Co-Treatment of Copper Oxide Nanoparticle and Carbofuran Enhances Cardiotoxicity in Zebrafish Embryos

Abstract

The use of chemicals to boost food production increases as human consumption also increases. The insectidal, nematicidal and acaricidal chemical carbofuran (CAF), is among the highly toxic carbamate pesticide used today. Alongside, copper oxide nanoparticles (CuO) are also used as pesticides due to their broad-spectrum antimicrobial activity. The overuse of these pesticides may lead to leaching into the aquatic environments and could potentially cause adverse effects to aquatic animals. The aim of this study is to assess the effects of carbofuran and copper oxide nanoparticles into the cardiovascular system of zebrafish and unveil the mechanism behind them. We found that a combination of copper oxide nanoparticle and carbofuran increases cardiac edema in zebrafish larvae and disturbs cardiac rhythm of zebrafish. Furthermore, molecular docking data show that carbofuran inhibits acetylcholinesterase (AChE) activity in silico, thus leading to impair cardiac rhythms. Overall, our data suggest that copper oxide nanoparticle and carbofuran combinations work synergistically to enhance toxicity on the cardiovascular performance of zebrafish larvae.

Keywords: CuO nanoparticle; carbofuran; cardiotoxicity; molecular docking; zebrafish.

Figure A1

Schematic diagram shows the experimental design for testing cardiac toxicity of CuO nanoparticles and carbofuran on zebrafish larvae. Embryos were exposed to CuO nanoparticles and carbofuran from 24 hpf (hour post-fertilization) onwards and the cardiac performance were assessed at 72 hpf.

Figure A2

General Ramachandran plot for acetylcholinesterase model for structural assessment. The Ramachandran plot is a plot of the torsional angles (phi (φ) and psi (ψ)) of the residues (amino acids) contained in a peptide.

Figure A3

Typical cardiac morphology after exposed to carbofuran in zebrafish embryos aged at 72 hpf (hour post-fertilization). Zebrafish have larger cardiac chamber size at different severity which is the characteristic of cardiac edema (A–C) compared to the control (D).

Figure A4

Poincare map of Zebrafish embryos after exposed to CuO nanoparticle and carbofuran. (A) Control, (B) CuO 0.6 mM + CAF 0 uM, (C) CuO 0 mM + CAF 9 uM, (D) CuO 0.6 mM + CAF 9 uM. (CuO = copper oxide nanoparticle (mM), CAR = carbofuran (µM)).

Figure 1

Analysis of physical properties of copper oxide nanoparticles. (A) X-ray diffraction (XRD) analysis; (B) Raman spectrum; (C) Fourier-transform infrared (FTIR) spectrum; (D) UV-Vis absorption spectrum; (E) Transmission electron Microscopy (TEM) observation and energy-dispersive X-ray spectroscopy (EDS); (F) Dynamic light scattering (DLS) analysis; (G) Zeta potential measurement.

Figure 2

Mortality rate of zebrafish embryos after incubation in several concentration of either (A) CuO nanoparticles, (B) carbofuran or (C) combination of both compounds (CuO = mM, CAF = µM).

Figure 3

Cardiac physiology alteration induced by CuO nanoparticle exposure in zebrafish larvae. (A) Stroke volume. (B) Heart rate of atrium. (C) Ejection fraction. (D) Cardiac output. (E) Heart rate of ventricle. (F) Shortening fraction. Data were statistically analyzed using ordinary one-way ANOVA with the Dunnett post-hoc test for multiple comparisons. * p < 0.05, *** p < 0.001.

Figure 4

Cardiac rhythm alteration induced by CuO nanoparticle exposure in zebrafish larvae. (A) Atrium SD1. (B) Ventricle SD1. (C) Atrium–ventricle interval. (D) Atrium SD2. (E) Ventricle SD2. (F) Ventricle–atrium interval. Data were statistically analyzed using a Brown–Fosythe one-way ANOVA with Dunnett post-hoc test for multiple comparisons. * p < 0.05, ** p < 0.005.

Figure 5

Cardiac physiology alteration induced by carbofuran in zebrafish larvae. (A) Stroke volume. (B) Heart rate of atrium. (C) Ejection fraction. (D) Cardiac output. (E) Heart rate of ventricle. (F) Shortening fraction. Data were statistically analyzed using ordinary one-way ANOVA with a Dunnett post-hoc test for multiple comparisons. * p < 0.05, ** p < 0.005.

Figure 6

Cardiac rhythm alteration induced by carbofuran in zebrafish larvae. (A) Atrium SD1. (B) Ventricle SD1. (C) Atrium–ventricle interval. (D) Atrium SD2. (E) Ventricle SD2. (F) Ventricle–atrium interval. Data were statistically analyzed using a Brown–Fosythe one-way ANOVA with a Dunnett post-hoc test for multiple comparisons. * p < 0.05.

Figure 7

Cardiac physiology alteration induced by CuO nanoparticle and carbofuran co-incubation in zebrafish larvae. (A) Stroke volume. (B) Heart rate of atrium. (C) Ejection fraction. (D) Cardiac output. (E) Heart rate of ventricle. (F) Shortening fraction. Data were statistically analyzed using the ordinary one-way ANOVA test with the Dunnett post-hoc test for multiple comparison analysis. * p < 0.05. (CuO = copper nanoparticle (mM), CAF = carbofuran (µM)).

Figure 8

Cardiac rhythm alteration induced by CuO nanoparticle and carbofuran co-incubation in zebrafish larvae. (A) Atrium SD1. (B) Ventricle SD1. (C) Atrium–ventricle interval. (D) Atrium SD2. (E) Ventricle SD2. (F) Ventricle–atrium interval. Data were statistically analyzed using the Kruskal–Wallis ANOVA test with Dunn’s post-hoc test for multiple comparison * p < 0.05, ** p < 0.005. (CuO = copper nanoparticle (mM), CAF = carbofuran (µM)).

Figure 9

Molecular docking poses comparing the binding mechanism of ACh and carbofuran to AChE chain A. (A) Full view of the ACh-AChE complex, (B) expanded view of ACh’s binding site with interacting residues, (C) full view of the carbofuran-AChE complex, (D) expanded view of carbofuran’s binding site with interacting residues.

Figure 10

Proposed model for cardiotoxicity triggered by CuO nanoparticle and carbofuran co-incubation in zebrafish larvae (A = atrium, V = ventricle). The solid line indicates direct evidence provide by our molecular docking data, while dotted line indicates indirect evidence collected from literature.