Exposure to 1,2-Propanediol Impacts Early Development of Zebrafish ( Danio rerio ) and Induces Hyperactivity

Abstract

The use of electronic cigarettes (e-cigarettes) is increasing as an alternative to tobacco burning cigarettes; however, their safety remains to be fully determined. The long-term effects of e-cigarettes are unknown, including the effects of maternal e-cigarette use on pre- and postnatal development. Additional research on the safety of e-cigarettes is needed. Especially useful would be information from high- and moderate-throughput economic model systems. This study investigates the effects of 1,2-propanediol, which was identified as the main component of e-cigarette liquid, on early development of zebrafish (an in vivo high-throughput model system that was recently proposed for the study of tobacco cigarette and e-cigarette toxicity). Zebrafish embryos were exposed to 1.25% or 2.5% 1,2-propanediol from 6 to 72 h post-fertilization (hpf). We show that exposure to 1,2-propanediol did not significantly affect mortality. Hatching success was significantly lower in 2.5% 1,2-propanediol-exposed embryos at 48 hpf, but at 72 hpf no significant differences were noted. Moreover, exposure to 1,2-propanediol reduced growth and increased the incidence of string heart, pericardial edema, and yolk sac edema. Most importantly, developmental exposure to 1.25% 1,2-propanediol caused hyperactive swimming behavior in larvae. This study demonstrates that 1,2-propanediol has adverse impacts on early development in zebrafish.

Introduction

The use of electronic cigarettes (e-cigarettes) has been increasing substantially in recent years as an alternative to tobacco cigarettes. E-cigarettes contain cartridges with liquid that is vaporized electronically without producing smoke. Despite the finding that e-cigarette vapor contains lower concentrations of a variety of potentially toxic compounds compared with tobacco cigarette smoke,¹ there are some chemicals found at higher concentrations in e-cigarettes. The e-cigarette liquid varies in composition, depending on the brand; however, >90% of e-cigarette liquid is composed of the humectants 1,2-propanediol (∼500–600 mg/mL) and glycerin (∼400–500 mg/mL).² In comparison, the concentration of nicotine in e-cigarette liquid is ∼9–23 mg/mL.³ Other chemicals, including various alcohols, polycyclic aromatic hydrocarbons, flavors, and so on, are also present in e-cigarettes, but in smaller concentrations (see Schober et al.² for more details on the composition of e-cigarette liquid).

It is estimated that from 2010 to 2013, the number of e-cigarette users in the United States grew from 1.8% to 13.0% of the total population.⁴ The use of e-cigarettes among high school students is especially worrisome, as it increased from 1.5% in 2011 to 16.0% in 2015.⁵ In fact, it has been suggested that the use of e-cigarettes may encourage tobacco cigarette use among U.S. adolescents.⁶ The increasing popularity of e-cigarettes raises concerns, since their safety has not been extensively studied. Accordingly, the FDA recently announced that it will examine more closely the information provided by e-cigarette manufacturers, to prevent misleading claims and to provide consumers with insights on the potential risks. Also, the sale of e-cigarettes (and other tobacco and nicotine products) to minors will likely be banned.⁷

E-cigarette safety is a controversial topic. E-cigarette vapor contains potentially toxic compounds; however, their concentrations are 9–450 times lower than in tobacco cigarette smoke, suggesting that substituting tobacco cigarettes with e-cigarettes would reduce exposure to toxicants.¹ A recent review on the safety of e-cigarettes concluded that although the long-term effects of e-cigarette use are unknown, they are likely less harmful than tobacco cigarettes, and could be recommended as a tool to help tobacco smokers to quit smoking.⁸ Moreover, the U.K. Department of Health recently released a study stating that e-cigarettes were 95% safer than tobacco cigarettes; however, this study was heavily criticized based on the chosen criteria to assess the level of harm of nicotine products and the scientific panel that did not include experts in tobacco research.⁹ Furthermore, analysis of the scientific literature on e-cigarettes revealed methodological problems, conflicts of interest, contradictory results, and lack of long-term follow-up studies, concluding that e-cigarettes cannot be considered harmless.¹⁰ The effects of maternal e-cigarette use on pre- and postnatal development are also unknown; however, it is known that maternal exposure to nicotine by itself produces neural and behavioral dysfunction in the offspring.^11–14

