Developmental toxicity of Clerodendrum cyrtophyllum turcz ethanol extract in zebrafish embryo

Abstract

Clerodendrum cyrtophyllum Turcz has been used in traditional medicine for the treatment of various diseases. In spite of its therapeutic applications, research on its toxicity and teratogenicity is still limited. Aim of the study: The study aimed to investigate the developmental toxicity of the ethanol extract of C. cyrtophyllum (EE) in zebrafish embryo model. Material and methods: Major compounds from crude ethanol extract of Clerodendron cyrtophyllum Turcz leaves were determined using HPLC-DAD-Orbitrap-MS analysis. The developmental toxicity of EE were investigated using zebrafish embryo model. Zebrafish embryos at 6 h post-fertilization (hpf) were treated with EE at different concentrations. Egg coagulation, mortality, hatching, yolk sac edema, pericardial edema and teratogenicity were recorded each day for during a 5-day exposure. At time point 120 hpf, body length, pericardial area, heartbeat and yolk sac area were assessed. In order to elucidate molecular mechanisms for the developmental toxicity of EE, we further evaluated the effects of the EE on the expression of genes involved on signaling pathways affecting fish embryo's development such as heart development (gata5, myl7, myh6, has2, hand2, nkx 2.5), oxidative stress (cat, sod1, gpx4, gstp2), wnt pathway (β-catenin, wnt3a, wnt5, wnt8a, wnt11), or cell apoptosis (p53, bax, bcl2, casp3, casp8, casp9, apaf-1, gadd45bb) using qRT-PCR analysis. Results: Our results demonstrated that three major components including acteoside, cirsilineol and cirsilineol-4'-O-β-D-glucopyranoside were identified from EE. EE exposure during 6-96 h post-fertilization (hpf) at doses ranging from 80 to 200 μg/mL increased embryo mortality and reduced hatching rate. EE exposure at 20 and 40 μg/mL until 72-120 hpf induced a series of malformations, including yolk sac edema, pericardial edema, spine deformation, shorter body length. Based on two prediction models using a teratogenic index (TI), a 25% lethality concentration (LD25) and the no observed-adverse-effect level (NOAEL), EE is considered as teratogenic for zebrafish embryos with TI (LC50/EC50) and LD25/NOAEC values at 96 hpf reaching 3.87 and 15.73 respectively. The mRNA expression levels of p53, casp8, bax/bcl2, gstp2, nkx2.5, wnt3a, wnt11, gadd45bb and gata5 were significantly upregulated by EE exposure at 20 and 40 μg/mL while the expression of wnt5, hand2 and bcl2 were downregulated.

The impact of differential lignin S/G ratios on mutagenicity and chicken embryonic toxicity

“…Moreover, a significantly increased MDA level, as a biomarker of lipid peroxidation, in the liver sample, was observed in the same group compared with the control (Kurantowicz et al, 2017). The one potential mechanism of adverse effects on embryonic development was related to oxidative stress damage (Nguyen et al, 2020). For the S/G = 2 treatments, the high mortality and malformation rates did not exist with alternated other developmental indexes or increased MDA level, which suggested that other mechanisms might play roles, such as anti‐angiogenic or apoptosis (Beedie et al, 2016).…”

Risk compounds, potential mechanisms and biomarkers of traditional Chinese medicine‐induced reproductive toxicity

“…Increased mortality and malformations, decreased body length and a delayed hatching period were related to XAP‐induced activation of the Wnt pathway and are associated with altered mRNA expression including β‐catenin, wnt3a, wnt8a and wnt11 (Li, Zhang, Liu, et al, 2018). Ethanol extract of C. cyrtophyllum induces toxicity of embryonic heart development in zebrafish that are associated with the Wnt pathway, including β‐catenin, wnt3a, wnt5, wnt8a and wnt11 (Nguyen et al, 2021). Protein kinases are a series of enzymes catalysing the process of protein phosphorylation.…”

“…XAP induced developmental toxicity through increased expression levels of oxidative stress‐related genes (cat, sod‐1 and gstp2), Wnt pathway‐related genes (β‐catenin, wnt3a and wnt8a) and Bcl‐2 (Li, Zhang, Liu, et al, 2018). Developmental toxicity of Clerodendrum cyrtophyllum Turcz is related to the up‐regulated expression of p53, caspase‐8, Bax/Bcl‐2, gstp2, nkx2.5, wnt3a, wnt11, gadd45bb and gata5, along with down‐regulating wnt5, hand2 and Bcl‐2 (Nguyen et al, 2021). Isoliquiritigenin could trigger oxidative stress‐induced apoptosis by down‐regulating genes like Nrf2, HO‐1, SOD‐1 and SOD‐2 (Song et al, 2020).…”

Insight into Lotusine and Puerarin in Repairing Alcohol-Induced Metabolic Disorder Based on UPLC-MS/MS

“…We selected zebrafish as an insensitive platform to investigate metabolic variation. Zebrafish, a small tropical freshwater fish, have been widely used in pharmacology and toxicology in recent years as a model organism with high homology to the human genome [ 24 , 25 ]. In contrast to rodents, zebrafish embryos are transparent, and their major organs are similar to those of mammals.…”