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Inhibitory effects of neurotoxin β-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus
Aquatic Toxicology 221 (2020) 105425
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Inhibitory effects of neurotoxin β-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus
T
Aifeng Lia,b,*, Jose Espinozac, Amro Hamdounc a
College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao 266100, China c Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093–0202, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: β-N-methylamino-L-alanine (BMAA) Sea urchin Lytechinus pictus Embryonic development Malformation Efflux assay
Neurotoxin β-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 μg L−1. The EC50 values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 μg L−1 (1.4 μmol L-1) and 329 μg L−1 (2.8 μmol L1 ), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 μg L−1, even up to 500 μg L−1. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 μg L−1. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.
1. Introduction Neuroscientists have paid attention to one derivative of alanine, βN-methylamino-L-alanine (BMAA), due to its toxicity to motor neurons (Rao et al., 2006; Buenz and Howe, 2007; Lobner et al., 2007). BMAA was inferred as an environmental trigger for neurodegenerative disease such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the Chamorro people in Guam (Spencer et al., 1987; Cox et al., 2003). It was a further evidence for this hypothesis that researchers detected BMAA in the brain tissues of two Canadians died from progressive neurodegenerative disease (Murch et al., 2004). So far the neurotoxin was reported in diverse cyanobacterial samples in freshwater or seawater environments (Cox et al., 2003; Johnson et al., 2008; Brand et al., 2010; Violi et al., 2019) and marine eukaryotic diatoms (Jiang et al., 2014; Réveillon et al., 2016a), which hints a risk of BMAA exposure to wild organisms in aquatic ecosystems. Additionally, BMAA has also been found in marine invertebrates, such as mussels, from different sea area (Beach et al., 2015; Réveillon et al., 2016b; Li et al., 2018). Experiments carried out on the arthropod
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Daphnia magna, and the mussels Mytilus galloprovincialis and Dreissena polymorpha, also demonstrated the accumulation of BMAA along food chains under laboratory conditions (Lürling et al., 2011; Baptista et al., 2015; Esterhuizen-Londt et al., 2015; Lepoutre et al., 2018). Negative effects of BMAA were tested on organisms in diverse trophic levels, including the macrophyte (Ceratophyllum demersum), the zooplankter (D. magna), and the brine shrimp (Artemia salina) (Lürling et al., 2011; Esterhuizen-Londt et al., 2011, 2015; Brooke-Jones et al., 2018; Purdie et al., 2009a). Therefore, the risk of BMAA exposure to marine animals should be considered and assessed to understand the ecological risk of BMAA to wild organisms. Developmental toxicity of BMAA to aquatic animals should be performed to the potential release of BMAA from its producers in water environments. For example, the neuromuscular abnormality occurred in zebrafish when the exposure concentration of BMAA was above 50 μg L−1, while the heart rate of zebrafish reduced when the exposure of BMAA increased above 500 μg L−1 (Purdie et al., 2009b). According to the animal model of developmental toxicity test, sea urchins have been accepted internationally as a standardized marine pollutant assay
Corresponding author at: College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (A. Li).
https://doi.org/10.1016/j.aquatox.2020.105425 Received 14 December 2019; Received in revised form 27 January 2020; Accepted 27 January 2020 Available online 30 January 2020 0166-445X/ © 2020 Elsevier B.V. All rights reserved.
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for more than 30 years due to some advantages such as simplicity, speed, sensitivity, unambiguity of results, uniformity and accuracy (Kobayashi, 1984). A standard ecotoxicology test procedure developed on the sea urchin embryos was adopted by Environmental Protection Agency, USA in 1995 (US EPA 600R95136). Sea urchin bioassays have been used to study diverse toxic chemicals, such as phosphate pollutants and mercuric chloride (Böttger and McClintock, 2001; Marc et al., 2002). The early development of sea urchins has also been used to study the effects of neurotoxins due to similarities between sea urchin and vertebrate development (Buznikov et al., 2001, 2007; Quiao et al., 2003). For example, the sea urchin embryos were used to test the effects of organophosphate pesticides on gene expression (Aluigi et al., 2008) and the effects of pesticide monocrotophos on serotonin metabolism (Xu et al., 2012). In addition to being a model for toxic exposure, the sea urchin embryo is also a model for cellular defense by ATP-binding cassette (ABC) transporters. More than half of the chemical defense genes that regulate ABC-transporters are expressed during embryonic or larval life stages (Goldstone et al., 2006). The apical transporters ABCB1a, ABCB4a, and ABCG2a overexpressed in the embryos of sea urchin (Stronglycentrotus purpuratus) can account for as much as 87 % of the observed efflux activity, providing a robust assay for their substrate selectivity (Gökirmak et al., 2012). The ABC transporters can be inhibited by ubiquitous pollutants found in fish and other foods due to specific binding of drug efflux transporter P-glycoprotein by pollutants, which argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals (Nicklisch et al., 2016). This study utilized the advantages of sea urchin (Lytechinus pictus) embryos to assess the effect of BMAA exposure to fertilization, development, and ABC transporter activity. Gametes and embryos were exposed to a series of BMAA concentrations and compared by microscopic observation. Possible effects of BMAA on ABC transporter activity were also assessed using a fluorescent substrate efflux assay.
