Developmental toxicity and neurotoxicity assessment of R-, S-, and RS-propylene glycol enantiomers in zebrafish (Danio rerio) larvae

Propylene glycol (PG) is widely used in the foods, pharmaceuticals, oil industry, animal feed, cosmetics and other industries. Because of the existence of a chiral carbon center, PG forms R (Rectus)- and S (Sinister)-enantiomers. Currently, the toxicity study of its R-, S-enantiomers is still very scarce. In this study, we have assessed the developmental toxicity and neurotoxicity of the R-, S-, and RS-PG enantiomers in zebrafish larvae. We found that exposure to R-, S-, and RS-PG enantiomers did not significantly affect the basic developmental endpoints of embryos or larvae (i.e., embryonic movement, hatching, mortality, malformation, heartbeat, body length), indicating that R-, S-, and RS-PG exposures did not exhibit the basic developmental toxicity in zebrafish larvae. The toxicity of three enantiomers was lower than that of ethanol, and there was no significant difference between them. However, R-, S-, and RS-PG exposures with high doses could significantly change the eye diameter and locomotor activity of larval zebrafish, indicating that R-, S-, and RS-PG enantiomers of high doses could potentially exhibit the neurotoxicity and ocular developmental toxicity in zebrafish larvae. Therefore, the potential neurotoxicity and ocular developmental toxicity of R-, S-, and RS-PG enantiomers for infants and toddlers should be considered.


Introduction
Propylene glycol (PG) is a colorless, tasteless, water-soluble liquid, which also known as 1,2-propanediol (Center for the Evaluation of Risks to Human Reproduction 2004). It belongs to the group of diols, which is a small aliphatic organic compound. Because of the existence of a chiral carbon center, PG forms R (Rectus)-and S (Sinister)-enantiomers (Ferreira et al. 2017). The S-enantiomer can be produced by microbes via the fermentation of L-rhamnose or L-fucose while the R-enantiomer can be produced from pentoses and hexoses via the methylglyoxal bypass (Ingvadottir et al. 2018). PG can dissolve hydrophobic substances; it is commonly present in poorly water-soluble intravenous medications and activated charcoal preparations (Greene and Krasowski 2020). Because of PG has very low toxicity and is currently considered as a non-carcinogenic or non-genotoxic compound, it is an important liquid for industrial and technological applications (Zaripov et al. 2020). As an important and safe intermediate, PG has been used in several industrial areas (Ferreira et al. 2017). It is widely used in foods, pharmaceuticals, oil industry, animal feed, cosmetics, and other industries (Zaripov et al. 2021). In 1999, 1083 million pounds of PG was produced in the USA with apparent consumption of 854 million pounds. About 170 million pounds of PG is consumed in human foods (Center for the Evaluation of Risks to Human Reproduction 2004). Thus, it is generally recognized as a safe food additive (Phillips et al. 2017). Besides, PG has numerous other applications in industry, including as a humectant, moisturizer, non-toxic antifreeze, and carrier in fragrance oils (Gaworski et al. 2010;Binks et al. 2013).
Several studies have examined the toxicity of RS-PG in the context of repeated exposure to these preparations. For instance, embryonic exposure to 0.625 or 1.25% RS-PG not only affects the behavioral parameters in zebrafish (Danio rerio) larvae, but cause persisting behavioral effects in adults (Massarsky et al. 2018). Besides, parenteral administration of RS-PG can cause certain behavioral effects in rodents, including locomotor suppression and anxiolytic effects (Lin et al. 1998;Silva & Elisabetsky, 2001;Harris et al. 2018). However, 0.45 and 2 g/kg RS-PG is intravenously administer for 28 days without exhibiting any discomfort or injury in rat (Pandey et al. 2017). Although the toxicity studies of RS-PG in different animal models have been very common, the toxicity study and safety evaluation of its R-, S-enantiomers are still very scarce.
Calcium/sodium ion conduction is an important process to maintain excitation-contraction coupling in neuron and muscle cells Zhang et al. 2013;Shen et al. 2020b). Calcium/sodium ion conduction disorders can disturb the normal function of cells and cause the defects of organs or tissues (Ranasinghe et al. 2020). In the central nervous system (CNS) of vertebrates, γ-amino butyric acid (GABA) can couple with the GABA receptors (GABARs) to regulate the locomotor behavior of organisms . Dysregulated genes expression of GABA and its receptors can seriously affect the locomotor behavior in organisms (Stehr et al. 2006;Assad et al. 2020).
Zebrafish is one of the most widely used model species in previous studies (Ge et al. 2015). Zebrafish has been used not only to assess effects on aquatic biota but also to bridge the gap to other vertebrates (Ma et al. 2019). It has many suitable features, which makes it an ideal vertebrate model for toxicology research Sun et al. 2020;. Due to its unique advantages, such as small size, rapid external development, high fecundity, transparency, easy access, and maintenance, make it easy to carry out experiments (Wang et al. 2018(Wang et al. , 2020aJia et al. 2020). In particular, its high homology with mammalian genes is favorable for assessing developmental toxicity and neurotoxicity of chemical substances (Jia et al. 2020;. Besides, using zebrafish embryos to address scientific questions is more suitable to meet current legislation (Teng et al. 2019).
In the present study, zebrafish embryos and larvae were employed to assess the developmental toxicity and neurotoxicity of R-, S-, and RS-PG enantiomers. Basic developmental endpoints (i.e., embryonic movement, hatching, mortality, malformation, heartbeat, body length, and diameter of eyes), locomotor activity, and relative transcripts of calcium/sodium ion conduction and GABARs-involved genes were assessed in zebrafish embryos or larvae. These results will play a vital role for the formulation of safety guidelines regarding potential hazards of PG in the early warnings to human health.