It is important to note that traditionally mammalian models have been used for the study of cigarette smoke toxicity, and more recently the toxicity of e-cigarettes. However, in recent years, the zebrafish model was proposed for the study of developmental toxicity of tobacco cigarettes and e-cigarettes.^15–19 The zebrafish is an important developmental model with a highly conserved gene function relative to humans, and it has been used extensively to model human disease.²⁰

This study aims to investigate the effects of one of the main components of e-cigarette liquid 1,2-propanediol (also known as propylene glycol) on early zebrafish development. Zebrafish embryos were exposed to 1,2-propanediol for 3 days, starting at 6 h post-fertilization (hpf). Several physiological parameters were assessed during or after the exposure, including viability, hatching success, growth rate, deformities, and behavior.

Materials and Methods

Chemicals

All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. 1,2-Propanediol was purchased from Sigma Aldrich (57-55-6, 99%). The stock solution of 1,2-propanediol was diluted to working concentrations in 30% Danieau's (in mM: 58 NaCl, 0.7 KCl, 0.4 MgSO₄, 0.6 Ca(NO₃)₂, 5 HEPES).

Zebrafish maintenance

Adult wild-type EkkWill zebrafish (EkkWill Waterlife Resources, Ruskin, FL) were maintained in holding tanks on a 14:10 h light–dark cycle (lights on at 9 AM) at 28°C in a circulating AHAB system (Aquatic Habitats, Apopka, FL) in 60 mg/L salt water (Instant Ocean, Foster & Smith, Rhinelander, WI). Fish were fed brine shrimp in the morning and Zeigler's Adult Zebrafish Complete Diet (Aquatic Habitats) in the afternoon. Breeding tanks were set at 5 PM, and embryos were collected the following morning within 1 h of spawning between 9 and 10 AM and kept in Danieau's at 28°C until they were separated into experimental groups. All procedures were approved by the Duke University Institutional Animal Care and Use Committee (A279-08-10).

Experimental setup

At 6 hpf, zebrafish embryos were screened under a dissecting microscope (SMZ1500; Nikon) and distributed into 6 cm glass petri dishes (15 embryos/dish), containing 14 mL Danieau's to which 1 mL 1,2-propanediol working solution was added. Each treatment group had four replicate dishes, such that each treatment had a total of 60 embryos per cohort. The final nominal concentrations of 1,2-propanediol were 1.25% and 2.5%. Initially, 5% and 10% 1,2-propanediol concentrations were also included, but were excluded in subsequent experiments, since 5% 1,2-propanediol resulted in severe deformities (Fig. 1A) and 10% 1,2-propanediol led to 100% mortality. The concentrations used in this study are ∼20–40 times lower than the concentration of 1,2-propandeiol found in e-cigarette liquid² (see the Discussion section for more details). Throughout the exposure, the embryos were kept in a 28°C incubator (Model 3326; Forma Scientific, Inc., Marietta, OH) on a 14:10 h light–dark cycle. The study was repeated at least three times, using independent zebrafish cohorts (the n-value refers to the number of cohorts rather than individual embryos/larvae).

FIG. 1.

Zebrafish larvae (72 hpf) after exposure to 1,2-propanediol. Larvae exposed to 5% 1,2-propanediol were severely deformed (A). The pericardial area was measured in control larvae (B), and larvae exposed to 1.25% (C) and 2.5% (D) 1,2-propanediol. (E) Summary of the fold change in pericardial area. Mean + SEM are presented (n = 6). The letters indicate significant differences that were assessed by using one-way ANOVA with a post hoc Tukey; p ≤ 0.05 was considered significant.

General physiology

Throughout the exposure, the embryos/larvae were monitored for mortality, starting at 24 hpf, and dead embryos/larvae were removed. Spontaneous contractions of the trunk were assessed at 24 hpf as the earliest biomarker of embryonic motor behavior that is mediated by the spinal circuit independently of the main neurotransmitter systems.²¹ The spontaneous contraction frequency was assessed by counting the number of contractions during a 2-min interval. Hatching success was assessed at 48 and 72 hpf. Heart rate was assessed at 48 hpf by counting the number of heartbeats within 15 s and extrapolating to heartbeats per minute. At 72 hpf, the larvae were screened for deformities, including yolk sac edema, pericardial edema, and string heart, to determine their incidence. Images of larvae from each treatment group were captured by using an SMZ1500 dissecting microscope that was equipped with Sight DS-Fi1 camera (Nikon, Tokyo, Japan), to measure the pericardial area and larval length by using Image J software (National Institutes of Health, Bethesda, MD). The images were also used to assess the somite number, to validate any potential effects on growth rate.

