Antioxidant vitamin E protects embryos of Xenopus tropicalis against lambda-cyhalothrin induced embryotoxicity

Pesticides are capable of increasing risks to the early development of nontarget organisms through oxidative stress. The supplementation of antioxidants could help to modulate the toxic effects of pesticides, but much remains to be understood in the interactions between pesticides and antioxidants in amphibians. In the present study, the embryotoxicity of a widely used pyrethroid, lambda-cyhalothrin (LCT), and the potential effect of α-tocopherol (TOC) on embryos of Xenopus tropicalis were evaluated. Exposure to LCT did not affect the hatch rate, survival, or body length of the embryos. However, environmentally relevant concentrations of LCT could induce significant malformations on the larvae. Exposure to LCT led to a concentration-dependent induction of oxidative stress and cytotoxicity that subsequently resulted in embryotoxicity. During the early developmental stages, vitamin E could work as a powerful protective antioxidant. The LCT-induced overproduction of reactive oxygen species and increased enzymatic activities were fully inhibited by treatment with 1 μg/L TOC. However, only supplementation with 100 μg/L TOC provided partial protection against the morphological changes caused by LCT. The results from the present study suggest that antioxidant vitamin E possesses protective potential against pyrethroid-induced embryotoxicity in amphibian embryos through the prevention of oxidative stress.

Keywords: Antioxidant; Embryotoxicity; Oxidative stress; Pyrethroid; Xenopus tropicalis

Billions of kilograms of pesticides are continuously used in agricultural and non-agricultural sectors each year around the world (Listed [22]). These pesticides help to efficiently control insects or weeds; however, once released into the environment, they also increase potential threats to nontarget organisms, leading to widespread impacts on the environment (Ye et al. [42]). Studies have documented that a broad range of pesticides could induce toxicities such as immunotoxicity, neurotoxicity, and endocrine disruption to various animal species (Pašková and Hilscherová [29]; Ullah [38]). As breeding and developmental stages of some aquatic organisms such as fishes and amphibians often parallel the seasonal applications of pesticides, these chemical stressors in the environment may cause early alterations to the embryos and thus be linked to later developmental toxicity (Oros and Werner [28]). However, the underlying mechanisms related to the embryotoxicity of pesticides to amphibians are still not fully understood.

It has been suggested that intracellular redox states have a causative role in the early development of organisms (Harvey et al. [18]). Disturbance in the cellular prooxidant-antioxidant system could result in increased production of free radicals and peroxides and lead to unfavorable outcomes for embryo development as a consequence of injury to macromolecules, including proteins, nucleic acids, and lipids (Devasagayam et al. [12]). A previous study indicated that endosulfan could increase the levels of lipid peroxidation and the activities of superoxide dismutase (SOD) and catalase (CAT) in freshwater juvenile cyprinid fish (Dar et al. [10]). It has been shown that DDT is capable of stimulating the excessive production of reactive oxygen species (ROS) in human blood mononuclear cells (Pérez-Maldonado et al. [30]). Growing evidence suggests that enhanced oxidative stress following pesticide exposure could contribute to embryotoxicity in aquatic organisms (Pašková and Hilscherová [29]).

