for the 96 hpf test.