Larval behavior

Larval behavior was assessed in control and 1.25% 1,2-propanediol-treated larvae, since most of the larvae in these two groups did not display any deformities (see section General Physiology for details), and only non-deformed larvae were chosen for the behavioral assay. The 72 hpf larvae were transferred into 50 mL beakers, containing 25 mL fresh Danieau's, and transported to Dr. Levin's laboratory, where they were kept under a 14:10 h light–dark cycle at 28°C. At 144 hpf, the larvae were randomly transferred to a 96-well plate (1 larvae/well, ∼30 wells/treatment) and allowed to acclimate in the dark for 1 h before being transferred to a DanioVision™ observation chamber (Noldus, Inc., Wageningen, The Netherlands). Swimming distance was monitored for 50 min in alternating 10 min dark (“0% illumination,” <1 lux) and light (“100% illumination,” 5000 lux) periods, starting with a 10 min habituation period in the dark. Larval motion was recorded at a sample rate of 30 times/s via a high-speed infrared camera. Video data were analyzed by computer tracking software EthoVision XT^® (Noldus, Wageningen, The Netherlands), to calculate total distance moved for each individual larva over the course of the session.

Statistical analysis

All data are presented as means and standard error of the mean (SEM). Statistical analyses were conducted by using SigmaPlot (SPW 12; Systat Software, Inc., San Jose, CA). One-way analysis of variance (ANOVA) with a post hoc Tukey method was used to assess significant differences in parameters related to general physiology and spontaneous contractions. Two-way ANOVA with a post hoc Tukey method was used to assess significant differences in larval behavior. The statistical tests are specified in Table and Figure legends. In all cases, an n-value (refers to number of independent cohorts) of at least 3 was used and p ≤ 0.05 was considered significant.

Results

General physiology

Exposure to 1,2-propanediol did not significantly affect mortality despite a trend for higher mortality at 2.5% 1,2-propanediol; mortality was below 5% in all treatment groups (Table 1). Hatching success at 48 hpf was significantly lower in 2.5% 1,2-propanediol-exposed embryos in comparison to control embryos; however, no significant differences in hatching were noted at 72 hpf (Table 1). The heart rate at 48 hpf was not significantly affected by 1,2-propanediol. Larval growth was impacted by 1,2-propanediol. Although larval length at 72 hpf did not differ significantly between exposed and control larvae, larvae exposed to 2.5% 1,2-propanediol were significantly shorter than those exposed to 1.25% 1,2-propanediol (Table 1), and the number of somites was significantly lower in larvae exposed to 2.5% 1,2-propanediol in comparison to control larvae (Table 1). The frequency of spontaneous contractions was significantly higher in 2.5% 1,2-propanediol-exposed embryos compared with control embryos (Table 1).

Table 1.

Summary of Physiological Parameters in Zebrafish

	Hours post-fertilization	Control	1.25%	2.5%
Cumulative mortality (%)	24	0.8 ± 0.5	0.4 ± 0.4	2.6 ± 0.7
	48	1.1 ± 0.5	0.4 ± 0.4	3.3 ± 1.3
	72	1.1 ± 0.5	0.8 ± 0.5	4.8 ± 2.1
Cumulative hatching (%)	48	27.8 ± 4.1^a	20.6 ± 4.4^ab	9.5 ± 2.7^b
	72	99.6 ± 0.4	99.3 ± 0.7	98.1 ± 1.5
Heart rate (beats/min)	48	144 ± 9	148 ± 7	143 ± 7
Larval growth
Length (mm)	72	3.2 ± 0.1^ab	3.3 ± 0.1^a	3.1 ± 0.1^b
Somite number	72	29.2 ± 0.3^a	28.5 ± 0.3^a	26.1 ± 0.7^b
Spontaneous contractions (contractions/min)	24	6.9 ± 0.9^a	9.3 ± 0.9^ab	13.2 ± 1.7^b
Deformities
String heart (%)	72	1.3 ± 1.3^a	0.0 ± 0.0^a	26.0 ± 9.5^b
Yolk sac edema (%)	72	1.0 ± 1.0^a	0.0 ± 0.0^a	38.1 ± 14.1^b
Pericardial edema (%)	72	1.0 ± 1.0^a	7.3 ± 1.5^a	77.0 ± 6.0^b