To maintain the physiological redox state, cells have evolved several biological defense mechanisms against oxidative stress caused by free radicals. One of the basic defense mechanisms is the endogenous antioxidant enzymes, such as SOD, CAT, and glutathione peroxidase (GPx), as well as other related enzymes, such as glutathione transferase (GST). These enzymes can effectively degrade free radicals and free radical–derived active products (López et al. [23]). There are also some exogenous nonenzymatic antioxidants such as vitamins and carotenoids that can prevent the oxidation of biomolecules by the oxidation of themselves (Sies and Stahl [34]). Antioxidants have a series of biological functions in protecting organisms against lipid oxidation and preventing oxidative chain reactions and thus can neutralize free radicals and attenuate oxidative stress (Abdollahi et al. [1]). Vitamins such as vitamins C and E are important nutrition for organisms to maintain normal activities and are also critical to protect against oxidative damage (Ching Kuang Chow [8]; Zaidi and Banu [46]). There are a number of studies that demonstrate the protective effects of vitamins against pesticide-induced toxicities. The increase in the levels of malondialdehyde (MDA), 4-hydroxynonenal, hydrogen peroxide, and oxidized glutathione was less pronounced when chlorpyrifos or methyl parathion was given to rats fed with a mixture of vitamins A, E, and C (Verma et al. [39]). Vitamin C treatment in imidacloprid-intoxicated male mice can decrease lipid peroxidation levels and normalize the activities of CAT and SOD (Aly et al. [3]). Thus, vitamins have been shown to be one of the most important antioxidants that modulate pesticide-induced oxidative stress in mammal models. However, essentially, no knowledge is available on the effect of vitamins against pesticide-induced toxicities on amphibians.

Pyrethroids are synthetic compounds similar to natural pyrethrins. Because of high efficiency to insects and generally low harm to human beings, pyrethroids are widely applied in agriculture, households, and public settings (Elliott et al. [15]). Research has shown that 149,537 lb of pyrethroids were used in Sacramento and San Joaquin Valley counties in 2003, and their residues were present in the aquatic systems (Oros and Werner [28]). The total occurrence of several kinds of pyrethroids in surface water worldwide has been detected up to 13,000 μg/L (Tang et al. [36]). Pyrethroids are degradable in soils or in water with half-lives ranging from 3 to 96 days aerobically and 5 to 430 days anaerobically (Laskowski [21]). Pyrethroids can persist for some time and possess different toxicities to various nontarget organisms (Mian and Mulla [26]). For example, allethrin has been shown to be cytotoxic to isolated rat testicular carcinoma cells due to alteration of antioxidant status and generation of free radicals (Madhubabu and Yenugu [24]). Bifenthrin can cause developmental toxicity, endocrine disturbance, and abnormal behavior in embryo-larval zebrafish (Jin et al. [20]). Cypermethrin can induce oxidative stress, inflammation, and apoptosis in the brains of carps (Arslan et al. [6]) and generate behavioral and morphological deformities in Duttaphrynus melanostictus at sublethal concentrations (David et al. [11]). The potential toxic effects induced by pyrethroids have been mostly investigated in fishes and mammals, with only a few studies conducted on the most threatened population of amphibians.

The aim of the present study was to investigate the toxic effects of pyrethroid, including the alteration of oxidative stress during the early development stages of amphibians, and to determine whether antioxidant supplementation could help to protect against pyrethroid-induced embryotoxicity. Lambda-cyhalothrin (LCT), which was widely used around the world and detected at levels of 346 ng/L to 1.8 μg/L in water column (Anderson et al. [4]; Tsaboula et al. [37]; Vryzas et al. [40]), was selected as a representative of pyrethroids. Vitamin E is a typical family member of the fat-soluble antioxidants, and its most abundant and biologically active form is α-tocopherol (TOC). Embryos of X. tropicalis were exposed to LCT, and co-exposure with TOC. Morphological changes and biomarkers of oxidative stress in embryos after LCT exposure with or without antioxidant were studied.

Analytical standards of LCT (≥98%, purity) and TOC (≥95.5%, purity) were purchased from Sigma Chemical (St. Louis, MO, USA). The test compounds were dissolved in the co-solvent dimethyl sulfoxide (DMSO, Sigma Chemical, St. Louis, MO, USA) and stored at 4 °C in the dark as the stock solutions. The exposure solutions were prepared from the stock solutions and diluted in Ringer's solution with a final DMSO concentration of 1‰ (V/V) immediately before exposure. Other chemicals used in the present study were of analytical grade.