albumin) were used to culture embryos. First, fifty healthy eggs of sea urchin were picked up and added to 9.5 mL FSW in each well. Then, BMAA stock solution was spiked to reach 10, 50, 100, 300 and 500 μg L−1 in 10 mL final volume. Meanwhile, a volume of 2 mmol L−1 HCl equivalent to the highest concentration of BMAA (500 μg L−1) was added to well as a solvent control treatment (denoted as 0 μg L−1 of BMAA). Finally, the diluted sperm was added to wells to start fertilization of eggs. Total larvae and active swimming larvae were counted under an inverted anatomical microscope after 96 h post fertilization (HPF). Every treatment was repeated in triplicate in one batch exposure test and total three batches of exposure tests were calculated in this experiment.
2. Materials and methods
2.6. Efflux assay for ABC transporter exposed to BMAA
2.1. Chemicals
Calcein-AM (CAM) is membrane permeable and non-fluorescent. After entry into the cell intercellular esterase activity cleaves the -AM moiety, leaving membrane impermeable calcein. Cells with active ABCB and ABC-C transporters efflux CAM, whereas cells with reduced ABC transporter activity accumulate fluorescent calcein. Embryos were incubated in CAM and intracellular calcein accumulation was measured using a Zeiss LSM-700 laser scanning confocal microscope (Jena, Germany) equipped with a Zeiss Plan APOChromat 20× air objective (numerical aperture, 0.8). At 5 HPF embryos were exposed to CAM and 50 μg L−1 BMAA or 2 mmol L−1 HCl for 90 min. The exposed embryos were washed 10 times with FSW to remove the background fluorescence and incubated for an additional 30 min before imaging. Two mmol L−1 HCl and 50 μg L-1 BMAA was added in treatment-1 and -2, respectively. Based on the results of treatment-1 and -2, additional ABCB transporter inhibitor 2 μmol L-1 PSC833 was added in the treatment-3 and -4, in order to calculate the efflux activity (discharge efficiency, DE, %). More than 15 embryos were imaged in each treatment. Micrographs were prepared and analyzed with ImageJ 1.47v (National Institutes of Health). The fluorescence intensity of micrographs of exposed embryos with and without PSC833 was calculated as FIEP and FIE, respectively. The DE value was calculated using the formula, DE = 100-(FIE/ FIEP)×100.
2.4. Effect of BMAA on the fertility rate of sea urchin sperm Healthy eggs from one female and healthy sperm from three different individuals were used to test the effect of BMAA on the fertility rate of sea urchins. Fifty healthy eggs were added to 10 mL FSW. Sperm was diluted as follows: 5 μL concentrated sperm was added to 15 mL FSW, and 5 mL of this dilution was added to 25 μL FSW, 2 mM HCl or 0.1 mg mL−1 BMAA, respectively. The sperm dilution was kept 5 and 10 min in room temperature before it was used to start fertilization. Fertilization rates of eggs were calculated using the numbers of twocells embryos for each treatment. 2.5. Observation for embryo development of sea urchins exposed to BMAA Sea urchin embryos exposed to 500 μg L−1 BMAA at different developmental stages were compared with embryos in the solvent control group using a laser scanning confocal microscope (LSM 700, Zeiss). Normal development morphology for sea urchin embryos at early cleavage division, blastula, gastrula, and prism larvae and pluteus larvae stages were recorded in this study. Abnormal development of sea urchin embryos exposed to BMAA were also carefully observed.