Zebrafish maintenance and embryonic acute toxicity test
Zebrafish maintenance and embryos collection were performed according to our previous studies Tang et al. 2020). The concentration of alcohol to be 1% and 5% has been used for the acute alcohol exposure to zebrafish in some previous studies (Chen et al. 2011;Tsang et al. 2018;Zindler et al. 2019). The positive control groups (1 and 5% ethanol, v/v) and test groups of R-, S-, and RS-PG (0.2, 1, and 5%, v/v, respectively) were prepared in zebrafish culture medium (1 L H 2 O dissolved 3.5 g NaCl and 0.05 g KCl, NaHCO 3 , and CaCl 2 , respectively). The control group contained zebrafish culture medium only. 40 embryos between 0.5 and 1.0 h post fertilization (hpf) were cultured in a 10-mL exposure solution (three replicates for each treatment) and renewed solutions once daily.

Embryonic movement, heartbeat, body length, and diameter of eyes assessment
Basic developmental endpoints (24 hpf embryonic movement, 72 hpf heartbeat, 72 hpf body length, and 72 hpf diameter of eyes) were assessed and indirectly analyzed using DanioScope 1.1 (Noldus IT, Wageningen, the Netherlands). The morphologically normal embryos and larvae were kept in the treatment plate, and were recorded videos or images. The embryonic movement (n ≥ 20 embryos for each treatment) and heartbeat (n ≥ 15 larvae for each treatment) were recorded videos in a 45 s and 30 s, respectively. The body length and diameter of eyes (n ≥ 18 larvae for each treatment) were measured from images.

Locomotor behavioral analysis
The detail locomotor behavioral analysis was implemented according to our previous study ). Briefly, the 5 days post fertilization (dpf) larvae treated with R-, S-, and RS-PG for 5 days were selected by simple random sampling and assigned gently into 24-well plates with one larva per well contained a 2-mL embryo medium (24 larvae for each treatment). Behavioral movements of larval zebrafish were video-recorded and analyzed using Ethovision XT 11.5 software (Noldus IT, Wageningen, the Netherlands).

Gene expression levels analysis
The total RNA of 5 dpf zebrafish larvae (n = 20 larvae/ sample, six biological replicates for each treatment) was extracted using a TRIzol Reagent (TaKaRa, Dalian, China). cDNA synthesis and gene expression levels analysis were performed according to our previous references ). The primer information used for mRNA expression analysis is listed in S- Table 1.