Adult X. tropicalis were obtained from Nasco (Fort Atkinson, WI, USA) and maintained according to Yu et al. ([44]). The experimental procedures obeyed the standards drawn up by the University Animal Care and Use Committee. Breeding was induced by injecting with human chorionic gonadotropin (HCG, Zhejiang, China) into the dorsal lymph sac of frogs. Each male or female was first injected with 20 IU hCG, and then 100 IU hCG after 36 h of the first injection. The injected pairs were kept in a quiet and dark environment without disturbance (Yu et al. [44]). Healthy gastrula embryos between Nieuwkoop and Faber (NF) stages 10 to 11 were selected for the following experiments (Nieuwkoop and Faber [27]). Exposures were performed in 90-mm-diameter clean dishes with 20 embryos and 40 mL solutions per dish at 25 °C. Seven selected concentrations (0.08, 0.4, 2, 10, 50, 250, and 1250 μg/L) were used to quantify the dose-response embryotoxicity of LCT. To study whether TOC could prevent the toxicity of LCT, a high LCT exposure concentration of 125 μg/L, which can cause significant teratogenic effects, was chosen, and 1, 10, and 100 μg/L TOC were studied both alone and in co-exposure with LCT. Four replications were performed for each group. The exposure solutions were renewed every 24 h, and dead embryos were removed. The percentages of hatching and survival were recorded.

After 48 h of exposure, the surviving embryos were anesthetized with 100 mg/L MS-222 and then preserved in 70% alcohol for morphological observations. Embryos were observed under an Olympus SZX 7 stereomicroscope (Tokyo, Japan), and pictures were taken with a ToupCam camera (Hangzhou, Zhejiang, China). The body length was measured from the tip of the snout to the tip of the tail in ImageJ software. Incidence and characterization of different phenotypes of malformations were carefully evaluated.

The cellular details of embryonic phenotypes were further analyzed using scanning electron microscopy. The surviving embryos from the control group and 250 μg/L LCT group were fixed in 2.5% glutaraldehyde. The embryos were rinsed with 0.1 mol/L phosphate buffer (pH 7.2–7.4) three times to wash out the glutaraldehyde and fixed in 1% osmium tetraoxide solutions for 1 h. Then, these embryos were dehydrated in a graded ethanol series of 30% to 100% and acetone, successively. The samples were critical-point dried with CO2 and finally sputter-coated with platinum. Embryos were examined with an S-4800 field emission scanning electron microscope (Hitachi Limited, Tokyo, Japan).

Analysis of pro-oxidative species such as ROS and antioxidant enzymes, which are equipped to protect from cellular pro-oxidative species, can provide valuable information on oxidative stress after pesticide exposure (Pašková and Hilscherová, [29]). To further investigate the redox mechanism of embryotoxicity, the effects of single LCT exposure and co-exposure with TOC on ROS and antioxidant enzymes were assessed. After 48 h of exposure, the surviving embryos were gathered and homogenized in ice-cold buffer. The tissue homogenates were centrifuged at 2000 rpm for 10 min at 4 °C, and the supernatant was collected for subsequent determination. The activities of important antioxidant enzymes, including SOD, CAT, and GST; the level of a lipid peroxidation product, malondialdehyde (MDA); and the production of ROS, were measured by commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) to investigate oxidative stress. Intracellular protein and an indicator of cytotoxicity lactate dehydrogenase (LDH) were also analyzed using test kits (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). All measurements were performed following the protocols of the kits.

Data were expressed as the mean ± standard error of mean (SEM), and statistical significance was analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 software followed by Tukey's statistically significant difference test. Differences among means were considered statistically significant at p<0.05, 0.01, or 0.001.