Hydrochloric acid (HCl) and L-BMAA hydrochloride (B107, C4H10N2O2·HCl, 10 mg) were purchased from Sigma-Aldrich (Oakville, ON, Canada). The solution of 2 mmol L−1 HCl was used to dissolve BMAA at 0.1 mg mL−1 as a stock solution. Calcein-AM (CAM), plasma membrane permeable substrate of ABCB/P-gp and ABCC/MRP-type ABC transporters of echinoderm eggs and embryos, was obtained from Biotium (Hayward, CA). ABC-B inhibitor PSC833 was purchased from Millipore Sigma (St. Loius, MO). Stock solutions of CAM and PSC833 were prepared in dimethyl sulfoxide (DMSO) and diluted to the final concentrations in filtered seawater (0.22 μm, FSW). The final DMSO concentration in the assays did not exceed 0.5 %. Pure water (18.2 MΩ cm resistivity or better) was purified by a Milli-Q ultrapure water system (Millipore Corp., Billerica, MA, USA). 2.2. Echinoderm collection, gamete gathering and embryo culture Adults of sea urchin (Lytechinus pictus) collected from La Jolla (CA, USA) were held between 10 °C and 12 °C in flow-through seawater aquaria. Selected animals were stimulated to spawn by intra-coelomic injection of 0.55 mol L−1 KCl. Eggs obtained from females were washed twice with 0.22 μm FSW and diluted to either a 1 % (v/v) suspension, or to 500 eggs per mL in FSW. They were fertilized in a final sperm dilution of 1:250,000.
2.7. Statistical analysis The experiments of embryos to BMAA were conducted and repeated in three batches with each batch test containing three repetitions of each treatment. The exposure experiments for sperm were carried out in six repetitions, and the efflux assay for ABC transporter was tested in
2.3. Exposure experiments for sea urchin embryos to BMAA Six-well plates previously washed with 1 % BSA (bovine serum 2
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Fig. 2. Fertility rate (%) of sea urchin (Lytechinus pictus) eggs caused by sperms exposed to solvent HCl (∼10 μmol L−1) or BMAA (∼500 μg L−1) for 5 and 10 min. Note: Five μL of original sperms was diluted by 15 mL of filtered seawater, and part of 5 mL of the diluent sperms was added by 25 μL of 2 mmol L−1 HCl and 0.1 mg mL−1 BMAA used as solvent control and BMAA exposure, respectively. Data of each treatment were obtained from six repetitions, and * means significantly different from the control at p < 0.05 (one-way ANOVA).
300 μg L−1, except for the highest concentration 500 μg L−1 (Fig. 1B). The EC50 values of active swimming larvae and total larvae indexes were roughly inferred by an exponential equation as 165 μg L−1 (1.4 μmol L-1) and 329 μg L−1 (2.8 μmol L-1), respectively. The fertilization ability of sea urchin sperm exposed to BMAA is shown in Fig. 2. No significant difference occurred between blank control and solvent control treatments, which indicates that the additional low concentration of HCl did not affect fertilization. However, the fertilization ability of sea urchin sperm was significantly decreased (ANOVA, p < 0.05) after exposure to BMAA at 500 μg L−1 for 10 min. Fig. 1. Mortality (%) of sea urchin (Lytechinus pictus) embryos (A) and the percent of active swimming larvae in the surviving embryos (B) exposed to different concentrations of BMAA at 96 HPF (hours post fertilization). Note: An equivalent volume of 2 mmol L−1 HCl to the exposure group of 500 μg L−1 was added into the culture well as solvent control group marked as the concentration of BMAA at zero. Data of each treatment were obtained from the average of three batches of experiments and each batch contained different treatments in triplicate. Different letter shows the significantly different from each treatment at p < 0.05 (one-way ANOVA).
3.2. No effect of BMAA on the ABC transporter activity of sea urchin embryos Accumulation of calcein dye and discharge efficiency of ABC transporter in sea urchin embryos are shown in Fig. 3. Fluorescence intensity, an indicator of calcein accumulation and low transport activity, extremely significantly (ANOVA, p < 0.01) increased when the ABC-B specific inhibitor PSC833 was present in treatment-3 and -4. But no significant difference was observed between the solvent control group (adding 2 mmol L−1 HCl) and BMAA exposure group (50 μg L−1) in three tests. A higher concentration of BMAA exposure, 500 μg L−1, was also analyzed in one test, but still no obvious difference for the accumulation of calcein in embryos (data not shown).