Statistical analysis
The data were analyzed using the Duncan's post hoc test after one-way analysis of variance (ANOVA) and performed using Graphpad Prism 7.0 software. All results were showed as the mean and standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of R-, S-, and RS-PG on the developmental endpoints in zebrafish
Survival rate.
Survival rate of zebrafish embryos or larvae after treatment with R-, S-, and RS-PG for 24, 48, 72, 96, and 120 h are shown in Table 1. Ethanol was employed as a positive control. Zebrafish embryos after treatment with 5% ethanol for 24 h and 5% R-, S-, and RS-PG for 72 h caused 100% mortality, which indicated that R-, S-, and RS-PG showed lower toxicity than ethanol. As shown in Table 1, zebrafish embryos treated with 5% S-, and RS-PG for 24 h compared with the blank control group decreased the survival rate to 75.08 ± 6.28% and 63.47 ± 4.68%, respectively. And zebrafish embryos treated with 5% R-, S-, and RS-PG for 48 h decreased the survival rate to 34.95 ± 3.22%, 48.26 ± 8.26%, and 13.01 ± 1.59%, respectively.
The effects of R-, S-, and RS-PG exposure on embryonic movement are shown in Fig. 1a. A 5% R-, S-, and RS-PG exposure resulted in significant decreases on embryonic movement at 24 hpf. However, 1% ethanol exposure significantly increased embryonic movement compared with the blank control group.
Hatching. The effects of R-, S-, and RS-PG exposure on hatching rate are shown in S- Fig. 2. Compared with the blank control group, the hatching rate was not affected at 60 and 72 hpf. Heartbeat.
Malformation. The effects of R-, S-, and RS-PG exposure on spinal curvature rate, pericardial edema rate, and yolk sac edema rate are shown in Fig. 2b, c, and d, respectively. When embryos were treated with 1% ethanol for 72 h, the malformation frequency of spinal curvature and pericardial edema were significantly increased. However, embryonic exposure to R-, S-, and RS-PG did not affect the spinal curvature rate and pericardial edema rate in 72 hpf larvae. Besides, the yolk sac edema rate was not affected in R-, S-, and RS-PG and ethanol treated group compared with the blank control.
Body length.
The effects of R-, S-, and RS-PG exposure on body length are shown in Fig. 3a. A significant decrease of body length was observed in 1% ethanol-treated group. However, a significant increase was observed in 0.2% S-PG-treated group.
Diameter of eyes. The effects of R-, S-, and RS-PG exposure on diameter of eyes X-and Y-axis are shown in Fig. 3b and 3c. Embryonic exposure to R-, S-, and RS-PG for 72 h could significantly increase the diameter of eyes X-axis of zebrafish larvae. A significant decrease of the diameter of eyes Y-axis was observed in 1% ethanol and 1% RS-PG-treated groups. However, a significant increase of the diameter of eyes Y-axis was observed in 0.2% R-PG-treated group.

Behavioral effects
Locomotor activity was implemented in 120 hpf larvae (Fig. 4). Our results showed that zebrafish larvae exposed with R-, S-, and RS-PG for 120 h displayed incremental total moved distance (Fig. 4a) and average movement speed (velocity) (Fig. 4b). However, exposure to 1% ethanol did not affect the total moved distance and velocity of larvae. The behavioral trajectory (Fig. 4c) of larvae was more chaotic in 1% ethanol, 1% R-, S-, and RS-PG-treated groups compared with the blank control group.

Gene expression analysis
The relative transcripts of genes on calcium/sodium ion conduction.
Calcium/sodium ion conduction related genes play a vital role in excitation-contraction coupling in skeletal muscle and neuron cells . To further understand the effects of R-, S-, and RS-PG on locomoter activity in larvae, the expression levels of genes involved with calcium/ sodium ion conduction were analyzed (Fig. 5a). The relative transcripts of three genes (cacna1aa, ryr3, and slc8a3) exhibited significant dysregulation compared with the blank control. The expression level of cacna1aa was significantly upregulated in 0.2% and 1% RS-PG-treated groups. The expression level of ryr3 was significantly upregulated in 0.2% RS-PG, 1% R-, S-, and RS-PG-treated groups. However, the expression level of slc8a3 was significantly downregulated in 0.2% R-and S-PG and 1% R-PG-treated groups.