After 48 h of exposure, the percentages of hatching and survival, the body length, and the total incidence of malformation were first analyzed to determine the effects of LCT on X. tropicalis embryos. No significant differences were observed in the percentage of hatching, the percentage of survival, or the body length between the control group and all LCT-treated groups (Table 1). Exposure to high concentrations of LCT induced behavioral abnormalities including twitching, spasms, and intermittent twisting. LCT also caused a concentration-dependent increase in embryonic malformation. Even at the lowest concentration of 0.08 μg/L, the total incidence of malformation significantly increased to 26.4%, which was 1.6-fold to that of the control group (p<0.01). Exposure to a higher concentration of 2 μg/L, 71.7% of embryos exhibited different malformations, and at the highest concentration of 1250 μg/L, the total incidence of embryonic malformation reached 100.0% (p<0.001). The EC50 of LCT for embryonic malformations was 0.62 μg/L. The phenotypes were further examined. The X. tropicalis larvae of the control group had well-developed notochord and pericardium, while LCT induced a variety of malformations, including bent notochord, hypopigmentation, and pericardial edema (Fig. 1). Exposure to LCT ranging from 2 to 1250 μg/L induced a significant bent notochord (Fig. 1). At a concentration of 10 μg/L, the incidence of bent notochord increased to 83.0%, which was 9.5-fold to that of the control group (p<0.001) (Table 1). At the highest concentration of 250 μg/L, the incidence increased to 10.4-fold to that of the control group, and at 1250 μg/L, the occurrence increased up to 100% (p<0.001). In the 2 μg/L and 10 μg/L groups, 50.5% and 89.3% of embryos exhibited obvious skin hypopigmentation, respectively (p<0.001) (Table 1). Surprisingly, at higher LCT concentrations from 50 to 1250 μg/L, embryonic hypopigmentation decreased from 51.0 to 17.8%. In addition, pericardial edema occurred significantly in embryos after exposure to LCT in a concentration-dependent manner, with more than 80% of embryos exhibiting this specific malformation in the 50 μg/L group. The results indicated that although there was no influence on hatch rate, survival, or body length, LCT could induce characteristic malformations in the early development of embryos even at low concentrations.

Effects of lambda-cyhalothrin exposure on hatching, survival, body length, and morphology of X. tropicalis embryos

Results are represented as the mean ± SEM of four replicates. A single asterisk, double asterisk, and triple asterisk indicate p<0.05, p<0.01, and p<0.001 compared to the control group by ANOVA, respectively

Graph: Fig. 1Teratogenic effects of lambda-cyhalothrin on X. tropicalis embryos after 48-h exposure. Abbreviations: n, notochord; sp, skin pigment; bn, bent notochord; hp, hypopigmentation; pe, pericardial edema. Scale bar=0.5 mm

To obtain details of phenotypes induced by LCT at the cell level, embryos of the control group and 250 μg/L groups were examined under scanning electronic microscope. The epidermal cells of the head were of normal shape with distinct outlines in the control group (Fig. 2a). In the 250 μg/L LCT exposure group, the cells of the head appeared irregular and could not be clearly distinguished, with some small juts observed on the surface of external cells (Fig. 2b). Moreover, the belly skin of the embryos in the control group was relatively smooth, and the surface pores were well distributed on the surface (Fig. 2c). However, in the LCT exposure group, some of the external pigment cells were extruded, and the surface pores became more obvious (Fig. 2d). In addition, compared to the control group, the surface of the tails of the exposure group was ragged and contained more tiny pores (Fig. 2e, f).

Graph: Fig. 2Scanning electron microscope photographs of specific malformations induced by lambda-cyhalothrin in X. tropicalis embryos. a, c, and e show normal embryos in the control group, and b, d, and f show multiple phenotypes in embryos exposure to 250 μg/L lambda-cyhalothrin. The arrowheads in the whole embryo box at the bottom left of each picture indicate the area of magnification. Abbreviations: h, head; sp, skin pigment; tf, tail fin