triplicate. Statistical analysis was performed using the IMB SPSS Statistics V25.0 software (IBM Corporation, New York, USA). If assumptions of homogeneity of variances were satisfied, one-way analysis of variance (ANOVA) followed by LSD’s test was used. A P-value < 0.05 was considered to be statistically significant. 3. Results
3.3. Abnormal division and malformation development of embryos exposed to BMAA
3.1. Inhibitory effects of BMAA on embryonic development and sperm fertilization
Normal morphologic images of sea urchin in the early 96 HPF development stage in the solvent control (10 μmol L−1 HCl) are shown in Fig. 4. The embryo started the first cleavage after about 2 HPF at room temperature 25 °C and finished the first three cleavage divisions in 3 HPF. Then the embryo developed to the blastula and gastrula stage at about 7 and 24 HPF, respectively. Finally, the embryo developed as a four-arm pluteus larva at 96 HPF. Selected images of sea urchin embryos in the solvent blank control group (10 μmol L−1 HCl) and BMAA exposure group (500 μg L-1) after 24 HPF and 31 HPF are shown in Fig. 5. Some selected abnormal division and malformation development of sea urchin embryos exposed to BMAA (500 μg L-1) are shown in Fig. 6.
The effect of chronic BMAA exposure on mortality and normal development was determined using a 96-h exposure to various BMAA concentrations (Fig. 1). The mortality was calculated by averaging three independent tests with each test containing triplicate treatments. No significant effects on mortality were found in the treatments of embryos exposed to BMAA at 100 μg L−1 or lower, while the mortality significantly (ANOVA, p < 0.05) increased when the concentration of BMAA increased above 300 μg L−1 (Fig. 1A). However, the percentage of swimming larvae in the survived embryos were not significantly (ANOVA, p > 0.05) reduced when BMAA concentrations increased to 3
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(Fig. 1). However, there was a significant effect on mortality in BMAA exposures at 300 and 500 μg L−1. Observation of embryos exposed to BMAA at the highest concentration 500 μg L−1 only found that the percent of actively swimming four-armed plutei in the surviving embryos significantly decreased, while the other positive treatments for BMAA (≤ 300 μg L−1) were not significantly different from the control group. This phenomenon hints that the primary motor nerves controlling ciliary beating were not obviously destroyed by BMAA at the blastula stage in the experiment. But the standard deviations of the percent of active swimming larvae amplified when the concentration of BMAA ≥ 50 μg L−1, which demonstrated that the swimming ability was slightly inhibited by higher concentrations of BMAA. The roughly calculated EC50 value of active swimming larvae (165 μg L−1) was much lower than that of larval mortality (329 μg L−1), which also indicates that BMAA reduced the development of active swimming larvae of sea urchin. Some larvae only in situ rotated like a peg-top in the culture cell with higher concentration of BMAA (1000 μg L−1) although they developed to the two-armed pluteus stage at 96 HPF. Wada et al. (1997) reported that the neurotransmitters dopamine decreased, but serotonin increased, the beat frequency averaged over the ciliated epithelium of sea urchins (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) at the four-armed pluteus stage, which suggests that both the stability and the direction of ciliary beating are under nervous control. The serotonergic nervous system was also suggested to regulate the ciliary movement in mollusks and pond snails due to the association of cilia and the end of axons from serotonergic cells (Uhler et al., 2000; Kuang and Goldberg, 2001; Kuang et al., 2002). However, the irreversible inhibitor of tryptophan 5-hydroxylase, p-chlorophenylalanine (CPA), exclusively perturbed synthesis of serotonin but not nervous system organization, and CPA-treated sea urchin (H. pulcherrimus) larvae did not swim, despite the occurrence of ciliary beating in culture chamber (Yaguchi and Katow, 2003). Thus serotonin is not essential for ciliary beating but is necessary for swimming behavior. It is possible that catecholaminergic neurons, such as dopaminergic neurons or peptidergic neurons, may be involved in ciliary beating (Yaguchi and Katow, 2003). The SH-SY5Y neuroblastoma cells exposed to BMAA showed that BMAA perturbed alanine, aspartate and glutamate metabolism pathway, arginine and proline metabolism pathway, as well as in specific intermediary metabolites such as GABA and taurine which were involving several neurotransmitters/neuromodulators (Engskog et al., 2017). Rats treated with BMAA displayed alterations in the levels of glutamate, GABA and taurine in the motor cortex, which hints the excitotoxic mechanism due to loss of GABAergic inhibition involved in ALS in humans (de Munck et al., 2015). While BMAA-fed flies demonstrated the neurotoxin prolonged the NMDAR open time, which would allow excessive Ca2+ entry into the postsynaptic cell and lead to neurodegenerative effects (Koenig et al., 2015). Therefore, we hypothesized that the neurotoxin BMAA possibly disrupted the synthesis of dopamine and serotonin in the early embryo of sea urchin in this study. Effect of BMAA on the activity of these synthesis pathways should be explored in future studies. Fertilization percentage was significantly decreased when sperm was exposed to BMAA at 500 μg L−1 for 10 min (Fig. 2). The sulfated polysaccharides in the jelly coat surrounding the egg play a role in an initial step to induce the acrosome reaction of sea urchins (Alves et al., 1997). A high level of internal ATP needs to be maintained causing the stable sperm binding to induce a successful fertilization event (Hirohashi and Lennarz, 1998). Faster sperm of sea urchin (Lytechinus variegatus) demonstrated higher fertilization rates than slower sperm (Levitan, 2000). The mitochondrion swelled and shifted to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface after the occurrence of acrosome reaction (Kazama et al., 2006). A significant cytotoxic effect of BMAA on fish immune cell line (CLC) was also confirmed (Sieroslawska and Rymuszka, 2019), which was characterized by the reduction of cell membrane integrity and an increasement of ATP level in cells exposure to BMAA. Therefore, the
Fig. 3. Fluorescence intensity of Calcein-AM accumulated by embryos (A) and discharge efficiency (B) tested by the efflux assay of sea urchin (Lytechinus pictus) ABC-transporter with or without BMAA exposure. Data of each treatment were obtained from triplicate tests, and very significantly difference between Treat 1 - Treat 3, and Treat 2 - Treat 4, respectively (A). Average efflux assay of ABC-transporter exposed to BMAA was not significantly different from the control (B), p > 0.05 (one-way ANOVA).
