Neurobehavioral effects of 1,2-propanediol in zebrafish (Danio rerio)

Abstract

The use of electronic cigarettes (e-cigarettes) is increasing despite insufficient information concerning their long-term effects, including the effects of maternal e-cigarette use on pre- and postnatal development. Our previous study demonstrated that developmental exposure to 1,2-propanediol (a principal component of e-cigarette liquid) affected early development of zebrafish, causing reduced growth, deformities, and hyperactive swimming behavior in larvae. The current study extends assessment of the developmental toxicity of 1,2-propanediol by examining additional long-term behavioral effects. We demonstrate that embryonic/larval exposure of zebrafish to 1,2-propanediol (0.625% or 1.25%) not only affected behavioral parameters in the larvae, but also caused persisting behavioral effects in adults after early developmental exposure. Additional parameters, including neural and vascular development in larvae, stress response in adults, and concentration of neurotransmitters dopamine and serotonin in adult brain were examined, in order to explain the behavioral differences. These additional assessments did not find 1,2-propanediol exposure to significantly affect Tg(Neurog1:GFP) or the transcript abundance of neural genes (Neurog1, Ascl1a, Elavl3, and Lef1). Vascular development was not found to be affected by 1,2-propanediol exposure, as inferred from experiments with Tg(Flk1:eGFP) zebrafish; however, transcript abundance of vascular genes (Flk1, Vegf, Tie-2, and Angpt1) was decreased. No statistically significant changes were noted for plasma cortisol or brain neurotransmitters in adult fish. Lastly, analysis of gene transcripts involved with 1,2-propanediol metabolism (Adh5, Aldh2.1, and Ldha) showed an increase in Adh5 transcript. This is the first study to demonstrate that developmental exposure to 1,2-propanediol has long-term neurobehavioral consequences in adult zebrafish, showing that e-cigarettes contain substances potentially harmful to neurodevelopment.

Keywords: 1,2-Propanediol (propylene glycol); Behavior; Electronic cigarettes; Neural and vascular development; Xenobiotic metabolism; Zebrafish.

Figure 1.

Larval locomotor activity in 144 hpf zebrafish exposed to 0% (control), 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. A. Total distance moved per minute over the course of the test. B. Average distance moved during each 10-min illumination phase. Letters indicate significant differences determined using two-way ANOVA with a post hoc Tukey test; P ≤ 0.05 was considered significant.

Figure 2.

Novel tank dive test in ~5 months old adult zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. A. Total distance moved per minute over the course of the test for males and females. B. Distance to bottom of the tank per minute over the course of the test for males and females. C. Dive recovery for males and females. See section 3.1.2.1 for details on statistical differences.

Figure 3.

Startle tap test in ~5 months old adult zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. Total distance moved in the 5 s pre (black bars) and post (white bars) each tap over the course of the test is presented for males (top panel) and females (bottom panel). See section 3.1.2.2 for details on statistical differences.

Figure 4.

Shoaling test in ~5 months old adult zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. A. Total distance moved per minute over the course of the test for males and females. B. Distance to zone per minute over the course of the test for males and females. C. Pre-post difference for males and females. See section 3.1.2.3 for details on statistical differences.

Figure 5.

Predator avoidance test in ~5 months old adult zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 4 hpf until 72 hpf. A. Total distance moved per minute over the course of the test for males and females. B. Distance to zone per minute over the course of the test for males and females. C. Flee response from blue (black bars) and red (white bars) stimuli for males and females. See section 3.1.2.4 for details on statistical differences.

Figure 6.

Neural development in zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. Tg(Neurog1:GFP) zebrafish were used to visualize the expression of Neurog1; brightfield (left) and fluorescence (right) images of zebrafish were captured at 72 hpf (A), and fluorescent signal in the head region was quantified using Image J (B). Transcript abundance of Neuorg1, Ascl1a, Elavl3, and Lef1 was assessed in wild type zebrafish at 72 hpf (C). No significant differences were detected.

Figure 7.

Vascular development in zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. Tg(Flk1:eGFP) zebrafish were used to visualize the expression of Flk1; fluorescence images of zebrafish were captured at 72 hpf (A), and fluorescent signal in the head and trunk regions was quantified using Image J (B). Transcript abundance of Flk1, Vegf, Tie-2, and Angpt1 was assessed in wild type zebrafish at 72 hpf (C). Letters indicate significant differences that were assessed using one-way ANOVA with a post hoc Tukey test; P ≤ 0.05 was considered significant.

Figure 8.

Markers of 1,2-propanediol metabolism in zebrafish exposed to 0%, 0.625% or 1.25% 1,2-propanediol from 6 hpf until 72 hpf. Transcript abundance of Adh5, Aldh2.1, and Ldha was assessed in wild type zebrafish at 72 hpf. Letters indicate significant differences that were assessed using one-way ANOVA with a post hoc Tukey test; P ≤ 0.05 was considered significant.