As alterations of the cellular redox state may be an important cause of embryotoxicity, various biomarkers of oxidative stress were analyzed in embryos exposed to LCT. As shown in Fig. 3a, with the increase in LCT concentration, the protein content of an individual embryo decreased significantly to 83.9% of the control in 250 μg/L groups (p<0.001). The development of embryos was affected by LCT in a concentration-dependent manner. LDH activity was significantly increased even at the lowest concentration of 0.4 μg/L (p<0.01) (Fig. 3b), which indicated that LCT could induce cytotoxicity to the embryos. CAT activity was also not significantly changed after exposure to LCT (Fig. 3c). However, the increase in LCT concentration led to higher SOD activity. At the concentrations of 10 μg/L and 250 μg/L, SOD activity was significantly induced to 1.79- and 1.84-fold to that of the control group, respectively (p<0.01) (Fig. 3d). Furthermore, the production of ROS in embryos was induced in a concentration-dependent manner. As shown in Fig. 3e, ROS production was significantly increased to 1.52-fold by 0.4 μg/L LCT (p<0.01) and induced to 2.51-fold to that of the control after exposure to 250 μg/L LCT (p<0.001). In addition, the activity of GST was significantly increased by treatment with LCT, with the highest GST activity of 2.78-fold in the 250 μg/L exposure group (p<0.001) (Fig. 3f). These results show that oxidative stress can be induced in response to LCT during early embryonic development.

Graph: Fig. 3Induction of oxidative stress in embryos after exposure to lambda-cyhalothrin. Embryos of X. tropicalis were treated with different concentrations of lambda-cyhalothrin, and the protein content (a), LDH activity (b), CAT activity (c), SOD activity (d), ROS production (e), and GST activity (f) were measured. Results are represented as the mean ± SEM. A single asterisk, double asterisk, and triple asterisk indicate p <0.05, p <0.01, and p <0.001 when compared to the control group, respectively

To determine whether TOC could protect against LCT-induced oxidative stress, related oxidative stress biomarkers were also analyzed in embryos of co-exposure groups and compared with the 125 μg/L LCT group. LDH activity increased to 1.5-fold to that of the control group (p<0.05) but was not significantly affected by TOC (Fig. 4a). However, co-exposure to TOC caused a significant rescue of the protein content that was decreased by LCT.

Graph: Fig. 4Inhibition of lambda-cyhalothrin (LCT)-induced oxidative stress by supplement with α-tocopherol (TOC) on embryos of X. tropicalis. Embryos were exposure to vehicle control, 125 μg/L LCT, and 100 μg/L TOC, as well as co-exposure to 1 μg/L, 10 μg/L, and 100 μg/L TOC along with 125 μg/L LCT. LDH activity (a), SOD activity (b), ROS production (c), and GST activity (d) were measured. A single asterisk and double asterisk indicate p <0.05 and p <0.01, relative to the control group. The capital letter above error bar indicates a significant difference of p <0.05 between LCT exposure group and co-exposure groups, and the lowercase letter indicates a significant difference of p <0.05 between the TOC exposure group and co-exposure groups

After co-exposure to 1 μg/L TOC, the activity of SOD that was increased by LCT had decreased by over 30.3%, and no significant difference was found between all co-exposure groups, the control group, and the TOC control group (Fig. 4b). Additionally, antioxidant TOC prevented LCT-induced production of ROS in a concentration-dependent manner. In the TOC control group, ROS generation was only 51.6% of the control group (p<0.01). At the co-exposure groups, the two higher concentrations (10 μg/L and 100 μg/L) of TOC significantly decreased ROS generation to the control group level, although they were still 5.8- to 7.2-fold higher than the TOC control group (p<0.05) (Fig. 4c). Moreover, the addition of TOC caused a decrease in GST activity. At a TOC concentration of 10 μg/L, the activity of GST decreased by 22.0% compared with the LCT exposure group (Fig. 4d). The results show that the antioxidant TOC can prevent LCT-induced decreases in protein content and significantly protect embryos from cellular oxidative stress.