4. Discussion The sea urchin has been adopted as a standard animal model for marine ecotoxicology (US EPA 600R95136). Just as in the mammalian central nervous system, sea urchin embryos can synthesize, store and release neurotransmitters like acetylcholine, serotonin (5 H T), dopamine and norepinephrine, and possess homologous populations of receptors and their downstream signaling cascades (Buznikov et al., 2007). The chemical reagents that target specific neurotransmitter mechanisms or signaling cascades in the mammalian brain have counterparts in “pre-nervous” morphogenetic anomalies in the developing sea urchin (Shmukier, 1993; Buznikov et al., 2001; Quiao et al., 2003). The effects of exogenous chemicals on the early development of sea urchin embryos could reflect drug-induced damage to mammalian neurons to some degree, and cell signaling during sea urchin development could be adopted to assess toxicity of environmental contaminants (Angelini et al., 2005). Considering that BMAA has adverse effects on motor neurons in some animal models (Rao et al., 2006; Buenz and Howe, 2007; Lobner et al., 2007), the early development of embryos of sea urchin (Lytechinus pictus) after exposure to the neurotoxin BMAA was observed in this study. No significant inhibitory effects were found on the fertilization and development of sea urchin embryos exposed to BMAA ≤ 100 μg L−1 4
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Fig. 4. Confocal microscopy observation for normal sea urchin (Lytechinus pictus) embryos in the early 96 h development stage, ‘HPF’ means hours post-fertilization, scale bar equals to 50 μm.
sea urchin blastulas at the early development stage. The sea urchin (L. pictus) embryos quickly developed in the control group at room temperature, 25 °C, in this study. The first three cleavage divisions started at 2 HPF and finished at 3 HPF, then the embryo entered into blastula stage at about 7 HPF (Fig. 4). The two-armed pluteus and four-armed pluteus could be clearly observed at 48 and 96 HPF, respectively (Fig. 4). Selected gastrulas at 24 HPF and early two-armed pluteus were compared in the control group and exposure group at 500 μg L−1 (Fig. 5). Under the observation of confocal microscopy, many primary mesenchyme cells (PMCs) could be seen to organize the skeletal rods in the gastrula stage (Fig. 5B) and two arms normally developed with the organization of skeletal rods (Fig. 5C and D) of sea urchin in the control group. However, fewer PMCs entered the blastocoel the skeletal rods did not form in the BMAA exposure group (Fig. 5E-H). In the control group the pigment granules were dispersed throughout the larva (Fig. 5C and D), but only a few black granules were distributed along the ring of the edge of sea urchin gastrula
ability of the mitochondrion to maintain high ATP levels is very important for high fertilization ability of sea urchin sperm. One possible mechanism for the reduction in fertilization percentage observed in the present study could be that BMAA inhibited the synthesis of ATP in sperm mitochondria. This mechanism should be investigated in future studies. CAM is a commonly used, membrane permeable substrate for ABC-B and ABC-C subfamily transporters in sea urchin research (Gökirmak et al., 2012, 2014). Cells with high ABC-B and ABC-C transporter activity would accumulate less calcein and be less fluorescent (Gökirmak et al., 2014). Without the ABC-B specific inhibitor PSC833 (treatments 1 and 2) sea urchin embryos accumulated very little calcein (Fig. 3A), however there was no significant difference between solvent control and BMAA exposed embryos. No significant difference in discharge efficiency was found between the sea urchin blastulas exposed to control solvent and BMAA (Fig. 3B). Results showed that BMAA did not affect the ABC-B and ABC-C transporter activity in the cytomembrane of 5
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Fig. 5. Confocal microscopy observation for sea urchin (Lytechinus pictus) embryos exposed to 10 μmol L−1 dissolvent HCl (A–D) and BMAA toxin at 500 μg L-1 (E–H) after 24 h (A, B, E, F) and 31 h (C, D, G, H) post-fertilization, scale bar equals to 50 μm.
