In the present study, we assessed the developmental toxicity and neurotoxicity of the R-, S-, and RS-PG enantiomers in zebrafish larvae. Basic developmental endpoints of embryos or larvae (i.e., embryonic movement, hatching, mortality, malformation, heartbeat, body length) did not significantly be affected after treatment with R-, S-, and RS-PG enantiomers. The toxicity of the three enantiomers was lower than that of ethanol, and there was no significant difference between them. However, exposure to R-, S-, and RS-PG enantiomers with high doses (0.2% and 1%) significantly changed the diameter of eyes -X and -Y axis and the locomotor activity of larval zebrafish. In addition, the relative transcripts of sixteen genes involved in GABARs and three genes associated with calcium/sodium ion conduction exhibited significant dysregulation compared with the blank control. Altogether, these results indicate that R-, S-, and RS-PG enantiomers of high doses can exhibit the neurotoxicity and ocular developmental toxicity in zebrafish larvae.
The behavior of an organism is directly associated with the feeding, mating, and survival (Wu et al., 2020). 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 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 is mainly caused by the hypoglutamatergic and hyperGABAergic alterations (Probst et al., 2020). As one of the main inhibitory neurotransmitters in the 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, gabbr1b, gabbr2, gabrb3, gabrb4, and gabrg1) involved in GABARs exhibited significant up-regulation 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 (Shen et al., 2020b).
Ryanodine receptors (RyRs) are calcium-dependent calcium release channels embedded in the sarcoplasmic/endoplasmic reticulum (SR/ER), which regulate calcium-dependent 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 that plays a crucial part in excitation-contraction coupling via interaction with ryr3 (Shen et al., 2020b). In the previous study, shen et al. has reported that increased ryr3 and cacna1aa expression could significantly stimulate the neuron-mediated contraction (Shen et al., 2020b). Currently, we found that the relative transcripts of ryr3 and cacna1aa genes were significantly up-regulated 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 Ca2+ homeostasis in excitable cells (Shen et al., 2020b). We found that the relative transcript of slc8a3 was significantly down-regulated in R-, S-, and RS-PG treated larvae, demonstrating that exposure to R-, S-, and RS-PG of high doses could disrupt the cellular Ca2+ 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 (Huang et al., 2013). 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 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 up-regulated 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 the visual sensory system.
From birth to three 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 is 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 non-toxic or non-carcinogenic compound for adults (Zaripov et al., 2020). However, newborns and infants are specially 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 the 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 the locomotor activity of larval zebrafish. Besides, the expression levels of sixteen 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 the 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 the potential neurotoxicity and ocular developmental toxicity of R-, S-, and RS-PG enantiomers for infants and toddlers should be considered.