To test whether TOC could rescue LCT-induced malformation, X. tropicalis embryos were cotreated with LCT and TOC. The mortality rates for the TOC exposure group and LCT cotreated group ranged from 0 to 1.3%, and no significant difference was found between the control group and these groups. The body length was also not significantly changed by supplementation with TOC. At a concentration of 125 μg/L, 86.8% of the embryos displayed significant changes in morphology (p<0.01). There was no significant difference between the TOC control group and the control group. Supplementation with 1 μg/L and 10 μg/L TOC did not decrease the incidence of malformation. The LCT and 100 μg/L TOC cotreated embryos showed a significantly lower malformation incidence of 63.8%, with a restoration percentage of 26.6% compared to the LCT exposure group (p<0.05) (Fig. 5a). The incidences of bent notochord and pericardial edema also decreased with increasing concentrations of TOC. With the supplementation of 100 μg/L TOC, 56.3% and 57.5% embryos displayed bent notochord and pericardial edema, which were 73.5% and 67.2% of the LCT exposure group, respectively (p<0.05) (Fig. 5b, c). However, even at the highest concentration of TOC, LCT-induced malformations could still not be completely reversed. Moreover, in the TOC control group, the occurrence of hypopigmentation was significantly averted, and 100 μg/L TOC could decrease the incidence of hypopigmentation to 3.8% of the LCT exposure group (p<0.05) (Fig. 5d). The results show that co-supplementation with a high concentration of TOC can moderately rescue the defects caused by LCT.

Graph: Fig. 5The protective effects of TOC on lambda-cyhalothrin (LCT)-induced phenotype malformations of embryos. Embryos were exposed to vehicle control, 125 μg/L LCT, and 100 μg/L TOC, as well as co-exposed to 1 μg/L, 10 μg/L, and 100 μg/L TOC along with 125 μg/L LCT. Incidence of malformation (a), bent notochord (b), pericardial edema (c), and hypopigmentation (d) were calculated. A double asterisk indicates p <0.01 compared to the control group. The capital letter indicates a significant difference of p <0.05 between LCT exposure group and co-exposure groups, and the lowercase letter indicates a significant difference of p <0.05 between the TOC exposure group and co-exposure groups

With the wide application all over the world, pyrethroids are ubiquitous in environmental matrices and accumulated in biota, and therefore become an elevated risk for organisms (Oros and Werner [28]). As amphibians are highly susceptible to pesticides, early contact with pyrethroids may lead to adverse effects on amphibian embryos. The current study demonstrated that LCT could induce various malformations and significant oxidative damage to embryos of X. tropicalis. Supplementing antioxidant vitamin E could effectively reduce LCT-induced oxidative stress in embryos, but early morphological development could not be fully rescued by high concentrations of TOC.

Growing evidence has indicated that LCT exposure is linked to toxicities on nontarget organisms, especially fishes. However, very few studies demonstrate the potential effects on amphibian embryos. The results of the current study showed that although early exposure to LCT did not significantly affect hatch rate, survival, or body growth of the embryos, different morphological deformities were caused by 0.08–1250 μg/L LCT, including bent notochord, hypopigmentation, and pericardial edema. The results were similar to those of previous study on D. melanostictus tadpoles, which reported disarray of coiled intestine and deformities in the axial and tail (David et al. [11]). Studies of other pyrethroids have shown embryotoxicity in different amphibians as well. Low concentrations of α-cypermethrin could significantly induce abnormal gut, axial and tail malformations, and edema in X. laevis embryos (Yu et al. [45]). Similar morphological changes have also been found in embryos of Bufo melanostictus and Leopard frog exposed to cypermethrin and esfenvalerate (Materna et al. [25]; Ghodageri and Pancharatna [17]). It seems that pyrethroid exposure can cause common malformations of bent notochord and tail, as well as pericardial edema. The differences in the malformation phenotypes may be due to the structure of pyrethroids, species of amphibians, and the developmental stages.