first cleavage division (Fig. 6A), and some embryos underwent the-third cleavage division with unnormal arrangement of cells (Fig. 6B). Some eggs leaked cytoplasm and did not successfully fertilize in the BMAA exposure group (Fig. E). Some tenacious embryos slowly developed to blastula and two-armed pluteus at 24 HPF (Fig. 6F) and 48 HPF
exposed to BMAA (Fig. 5G and H). Some embryos treated with BMAA at 500 μg L−1 developed to the gastrula stage by 24 HPF, but few embryos had become two-armed plutei by 31 HPF. Most embryos exposed to 500 μg L−1 BMAA were arrested at different developmental stage with various malformation (Fig. 6). Some embryos could not complete the 6
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Fig. 6. Confocal microscopy observation for abnormal sea urchin (Lytechinus pictus) embryos exposed to BMAA of 500 μg L−1 in the early 48 h development stage, ‘HPF’ means hours post-fertilization, scale bar equals to 50 μm.
hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices, which demonstrated a significant decrease of PP2A activity and a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites such as Tyr307. BMAA has been directly proven to be transferred into astrocytes and neurons cells through cystine/glutamate antiporters (system xc−), which resulted in the imbalance of cystine uptake and a reduction of glutathione levels (Albano and Lobner, 2018). Therefore, one explanation for our data is that BMAA possibly affected the stability of microtubules in the mitotic spindle. This toxicological mechanism should be confirmed in future studies. It is unclear whether BMAA exposure would affect the differentiation on the neurogenic ectoderm. The Onecut gene is exclusively expressed in cells of the emerging cilliary band from late blastula stage. In later development Onecut is predominantly expressed in the cilliary band, but is also expressed in the apical organ and the elongating arms in the early pluteus stage (Poustka et al., 2004). Prominent, beating cilia were still observed in the abnormal gastrulas or pluteus exposed to BMAA, which suggested that the differentiation of the neurogenic ectoderm and ciliary band were not affected by BMAA in the early
(Fig. 6G-I), respectively. However, these tenacious embryos did not develop healthy organs such as mouth, stomach, and anus due to BMAA exposure. BMAA has been reported to affect all the main glutamate receptors, i.e., NMDA, AMPA/kainate, and metabotropic receptors (Lobner, 2009). In this study, BMAA had the following adverse effects on sea urchin development: (1) the fertilization process was arrested, (2) the first three cleavage divisions could not be normally arranged for embryos, and (3) late development of sea urchin blastulas was obviously inhibited by the neurotoxin BMAA. Exposure of sperm to BMAA decreased the fertilization rate, possibly by inhibiting intercellular ATP synthesis (Kazama et al., 2006). Our observations suggested that mitosis could not occur normally in many of the embryos exposed to BMAA. The mitotic spindle, the machinery for the separation of chromosomes during mitosis, mainly consists of microtubules (MTs) (Collins and Vallee, 1986). It has been well documented that MT disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed (Larkin and Danilchik, 1999). Arif et al. (2014) studied the effect of BMAA on PP2A activity and tau 7
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development of sea urchin. Sea urchin embryos can synthesize, store and release neurotransmitters like acetylcholine, serotonin, dopamine and norepinephrine, which take part in cell differentiation and morphological assembly (Buznikov et al., 2007). The initial cleavage divisions of the sea urchin embryo are controlled in part by serotonin responsive mechanisms (Shmukler, 1993). The toxic effects of BMAA that we have observed in the early development of sea urchin embryos possibly occur by disrupting the synthesis or function of neurotransmitters, especially of serotonin. This mechanism will be confirmed in a future study.