Zebrafish were exposed to 1.25% or 2.5% 1,2-propanediol; mortality, hatching, heart rate, growth, spontaneous contractions, and deformities (string heart, yolk sac edema, and pericardial edema) were assessed at various times during the exposure. Mean ± SEM are presented (n = 4–6). The letters indicate significant differences that were assessed by using one-way ANOVA with a post hoc Tukey test; p ≤ 0.05 was considered significant.

Moreover, exposure to 1,2-propanediol led to pericardial edema. The pericardial area was significantly larger in 2.5% 1,2-propanediol-exposed larvae in comparison to control and 1.25% groups (Fig. 1B–E). The incidence of string heart, yolk sac edema, and pericardial edema was also significantly higher in 2.5% 1,2-propanediol-exposed larvae in comparison to control and 1.25% groups (Fig. 2 and Table 1).

FIG. 2.

Incidence (%) of string heart, yolk sac edema, and pericardial edema in zebrafish larvae (72 hpf) after exposure to 1,2-propanediol. Each dot represents an average incidence from each cohort (n = 4–5). For significant differences, refer to Table 1.

Larval behavior

Exposure to 1.25% 1,2-propanediol altered swimming behavior, such that the exposed larvae showed locomotor hyperactivity compared with control (Fig. 3). Distance swum was significantly higher during the dark sessions in both control and 1,2-propanediol-treated larvae, which is the expected outcome in dark/light conditions for zebrafish larvae. There was also a significant 1,2-propanediol exposure × light/dark interaction. Follow-up tests of the simple main effects of 1,2-propanediol exposure during the light and dark sessions showed that there was a significant 1,2-propanediol-induced hyperactivity in both of the light sessions and the last dark session (Fig. 3B). No significant differences were noted during the initial dark adaptation session.

FIG. 3.

Swimming behavior in zebrafish larvae. Zebrafish were exposed to 1.25% 1,2-propanediol until 72 hpf. The larvae were transferred to clean Danieau's and kept at 28°C. Swimming behavior was assessed in 144 hpf larvae. (A) Distance swum during the dark and light time sessions throughout the entire 50 min trial. Mean + SEM are presented (n = 4). (B) Average distance swum during each of the dark/light sessions. Each dot represents an average distance swum from each cohort (n = 4). The asterisks indicate significant differences between 1,2-propanediol-treated and control larvae. Two-way ANOVA with a post hoc Tukey was used to assess statistical differences; p ≤ 0.05 was considered significant.

Discussion

The increased use of e-cigarettes raises concerns, since their safety has not been studied adequately. E-cigarette liquid contains potentially toxic compounds and, thus, cannot be considered harmless. The long-term effects to e-cigarette users, as well as the effects of maternal e-cigarette use on pre- and postnatal development, are largely unknown. This study investigated the developmental toxicity of 1,2-propanediol (the main component of e-cigarette liquid), using the zebrafish embryo model. Several physiological parameters, including hatching success, growth rate, incidence of deformities, and larval behavior, were affected by exposure to 1,2-propanediol.

Several studies examined the effects of 1,2-propanediol on zebrafish in the context of cryoprotectants. For example, Lahnsteiner²² exposed zebrafish to 0.125%–2.5% 1,2-propanediol for 48 h and reported several effects, as discussed later. The compatibility of 1,2-propanediol as a solvent for chemical exposures in zebrafish embryos and larvae was also studied. For example, Maes et al.²³ showed that embryos 4–48 hpf can tolerate 1,2-propanediol concentrations <2.5% during a 24-h exposure. Such studies mainly focused on viability after a relatively short exposure duration. To the best of our knowledge, only one study examined the toxicity of e-cigarette extract in developing zebrafish. Palpant et al.¹⁶ exposed zebrafish to tobacco or e-cigarette extracts at concentrations corresponding to 6.8, 13.7, and 34 μM nicotine, and they concluded that e-cigarette extract was less toxic than tobacco cigarette extract. The latter study used nicotine concentrations as a baseline to quantify the amount of e-cigarette extract. Assuming similar ratios of nicotine to 1,2-propanediol in e-cigarette vapor as previously reported,² the aforementioned concentrations of nicotine would correspond roughly to 0.003, 0.005, and 0.012% 1,2-propanediol, respectively.