Discussion
The behavior of an organism is directly associated with feeding, mating, and survival ). Locomotor behaviors have frequently been employed to study the neurodevelopmental effects of various chemicals (Ding et al. 2020). Usually, animals exposed to hazardous chemicals can change their behavior and these changes may affect their survival, growth, and reproduction (Wang et al. 2018). Previous study has been reported that embryonic exposure to PG can result in behavioral changes in zebrafish larvae (Massarsky et al. 2018). In the present Fig. 2 The effects of R-, S-, and RS-PG exposure on the malformation of larval zebrafish (72 hpf). a typical malformation pictures, b spinal curvature rate, c pericardial edema rate, d yolk sac edema rate.
Red arrows indicate spinal curvature or pericardial edema. Data presented as mean ± SE. *** P < 0.001. Scale bar = 500 μm study, we found that embryonic exposure to R-, S-, and RS-PG of high doses could significantly induce the locomotor hyperactivity of zebrafish larvae, demonstrating that R-, S-, and RS-PG of high doses could cause the neurodevelopmental effect in zebrafish larvae. The neurobehavioral abnormalities as a major pathophysiological hallmark are mainly caused by the hypoglutamatergic and hyperGABAergic alterations (Probst et al. 2020). As one of the main inhibitory neurotransmitters in vertebrate brain, GABA plays a critical role for regulating the circuitry underlying locomotor behavior (Yan et al. 2017). GABA displays rapid inhibitory action through the GABARs (Shen et al. 2020b). We found that the relative transcripts of several genes (gabra1, gabra2a, gab-br1b, gabbr2, gabrb3, gabrb4, and gabrg1) involved in GABARs exhibited significant upregulation in R-, S-, and RS-PG-treated larvae. Increased gene expression levels of GABARs could regulate the vast majority of rapid inhibitory synaptic transmission in the CNS .
Ryanodine receptors (RyRs) are calcium-dependent calcium release channels embedded in the sarcoplasmic/ endoplasmic reticulum (SR/ER), which regulate calciumdependent signal transduction in neurons or skeletal muscles (Frank et al. 2018;Tanaka et al. 2018). RyRs subunits contain a calcium-binding site that mediates calcium release and triggers intracellular calcium-induced calcium release, which is vital for muscle contraction (Ouyang et al. 2019;Wang et al. 2020b). RyRs have been characterized in many vertebrates including fish, birds, and amphibians (Darbandi & Franck, 2009;Murayama & Kurebayashi, 2011;Wang et al. 2020b). The ryr3 gene is mainly expressed in brain tissue, and low level in mammalian skeletal muscle (Darbandi & Franck, 2009). Calcium voltage-gated channel subunit alpha1A (cacna1a) is mainly expressed in neuronal tissue Fig. 3 The effects of R-, S-, and RS-PG exposure on the body length and eye diameter of larval zebrafish (72 hpf). a Body length; b diameter of eyes, X-axis; c diameter of eyes, Y-axis. Data presented as mean ± SE (n ≥ 18 per treatment). * P < 0.05, ** P < 0.01, *** P < 0.001 that plays a crucial part in excitation-contraction coupling via interaction with ryr3 ). In the previous study,  has reported that increased ryr3 and cacna1aa expression could significantly stimulate the neuron-mediated contraction. Currently, we found that the relative transcripts of ryr3 and cacna1aa genes were significantly upregulated in R-, S-, and RS-PG-treated larvae, indicating that the intracellular calcium release was significantly increase, and neuron-mediated contraction might be stimulative. Solute carrier family 8 member A3 (slc8a3) is mainly expressed in brain and muscle tissue, which contribute to cellular Ca 2+ homeostasis in excitable cells (Shen et al. 2020b). We found that the relative transcript of slc8a3 was significantly downregulated in R-, S-, and RS-PG-treated larvae, demonstrating that exposure to R-, S-, and RS-PG of high doses could disrupt the cellular Ca 2+ homeostasis of neuron and muscles.