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Engskog, M.K.R., Ersson, L., Haglöf, J., Arvidsson, T., Pettersson, C., Brittebo, E., 2017. β‑N‑methylamino‑l‑alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling. Amino Acids 49, 905–919. Esterhuizen-Londt, M., Pflugmacher, S., Downing, T.G., 2011. β-N-Methylamino-L-alanine (BMAA) uptake by the aquatic macrophyte Ceratophyllum demersum. Ecotoxicol. Environ. Safe. 74 (1), 74–77. Esterhuizen-Londt, M., Wiegand, C., Downing, T.G., 2015. β-N-methylamino-L-alanine (BMAA) uptake by the animal model, Daphnia magna and subsequent oxidative stress. Toxicon 100, 20–26. Gökirmak, T., Campanale, J.P., Shipp, L.E., Moy, G.W., Tao, H., Hamdoun, A., 2012. Localization and substrate selectivity of sea urchin multidrug (MDR) efflux transporters. J. Biol. Chem. 287 (52), 43876–43883. Gökirmak, T., Shipp, L.E., Campanale, J.P., Nichlisch, S.C.T., Hamdoun, A., 2014. Transport in technicolor: Mapping ATP-binding cassette transporters in sea urchin embryos. Mol. Reprod. Dev. 81, 778–793. Goldstone, J.V., Hamdoun, A., Cole, B.J., Howard-Ashby, M., Nebert, D.W., Scally, M., Dean, M., Epel, D., Hahn, M.E., Stegeman, J.J., 2006. The chemical defensome: Environmental sensing and response genes in theStrongylocentrotus purpuratus genome. Dev. Biol. 300, 366–384. Hirohashi, N., Lennarz, W.J., 1998. Sperm–egg binding in the sea urchin: a high level of intracellular ATP stabilizes sperm attachment to the egg receptor. Dev. Biol. 201, 270–279. Jiang, L., Eriksson, J., Lage, S., Jonasson, S., Shams, S., Mehine, M., Ilag, L.L., Rasmussen, U., 2014. Diatoms: a novel source for the neurotoxin BMAA in aquatic environments. PLoS One 9 (1), e84578. Johnson, H.E., King, S.R., Banack, S.A., Webster, C., Callanaupa, W.J., Cox, P.A., 2008. Cyanobacteria (Nostoc commune) used as a dietary item in the Peruvian highlands produce the neurotoxic amino acid BMAA. J. 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5. Conclusions A toxic effect of BMAA on the early development of sea urchin embryos was found and discussed for the first time in this study. Fertilization and development of sea urchin embryos were significantly inhibited by BMAA above 300 μg L−1. The EC50 values calculated for active swimming larvae and larval mortality at 96 HPF was 165 μg L−1 (1.4 μmol L−1) and 329 μg L−1 (2.8 μmol L-1), respectively. Fertilization ratio also significantly decreased resulting from sperm exposed to BMAA for 10 min. Abnormal divisions and development arrest occurred at different developmental stages in the treatment of BMAA at 500 μg L−1. Results suggested that the intercellular ATP synthesis of sperm, cell division, and the function of neurotransmitters such as serotonin of embryos were possibly affected and disrupted by BMAA. Multiple mechanisms may underlie the effects of BMAA on the early development of sea urchin embryos. CRediT authorship contribution statement Aifeng Li: Conceptualization, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Jose Espinoza: Methodology, Writing - review & editing. Amro Hamdoun: Methodology, Resources, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant # 41676093) and the National Study Abroad Fund Sponsored by China Scholarship Council (Grant # 201706335006). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquatox.2020.105425. References Albano, R., Lobner, D., 2018. Transport of BMAA into neurons and astrocytes by system xc−. Neurotox. Res. 33, 1–5. Aluigi, M.G., Angelini, C., Corte, G., Falugi, C., 2008. The sea urchin, Paracentrotus lividus, embryo as a “bioethical” model for neurodevelopmental toxicity testing, Effects of diazinon on the intracellular distribution of OTX2-like proteins. Cell Biol. Toxicol. 24, 587–601. Alves, A.P., Mulloy, B., Diniz, J.A., Mourao, P.A.S., 1997. Sulfated polysaccharides from the jelly layer are species-specific inducers of acrosome reaction in sperms of sea urchins. J. Biol. Chem. 272, 6965–6971. Angelini, C., Aluigi, M.G., Sgro, M., Trombino, S., Thielecke, H., Falugi, C., 2005. Cell signalling during sea urchin development: a model for assessing toxicity of environmental contaminants (book chapter). In: Matranga, V. (Ed.), Progress in Molecular and Subcellular Biology, pp. 45–70 39. Arif, M., Kazim, S.F., Grundke-Iqbal, I., Garruto, R.M., Iqbal, K., 2014. Tau pathology