In the current study, we exposed zebrafish embryos to 1.25% and 2.5% 1,2-propanediol from 6 to 72 hpf. These nominal concentrations correspond to the higher range of concentrations used by Lahnsteiner,²² and they are ∼20–40 times lower than the concentration of 1,2-propanediol found in e-cigarette liquid.² Although the blood/plasma concentration of 1,2-propanediol in e-cigarette users is yet to be measured, it has been shown that 1,2-propanediol is the most abundant volatile compound in e-cigarette vapor. Indeed, 1,2-propanediol concentrations were 110–395 μg/m³ in indoor air after 2-h vaping sessions.² Moreover, it is unclear how much 1,2-propanediol is actually taken up by the embryos/larvae (i.e., physiologically available dose), and its half-life in zebrafish is not known. In mammals, 1,2-propanediol is metabolized quite rapidly (a few hours) in the liver by alcohol dehydrogenase (ADH) to form lactaldehyde, which is, subsequently, metabolized into lactate.²⁴ Zebrafish possess three ADH isoforms (ADH3, ADH8A, and ADH8B) that share 68%–81% amino acid sequence similarity with the human ADH sequences.²⁵ Notably, ADH8A metabolizes ethanol at a similar rate to that of human ADH1.²⁶ Presumably, zebrafish ADHs can metabolize 1,2-propanediol at a similar rate to that of human ADHs, and our future research will examine whether ADH transcript abundance and/or activity change in response to 1,2-propanediol exposure.

We demonstrate that a 72-h exposure to 1.25 or 2.5% 1,2-propanediol starting at 6 hpf did not significantly affect survival; however, 5% 1,2-propanediol resulted in severe deformities, and 10% 1,2-propanediol led to 100% mortality. These results differ from Lahnsteiner,²² who observed mortality at much lower concentrations and reported an LD₀ (maximum dose without significant effect on mortality) of 0.75% 1,2-propanediol when embryos were exposed for 48 h starting at 1 hpf. This difference suggests that earlier exposure time leads to greater mortality. Palpant et al.¹⁶ also showed a decrease in survival rate at the highest concentration of e-cigarette extract (34 μM nicotine ≈0.012% 1,2-propanediol); however, e-cigarette extract likely contains several chemicals in addition to 1,2-propanediol, complicating the comparisons to 1,2-propanediol alone. Moreover, there was a slight, but statistically significant, delay in hatching success at 48 hpf at 2.5% 1,2-propanediol, but most embryos hatched by 72 hpf regardless of treatment.

The larval length and somite number were also assessed, to estimate the effects of 1,2-propanediol on growth. Although no significant differences were noted for larval length between the control and 1,2-propanediol-exposed groups, the larvae that were exposed to 2.5% 1,2-propanediol were significantly shorter than those exposed to 1.25% 1,2-propanediol, and the somite number was significantly lower in larvae exposed to 2.5% 1,2-propanediol in comparison to control larvae. It should be noted that Lahnsteiner²² or Palpant et al.¹⁶ did not assess larval growth. Although the effects of 1,2-propanediol or e-cigarette extract on zebrafish growth are unknown, the exposure of zebrafish to total particulate matter (TPM; the particulate phase of tobacco cigarette smoke) did decrease growth.¹⁷ However, TPM is composed of several hundreds of chemicals; thus, the mechanisms of toxicity might be quite different.

Furthermore, no significant differences were noted for heart rate at 48 hpf. Palpant et al.¹⁶ also showed no significant changes in heart rate in zebrafish exposed to e-cigarette extract. Although the heart rate was not significantly affected at 48 hpf, exposure to 2.5% 1,2-propanediol resulted in a significantly higher pericardial area at 72 hpf, which coincided with a higher incidence of pericardial edema and string heart. This suggests that the cardiac development may not be affected initially (48 hpf), but cardiac function could be impacted by prolonged exposure (>72 hpf) to 1,2-propanediol. Similarly, Palpant et al.¹⁶ reported a higher incidence of cardiac deformities, including pericardial edema and looped heart. In addition, the incidence of yolk sac edema was significantly higher in larvae exposed to 2.5% 1,2-propanediol.

