Cannabidiol exerts teratogenic effects on developing zebrafish through the sonic hedgehog signaling pathway

Abstract

The increasing legalization and decriminalization of cannabis for therapeutic and recreational purposes in various regions have influenced public perceptions and attitudes toward cannabidiol (CBD)-containing products, including their use during pregnancy. However, there is a knowledge gap regarding the effect of CBD on fetal development, and the mechanisms by which CBD induces developmental deficits have not been well studied. In this study, we investigated whether the teratogenic effects of CBD on developing zebrafish are mediated through Sonic hedgehog signaling (Shh), a critical pathway in development. Embryonic exposure to CBD reduced hatching and survival rates and suppressed Shh pathway activity, leading to decreased ptch2 expression-a regulatory receptor expressed in response to Shh signaling. Larval swimming activity was impaired by CBD exposure. However, coexposure to CBD and a synthetic Shh pathway activator significantly improved developmental outcomes, including decreased mortality, increased hatching rates, elevated ptch2 expression, and increased locomotor activity. These findings underscore the developmental risks associated with CBD use during pregnancy and highlight the involvement of the Shh signaling pathway in driving these effects. The results of this study can inform regulations for cannabis use during pregnancy and emphasize the need to develop therapeutic guidelines for the safe use of CBD-based treatments.

Keywords: Cannabidiol; Early development; Gastrulation period; Sonic hedgehog signaling; Zebrafish.

Fig. 1

Effect of cannabidiol (CBD) on developing zebrafish. (A) Experimental outline demonstrating the exposure paradigm used to investigate whether the sonic hedgehog (Shh) pathway plays a role in CBD-induced effects on developing zebrafish. The embryos were exposed to purmorphamine (PM) or its vehicle, dimethyl sulfoxide (DMSO), at 4.75 hpf for 30 min prior to gastrulation. During gastrulation, embryos were coexposed to CBD or the CBD vehicle, methanol (MeOH). The treated embryos were preserved at 10.75 hpf and 1 dpf to analyze the ptch2 expression pattern via in situ hybridization and qPCR. The expression of ptch2 among the treatment groups was also examined at 1 dpf via confocal microscopy in the transgenic zebrafish ptch2: Kaede line. All treatment groups were monitored daily from 1 to 5 dpf to assess hatching and survival rates. At 5 dpf, the locomotor activity of the embryos was also assessed (zebrafish illustrations were generated using BioRender.com). (B,C) Line graphs showing the survival and hatching rates of developing zebrafish exposed to different treatments: control, vehicle (0.2% DMSO + 0.3 MeOH), 3 mg/L CBD, 20 µM PM, and CBD + PM (n = 125). Asterisks indicate treatment groups that are significantly different from each other (Kruskal–Wallis test, followed by Dunn’s multiple comparisons test with Bonferroni correction, adjusted alpha ≤ 0.025). The error bars represent the standard error of the mean (SEM).

Fig. 2

Exposure to CBD during gastrulation altered the expression pattern of ptch2 in 10.75 hpf embryos, ascertained via in situ hybridization, and PM rescued the CBD-induced effects. Zebrafish embryos were treated with (A) embryo medium (control), (B) 0.3% methanol (MeOH) or 0.2% dimethyl sulfoxide (DMSO) as a vehicle, (C) 3 mg/L CBD, D) 3 mg/L CBD + 20 µM PM, or E) 20 µM PM during gastrulation (5.25–10.75 hpf), and at the end of gastrulation, in situ hybridization was performed to probe for ptch2 (n = 10–20). The arrows indicate adaxial cell organization in representative ptch2 in situ hybridization images of different treatments. (F) Bar graph showing the transcript abundance of ptch2 in embryos, via qPCR, collected at 10.75 hpf after being exposed to CBD, CBD + PM, PM, MeOH + DMSO, or the control during gastrulation (n = 5). Different lower-case letters indicate significant differences (one-way ANOVA was performed followed by Tukey’s post hoc test, p ≤ 0.05, error bars: ± SEM).

Fig. 3

The expression of ptch2 in 1 dpf embryos, via in situ hybridization, was affected following CBD exposure during gastrulation. Zebrafish embryos were treated with (A) embryo medium (control), (B) 0.3% methanol (MeOH) and 0.2% dimethyl sulfoxide (DMSO) as vehicle, (C) 3 mg/L CBD, (D) 3 mg/L CBD + 20 µM PM, or (E) 20 µM PM during gastrulation (5.25–10.75 hpf). At the end of gastrulation, the embryos were transferred to clean embryo media, and in situ hybridization was performed to probe for ptch2 at 1 dpf (n = 10–20). (F) Bar graph showing the transcript abundance of ptch2 in embryos, via qPCR, collected at 1 dpf after being exposed to CBD, CBD + PM, PM, MeOH + DMSO, or the control during gastrulation (n = 5). Different lower-case letters indicate significant differences (one-way ANOVA was performed followed by Tukey’s post hoc test, p ≤ 0.05, error bars: ± SEM).

Fig. 4

The expression of ptch2 in transgenic ptch2: Kaede embryos (1 dpf) was affected by CBD exposure during gastrulation. Confocal microscopy images of 1 dpf zebrafish embryos demonstrating how exposure to (A) embryo medium (control, n = 16), (B) 0.3% methanol (MeOH) or 0.2% dimethyl sulfoxide (DMSO) as a vehicle (n = 13), (C) 3 mg/L CBD (n = 16), (D) 3 mg/L CBD + 20 µM PM (n = 15), or (E) 20 µM PM (n = 14) during gastrulation alters the expression of ptch2. Images were captured at 40X as maximum intensity z-stack compilations. (F) Box plot showing the fluorescence intensity measured from confocal images as an indicator of ptch2 expression after different treatments. The horizontal line within the boxplot represents the median value and is surrounded by the interquartile range. The error bars represent the maximum and minimum values. Different lower-case letters indicate significant differences among treatments (Kruskal–Wallis test, followed by Dunn’s multiple comparisons test with Bonferroni correction, adjusted alpha ≤ 0.025).

Fig. 5

Exposure to CBD during gastrulation altered zebrafish larvae free swimming activity (locomotion) at 5 dpf. Zebrafish embryos were treated with embryo medium (control), 0.3% MeOH or 0.2% DMSO as a vehicle, 3 mg/L CBD, 3 mg/L CBD + 20 µM PM, or 20 µM PM during gastrulation, and fish locomotion was examined at 5 dpf (n = 21–54). Boxplots showing changes in (A) total distance moved (mm), (B) locomotion velocity during active periods (cm/s), and (C) locomotor activity (percent of pixel changes within an individual well over the period of 1 h). The horizontal line within the boxplot represents the median value and is surrounded by the interquartile range. The error bars represent the maximum and minimum values. Different lower-case letters indicate significant differences among treatments (Kruskal–Wallis test, followed by Dunn’s multiple comparisons test with Bonferroni correction, adjusted alpha ≤ 0.025).