Dataset Information

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

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 tub?1b, for the 48 h post fertilization (hpf) zebrafish embryo mitoxantrone test, while both eef1a1l1 and rpl13? 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 ?g/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 rpl13? are proposed as that for the 96 hpf test.

SUBMITTER: Liu L

PROVIDER: S-EPMC6274943 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Liu Lili L Zhu Hua H Yan Yanchun Y Lv Peng P Wu Wei W

International journal of molecular sciences 20181108 11

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>, followe ...[more]

PMID: 30413070

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Project description:BackgroundPer- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS.ObjectiveWe aimed to group PFAS based on shared toxicity phenotypes.MethodsZebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0-5 d post fertilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated.ResultsPFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity.ConclusionsAll emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals. https://doi.org/10.1289/EHP5843.

Project description:Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

Dataset Information

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

Publications

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

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