In contrast to the developmental effects reported here, studies that examined developmental toxicity of 1,2-propanediol in mammalian models did not show effects on fetal morphology. For example, Enright et al.²⁷ performed two studies in rats and rabbits. In the first study, female rats were administered 1,2-propanediol orally (1 g/kg) before/during mating, and on gestation day (GD) 0–7 and the embryonic development was assessed on GD 14. In the second study, pregnant rats and rabbits were exposed to the same dose of 1,2-propanediol during the period of organogenesis (rat: GD 6–17; rabbit: GD 7–19) and the fetal development was assessed. No significant differences in fetal morphology were noted in either study. The contrasting effects of 1,2-propanediol on developing zebrafish and developing rat/rabbit could be due to several factors, including differences in modes of exposure, potential differences in 1,2-propandiol metabolism/pharmacokinetics, and species sensitivity. It is also difficult to compare the concentrations of 1,2-propandiol that were used. It is possible that the waterborne exposure of zebrafish embryos to 1,2-propanediol results in higher physiological concentrations in comparison to the maternal oral exposure of rat/rabbit.

Exposure of zebrafish embryos to 1,2-propanediol also appeared to have neurological effects. The frequency of spontaneous contractions at 24 hpf was significantly higher in embryos exposed to 2.5% 1,2-propanediol; the spontaneous contractions were almost twice as frequent at this concentration. Spontaneous contractions are one of the earliest forms of motor function in zebrafish. The contractions are initiated by the spinal nerve, rather than by the release of neurotransmitters.²¹ Neither Lahnsteiner²² nor Palpant et al.¹⁶ assessed spontaneous contractions. It is noteworthy that increased spontaneous contractions in embryos exposed to TPM, but not nicotine, were reported.¹⁷ These early signs of neurological changes due to 1,2-propanediol exposure are important and should be investigated further.

Perhaps the most notable observation in this study was the effect of 1,2-propanediol on larval behavior at 144 hpf. Specifically, larvae exposed to 1.25% 1,2-propanediol displayed swimming hyperactivity; this effect was statistically significant during both light sessions and the last dark session. Since the hyperactivity was observed during the light and dark sessions, the possibility of general hyperkinesis cannot be ruled out. It is unclear how exposure to 1,2-propanediol impacts swimming activity; however, several chemicals were able to increase swimming activity in zebrafish. Guo et al.²⁸ reported that exposure to ethanol (0.1%–1%) increased swimming distances during dark sessions, whereas 2.0% ethanol increased activity in both light and dark sessions. The concentrations of neurotransmitters involved with norepinephrine, dopamine, and serotonin pathways were also affected in response to ethanol exposure.²⁸ Whether or not 1,2-propanediol and ethanol act through similar mechanisms to induce hyperactivity in larval zebrafish is unknown, but in mammals (including humans) propylene glycol can affect the central nervous system (CNS), resulting in “alcohol-like” effects. Specifically, infants are more susceptible to CNS effects of 1,2-propanediol due to their lower metabolic capacity in comparison to that of adults.²⁹

Several other compounds have been shown to induce hyperactivity in larval zebrafish. For example, Maximino et al.³⁰ reported that exposing zebrafish to fluoxetine, a selective serotonin reuptake inhibitor, resulted in anxiogenic effects during the light and dark sessions. The neuroactive chemical, pentylenetrazole (PTZ) also induced hyperactivity in larval zebrafish.³¹ Moreover PTZ, aconitine, and 4-aminopyridine were shown to affect the response to the light/dark by affecting gamma-aminobutyric acid, sodium, and potassium channels, respectively.³² PTZ, in particular, was shown to reverse the normal patterns to day and night cycles. Aconitine resulted in an almost complete elimination of the dark/light cycle coupled with an increase in activity. Interestingly, TPM-induced zebrafish locomotor hyperactivity was also reported, but only during the dark sessions.¹⁷ Since larval hyperactivity can be elicited via several neurological pathways as demonstrated in the aforementioned studies, follow-up studies should investigate in more detail the effects of 1,2-propanediol on various neurotransmitters, to shed light on possible mechanisms.

