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. 2022 Jul 29;23(15):8438.
doi: 10.3390/ijms23158438.

Cardiotoxicity of Zebrafish Induced by 6-Benzylaminopurine Exposure and Its Mechanism

Mengying Yang  1 , Jialu Luan  1 , Yixin Xu  1 , Chengtian Zhao  1 , Mingzhu Sun  2 , Xizeng Feng  1
Affiliations

Cardiotoxicity of Zebrafish Induced by 6-Benzylaminopurine Exposure and Its Mechanism

Mengying Yang et al. Int J Mol Sci. .

Abstract

6-BA is a common plant growth regulator, but its safety has not been conclusive. The heart is one of the most important organs of living organisms, and the cardiogenesis process of zebrafish is similar to that of humans. Therefore, based on wild-type and transgenic zebrafish, we explored the development of zebrafish heart under 6-BA exposure and its mechanism. We found that 6-BA affected larval cardiogenesis, inducing defective expression of key genes for cardiac development (myl7, vmhc, and myh6) and AVC differentiation (bmp4, tbx2b, and notch1b), ultimately leading to weakened cardiac function (heart rate, diastolic speed, systolic speed). Acridine orange staining showed that the degree of apoptosis in zebrafish hearts was significantly increased under 6-BA, and the expression of cell-cycle-related genes was also changed. In addition, HPA axis assays revealed abnormally expressed mRNA levels of genes and significantly increased cortisol contents, which was also consistent with the observed anxiety behavior in zebrafish at 3 dpf. Transcriptional abnormalities of pro- and anti-inflammatory factors in immune signaling pathways were also detected in qPCR experiments. Collectively, we found that 6-BA induced cardiotoxicity in zebrafish, which may be related to altered HPA axis activity and the onset of inflammatory responses under 6-BA treatment.

Keywords: 6-benzylaminopurine; HPA axis activity; cardiotoxicity; inflammation; zebrafish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
6-BA induced malformation of zebrafish heart development. (A) Cardiac developmental status and fluorescence images of zebrafish larvae at different stages under 6-BA exposure. (B) Schema of the atria and ventricular position. (C) Schematic diagram of measurement area of pericardial edema area. (D) Representative diagram of the measured BA–SV length. (E–G) Statistical analysis of pericardial edema area (E), BA–SV distance (F), and cardiac malformation rate (G) in zebrafish over time. Values are expressed as mean ± standard error (SEM). N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
Abnormal gene expression and cardiac dysfunction in zebrafish exposed to 6-BA at 72 hpf. (A) Diastolic and systolic states of larval hearts under bright field and green fluorescence. (B–D) Statistical results of larval cardiac function parameters, including heart rate, diastolic velocity, and systolic velocity. (E) Image of zebrafish heart after o-dianisidine staining. (F) Quantitative analysis of cardiac staining results. (G) Expression of the myocardial regionalization marker genes myl7, amhc, and vmhc in zebrafish. (H) Expression levels of the atrioventricular canal (AVC) marker genes bmp4, tbx2b, and notch1b in zebrafish. Values are expressed as mean ± standard error (SEM). N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Apoptosis assessment and expression of cycle-related genes in 72 hpf zebrafish. (A) Fluorescence image of zebrafish after acridine orange staining under 6-BA treatment. (B) Quantitative statistics of apoptotic cells in larval hearts after acridine orange staining. Apoptotic cells appear as bright green spots. (C–F) The mRNA expression levels of the apoptosis-related genes bax, bcl2, caspase3, and p53. (G) Expression of cell-cycle-related genes, including ccnd1, ccne1, cdk2, cdk6, c-myc, and gata3. Values are expressed as mean ± standard error (SEM). N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
6-BA induced inflammation in zebrafish at 72 hpf. (A–C) Expression of IL-6, IL1-β, and TNF-α, the hallmark proinflammatory factors of inflammatory response in zebrafish treated with 6-BA. (D–F) The mRNA levels of the landmark anti-inflammatory factors IL-10, IL-4, and receptor IL-4r in zebrafish. (G) Heatmap of inflammatory-response-related genes. Red indicates higher values than the control, and darker colors indicate larger values compared with the control. (H) Relative mRNA expression of stat3, a downstream target gene of IL-4. Values are expressed as mean ± standard error (SEM). N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
Figure 5
6-BA treatment affected HPA axis activity and thigmotaxis behavior in zebrafish. (A–E) Expression patterns of the HPA axis genes crha (A), crhb (B), pomca (C), pomcb (D), and nr3c1 (E) in larval zebrafish after 6-BA exposure. (F) The effect of 6-BA on HPA axis hormone (cortisol) levels in larval zebrafish. (G) Zebrafish exhibited thigmotaxis under 6-BA stimulation, and larvae with only their heads in contact with the petri dish were defined as having positive thigmotaxis. (H) Overall distribution of zebrafish larvae in petri dishes under different treatments. Larvae exhibiting thigmotaxis are indicated by red dots on the edge of the petri dish, and the rest are indicated by blue dots. (I) Statistics of zebrafish larvae thigmotaxis under different treatments. Values are expressed as mean ± standard error (SEM). N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05, ### p < 0.001.
Figure 6
Figure 6
Diagram of the underlying mechanisms by which 6-BA affects zebrafish heart formation and function. 6-BA led to a defective cardiac phenotype that prevented cardiac morphogenesis, allowing abnormal cardiac cyclization and reduced cardiac contractility, thereby affecting cardiac function. In addition, mRNA levels of HPA-axis-related genes and cortisol levels differed significantly compared with controls, which was consistent with the anxious behavior observed in zebrafish at 3 dpf. Signature cytokines of the inflammatory response were also altered by 6-BA induction. In general, the study found that 6-BA induced cardiotoxicity in zebrafish, which may be related to the change of HPA axis activity and the occurrence of inflammatory response under 6-BA treatment.
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