Oxidative stress is shown to contribute to the embryotoxicity generated by some widely used pesticides (Pašková and Hilscherová [29]). Excessive production of ROS is known to induce oxidative stress and result in damage to proteins, DNA, lipids, and other vital components of the cells (Schieber and Chandel [32]). SOD can prevent the accumulation of superoxide and detoxify ROS, and GSTs are a group of important enzymes involved in the detoxification of chemical toxicity and protection of cells against oxidative stress (Flohé and ötting [16]; Agianian et al. [2]; Apel and Hirt [5]). Thus, our results showed that LCT could perturb the cellular redox balance of the embryos toward oxidative stress. A similar influence was also found in embryos of D. melanostictus and R. nigromaculata after exposure to cypermethrin and α-cypermethrin (David et al. [11]; Xu and Huang [41]). A gradual increase in LDH activity showed that LCT could induce cytotoxicity in the embryos in the present study. The results were inconsistent with the study of X. laevis tadpoles, which indicated that LCT exposure caused a significant decrease in GST and LDH (Aydin-Sinan et al. [7]). The different effect on GST and LDH may be influenced by the species and the developmental stage. Coupled with the cellular changes and embryonic malformations, the results of the present study suggested that LCT was capable of inducing embryotoxicity through oxidative stress and cytotoxic responses.

As an antioxidant nutrient, vitamin E has been reported to prevent free radical damage and oxidative chain reactions, and plays an important role in the reduction of pesticide-induced oxidative stress (Ching Kuang Chow [8]; Singh et al. [35]). Results from previous studies have showed that LCT-induced GST and SOD depletion could be ameliorated by vitamin E in rabbit semen and erythrocytes (El-Demerdash [13]; Yousef [43]). Vitamin E was helpful in repairing oxidative damage induced by other pyrethroids in rat liver and the nematode C. elegans (El-Demerdash et al. [14]; Shashikumar and Rajini [33]). As observed in the present study, all concentrations of TOC significantly reduced LCT-induced protein content, SOD activity, GST activity, and ROS production in embryos compared to the control group. With increasing concentrations of TOC, more beneficial effects on the oxidative state of the embryos occurred. The findings indicated that supplementation with vitamin E could also effectively prevent oxidative stress caused by LCT in embryos of amphibian. It is remarkable that the elevatedLDH activity was not attenuated with the addition of TOC, showing that vitamin E exposure could not reduce the cytotoxicity induced by LCT. Therefore, vitamin E could successfully reduce LCT-induced oxidative stress but did not protect against cellular toxicity.

It is notable that antioxidant vitamin E could protect against the toxicities of various pollutants (Hassaan et al. [19]). Pretreatment with vitamin E led to significant improvement of pigmentation and reduction of axis malformation caused by endosulfan in zebrafish (Dale et al. [9]). Interestingly, although the protective effect of antioxidant vitamin E on oxidative stress was clearly observed during LCT exposure, only moderate alterations of embryonic morphology were found after the addition of vitamin E. No LCT-induced defects of bent notochord and pericardial edema were ameliorated except for the highest TOC concentration of 100 μg/L. It seems that the additional amount of TOC was far from sufficient to prevent embryotoxicity induced by LCT. These results could be reasonable as the cytotoxicity caused by LCT in the embryos was not rescued by TOC. These results were similar to the previous research showing that antioxidants could reduce reactive oxygen species but not embryotoxicity in the metabolic Danio rerio test (Pype et al. [31]). Thus, oxidative stress–induced damage could result in embryotoxicity but is not the only reason for the phenotypic changes in embryos of X. tropicalis. The results demonstrated that molecular pathways other than oxidative stress were changed by LCT, and the underlying mechanisms for the embryotoxicity of LCT should be further investigated.

In conclusion, the results of the present study suggested that low concentrations of LCT could cause malformations including bent notochord, hypopigmentation, and pericardial edema through induction of oxidative stress and cytotoxicity to embryos of X. tropicalis. In the early developmental stages, vitamin E could function as a powerful protective antioxidant and rescue LCT-induced oxidative stress. However, only high concentrations of vitamin E provided partial protection against morphological changes. This result corroborates the hypothesis that antioxidants could protect embryos against pyrethroid-induced embryotoxicity by decreasing oxidative stress and highlights the need for more investigations on the interactions between pesticides and antioxidants on amphibians.

This work was supported by the National Natural Science Foundation of China (No. 21407051), and Science and Technology Commission of Shanghai Municipality (Nos. 13ZR1453800, 17295810603, 18295810400).

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.