It is noteworthy that nicotine, the psychoactive ingredient in some e-cigarettes, was shown to decrease larval swimming.^33,34 It was suggested that nicotine-induced paralysis in zebrafish larvae may occur via alteration of axonal pathfinding in motoneurons and inactivation of acetylcholine receptors in the muscle.³³ Since 1,2-propanediol and nicotine appear to have opposite effects on larval hyperactivity, future studies should compare the effects of 1,2-propanediol on swimming behavior in the presence and absence of nicotine; however, the concentration of 1,2-propanediol in e-cigarette liquid is ∼20 times higher than that of nicotine, suggesting that the hyperactive effect of 1,2-propandiol could remain.

In summary, this study demonstrates that 1,2-propanediol impacts zebrafish development and behavior. Although more research is necessary to better understand how 1,2-propanediol impacts early development, and whether or not the observed effects in zebrafish are relevant to humans, this study does support the idea that e-cigarettes may not be harmless, and that the research on the toxicity of e-cigarettes and their main constituents should be pursued in more detail, especially since the effects of maternal e-cigarette use on the offspring are still largely unknown. It would also be important to study the interactions of 1,2-propanediol and nicotine. These two components are co-administered during e-cigarette use, and nicotine has been shown to cause neurobehavioral effects. The effects of glycerol, the other major component of e-cigarette liquid, should also be addressed.

Footnotes

Acknowledgments

The authors thank all members of the Di Giulio lab, as well as M. Chernick for technical assistance. This research was supported by RJR-Leon Golberg Postdoctoral Fellowship to A. Massarsky.

Disclosure Statement

No competing financial interests exist.

References

Goniewicz

, Knysak

, Gawron

, Kosmider

, Sobczak

, Kurek

, et al. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob Control, 2014; 23:133–139.

Schober

, Szendrei

, Matzen

, Osiander-Fuchs

, Heitmann

, Schettgen

, et al. Use of electronic cigarettes (e-cigarettes) impairs indoor air quality and increases FeNO levels of e-cigarette consumers. Int J Hyg Environ Health, 2014; 217:628–637.

Cameron

, Howell

, White

, Andrenyak

, Layton

, Roll

. Variable and potentially fatal amounts of nicotine in e-cigarette nicotine solutions. Tob Control, 2014; 1:77–78.

McMillen

, Gottlieb

, Shaefer

RMW

, Winickoff

, Klein

. Trends in electronic cigarette use among U.S. adults: use is increasing in both smokers and nonsmokers. Nicotine Tob Res, 2015; 17:1195–1202.

Singh

, Arrazola

, Corey

, Husten

, Neff

, Homa

, et al. Tobacco use among middle and high school students—United States, 2011–2015. MMWR Morb Mortal Wkly Rep, 2016; 65:361–367.

Dutra

, Glantz

. Electronic cigarettes and conventional cigarette use among US adolescents. JAMA Pediatr, 2014; 168:610–617.

Perrone

. FDA will require e-cigarettes and contents to be reviewed. Associated Press. 2016. Available at: http://bigstory.ap.org/article/9ae0c0e7415549e29cb21fbaf7e401b3/fda-brings-e-cigarettes-under-federal-authority (Accessed June 18, 2016).

Hajek

, Etter

J-F

, Benowitz

, Eissenberg

, McRobbie

. Electronic cigarettes: review of use, content, safety, effects on smokers and potential for harm and benefit. Addiction, 2014; 109:1801–1810.

Kirby

. E-cigarettes declared 95% less harmful than tobacco by UK health body. Lancet Respir Med, 2015; 3:750–751.

10.

Pisinger

, Døssing

. A systematic review of health effects of electronic cigarettes. Prev Med, 2014; 69:248–260.

11.

Levin

, Slotkin

. Developmental neurotoxicity of nicotine. In: Handbook of Developmental Neurotoxicology. Slikker

Jr. and Chang

(eds), pp. 587–615, Academic Press, San Diego, 1998.

12.

Levitt

. Prenatal effects of drugs of abuse on brain development. Drug Alcohol Depend, 1998; 51:109–125.