Observed teratogenic effects such as small eyes and eye diameter, high ocular distance, and large inter-eye distance are characteristic of eye defects (Cadena et al. 2020). These eye defects such as microphthalmia, coloboma, anophthalmia, retinal dystrophies, and congenital cataract can occur in the prenatal and perinatal periods (Kim et al. 2019). Previous studies show that zebrafish exposed to ethanol can cause severe eye defects including microphthalmia and abnormal photoreceptor differentiation (Muralidharan et al. 2015(Muralidharan et al. , 2018. The microphthalmia is a small eye normally defined by corneal diameter or axial length . Many behaviors are correlative with visual function in vertebrates (Shi et al. 2019). Decreased eye diameter can affect the visual function and lead to a reduced ability to capture prey (Qian et al. 2021). In the present study, we found that embryonic exposure to R-, S-, and RS-PG of high doses could significantly affect the eye diameter of zebrafish Fig. 4 Locomotor activity of zebrafish larvae exposed to R-, S-, and RS-PG at 5 dpf. a Total moved distance, b mean velocity, c behavioral trajectory. Data presented as mean ± SE (n = 24 per treatment). ** P < 0.01, *** P < 0.001 larvae, indicating that R-, S-, and RS-PG could impair the visual function of larvae. Besides, RyRs signaling pathway has been directly linked to visual functionality (Ma et al. 2015;Frank et al. 2019). We found that the relative transcript of ryr3 was significantly upregulated in R-, S-, and RS-PG-treated larvae, demonstrating that exposure to R-, S-, and RS-PG of high doses have the potential to affect visual sensory system. From birth to 3 years old is a crucial window for the promotion of optical growth, health, and development (Ojuri et al. 2018). Especially in infants below 16 weeks of age, the enzymes involved in the metabolism of exogenous substances are not as efficient as in adults (Nougadère et al. 2020). The development processes of this period are also more likely disturbed (Nougadère et al. 2020). Infants and toddlers have differing diet patterns than adults, consequently, different intake scenario are required in risk assessment (Stroheker et al. 2019). The variety of foods is ceaselessly growing and changing for infants and toddlers, which can result in specific dietary exposure (Chekri et al. 2019). Processed cereal-based foods and other infant foods should be free from chemical and biological hazards (Ojuri et al. 2018). Currently, PG is generally recognized as a safe food additive, which is oversupplied in Chinese market (Phillips et al. 2017;Tao et al. 2020;Zhao et al. 2020). PG has very low toxicity and is considered to be a nontoxic or non-carcinogenic compound for adults (Zaripov et al. 2020). However, newborns and infants are especially susceptible to the effects of PG (Massarsky et al. 2018). In the present study, our results showed that R-, S-, and RS-PG enantiomers of high doses can exhibit the neurotoxicity and ocular developmental toxicity in zebrafish larvae, indicating that R-, S-, and RS-PG exposures of high doses have potential neurotoxicity and ocular developmental toxicity for infants and toddlers. In the present study, we demonstrate that exposure to R-, S-, and RS-PG enantiomers of high doses could significantly change the eye diameter and locomotor activity of larval zebrafish. Besides, the expression levels of 16 genes involved in γ-amino butyric acid receptors (GABARs) and three genes associated with calcium/sodium ion conduction exhibited significant dysregulation, indicating that R-, S-, and RS-PG enantiomers of high doses can affect the CNS and visual sensory system. However, the toxicity of three enantiomers was lower than that of ethanol, and there was no significant difference between them. Taken together, our results indicate that R-, S-, and RS-PG enantiomers of high doses can exhibit the neurotoxicity and ocular developmental toxicity in zebrafish larvae. Therefore, we suggest that potential neurotoxicity and ocular developmental toxicity of R-, S-, and RS-PG enantiomers for infants and toddlers should be considered.