13.

Slikker

Jr , Xu

, Levin

, Slotkin

. Mode of action: disruption of brain cell replication, second messenger, and neurotransmitter systems during development leading to cognitive dysfunction—developmental neurotoxicity of nicotine. Crit Rev Toxicol, 2005; 35:703–711.

14.

Slotkin

, Pinkerton

, Seidler

. Perinatal environmental tobacco smoke exposure in rhesus monkeys: critical periods and regional selectivity for effects on brain cell development and lipid peroxidation. Environ Health Perspect, 2006; 114:34–39.

15.

Ellis

, Soo

, Achenbach

, Morash

, Soanes

. Use of the zebrafish larvae as a model to study cigarette smoke condensate toxicity. PLoS One, 2014; 9:e115305.

16.

Palpant

, Hofsteen

, Pabon

, Reinecke

, Murry

. Cardiac development in zebrafish and human embryonic stem cells is inhibited by exposure to tobacco cigarettes and e-cigarettes. PLoS One, 2015; 10:e0126259.

17.

Massarsky

, Jayasundara

, Bailey

, Oliveri

, Levin

, Prasad

, et al. Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine. Neurotoxicol Teratol, 2015; 51:77–88.

18.

Massarsky

, Bone

, Dong

, Hinton

, Prasad

, Di Giulio

. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos. Toxicol Appl Pharmacol, 2016; 309:63–76.

19.

Folkesson

, Sadowska

, Vikingsson

, Karlsson

, Carlhäll

C-J

, Länne

, et al. Differences in cardiovascular toxicities associated with cigarette smoking and snuff use revealed using novel zebrafish models. Biol Open, 2016; 5:970–978.

20.

Lieschke

, Currie

. Animal models of human disease: zebrafish swim into view. Nature, 2007; 8:353–367.

21.

Saint-Amant

, Drapeau

. Motoneuron activity patterns related to the earliest behavior of the zebrafish embryo. J Neurosci, 2000; 20:3964–3972.

22.

Lahnsteiner

. The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos. Theriogenology, 2008; 69:384–396.

23.

Maes

, Verlooy

, Buenafe

, de Witte

PAM

, Esguerra

, Crawford

. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. PLoS One, 2012; 7:e43850.

24.

European Medicines Agency. Background review for the excipient propylene glycol. Available at: www.ema.europa.eu/docs/en_GB/document_library/Report/2014/12/WC500177937.pdf. 2014 (Accessed December 23, 2016).

25.

Tran

, Nowicki

, Chatterjee

, Gerlai

. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver. Prog Neuropsychopharmacol Biol Psychiatry, 2015; 56:221–226.

26.

Reimers

, Hahn

, Tanguay

. Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics. J Biol Chem, 2004; 279:38303–38312.

27.

Enright

, McIntyre

. Assessment of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-β-cyclodextrin for use in developmental and reproductive toxicology studies. Birth Defects Res B Dev Reprod Toxicol, 2010; 89:504–516.

28.

Guo

, Lin

, Peng

, Chen

, Zhang

, Liu

, et al. Influences of acute ethanol exposure on locomotor activities of zebrafish larvae under different illumination. Alcohol, 2015; 49:727–737.

29.

Fowles

, Banton

, Pottenger

. A toxicological review of the propylene glycols. Crit Rev Toxicol, 2013; 43:363–390.

30.

Maximino

, da Silva

AWB

, Gouveia

, Herculano

. Pharmacological analysis of zebrafish (Danio rerio) scototaxis. Prog Neuropsychopharmacol Biol Psychiatry, 2011; 35:624–631.

31.

Berghmans

, Hunt

, Roach

, Goldsmith

. Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res, 2007; 75:18–28.

32.

Ellis

, Seibert

, Soanes

. Distinct models of induced hyperactivity in zebrafish larvae. Brain Res, 2012; 1449:46–59.

33.

Svoboda

, Vijayaraghavan

, Tanguay

. Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine. J Neurosci, 2002; 22:10731–10741.

34.

Crosby

, Bailey

, Oliveri

, Levin

. Neurobehavioral impairments caused by developmental imidacloprid exposure in zebrafish. Neurotoxicol Teratol, 2015; 49:81–90.