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The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity. Environ. Health Persp. 111 (14), 1730–1735. Rao, S.D., Banack, S.A., Cox, P.A., Weiss, J.H., 2006. BMAA selectively injures motor neurons via AMPA/kainate receptor activation. Exp. Neurol. 201, 244–252. Réveillon, D., Séchet, V., Hess, P., Amzil, Z., 2016a. Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum, Chaetoceros sp., Chaetoceros calcitrans and, Thalassiosira pseudonana) and bacteria isolated from a diatom culture. Harmful Algae 58, 45–50. Réveillon, D., Séchet, V., Hess, P., Amzil, Z., 2016b. Systematic detection of BMAA (b-Nmethylamino-L-alanine) and DAB (2,4-diaminobutyric acid) in mollusks collected in shellfish production areas along the French coasts. Toxicon 110, 35–46. Shmukier, Y.B., 1993. Possibility of membrane reception of neurotransmitter in sea urchin early embryos. Comp. Biochem. Phys. C 106, 269–273. Sieroslawska, A., Rymuszka, A., 2019. Assessment of the cytotoxic impact of cyanotoxin beta-N-methylamino-L-alanine on a fish immune cell line. Aquat. Toxicol. 212, 214–221. Spencer, P.S., Nunn, P.B., Hugon, J., Ludolph, A.C., Ross, S.M., Roy, D.N., Robertson, R.C., 1987. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science 237 (4814), 517–522. Uhler, G.C., Huminski, P.T., Les, F.T., Fong, P.P., 2000. Cilia-driven rotational behavior in gastropod (Physa elliptica) embryos induced by serotonin and putative serotonin reuptake inhibitors (SSRIs). J. Exp. Zool. 286, 414–421. Violi, J.P., Mitrovic, S.M., Colville, A., Main, B.J., Rodgers, K.J., 2019. Prevalence of βmethylamino-L-alanine (BMAA) and its isomers in freshwater cyanobacteria isolated from eastern Australia. Ecotoxicol. Environ. Safe. 172, 72–81. Wada, Y., Mogami, Y., Baba, S.A., 1997. Modification of ciliary beating in sea urchin larvae induced by neurotransmitters: beat-plane rotation and control of frequency fluctuation. J. Exp. Biol. 200 (1), 9–18. Xu, L., Tian, H., Wang, W., Ru, S., 2012. Effects of monocrotophos pesticide on serotonin metabolism during early development in the sea urchin, Hemicentrotus pulcherrimus. Environ. Toxicol. Pharmacol. 34, 537–547. Yaguchi, S., Katow, H., 2003. Expression of tryptophan 5-hydroxylase gene during sea urchin neurogenesis and role of serotonergic nervous system in larval behavior. J. Comp. Neurol. 466, 219–229.
F., Faassen, E., Geffard, A., Lance, E., 2018. Genotoxic and cytotoxic effects on the immune cells of the freshwater bivalve Dreissena polymorpha exposed to the environmental neurotoxin BMAA. Toxins 10 (3), 106. Levitan, D.R., 2000. Sperm velocity and longevity trade off each other and influence fertilization in the sea urchin Lytechinus variegatus. P. Roy. Soc. B Biol. Sci. 267 (1443), 531–534. Li, A., Hu, Y., Song, J., Wang, S., Deng, L., 2018. Ubiquity of the neurotoxin β-N-methylamino-L-alanine and its isomers confirmed by two different mass spectrometric methods in diverse marine mollusks. Toxicon 151, 129–136. Lobner, D., 2009. Mechanisms of beta-N-methylamino-L-alanine induced neurotoxicity. Amyotroph. Lateral Scler. 10 (Suppl 2), 56–60. Lobner, D., Piana, P.M.T., Salous, A.K., Peoples, R.W., 2007. β-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. Neurobiol. Dis. 25, 360–366. Lürling, M., Faassen, E.J., Eenennaam, J.S.V., 2011. Effects of the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) on the survival, mobility and reproduction of Daphnia magna. J. Plankton Res. 33, 333–342. Marc, J., Maguer, C., Bellé, R., Mulner-Lorillon, O., 2002. Sharp dose- and time-dependent toxicity of mercuric chloride at the cellular level in sea urchin embryos. Arch. Toxicol. 76, 388–391. Murch, S.J., Cox, P.A., Banack, S.A., Steele, J.C., Sacks, O.W., 2004. Occurrence of βmethylamino-L-alanine (BMAA) in ALS/PDC patients from Guam. Acta Neurol. Scand. 110, 267–269. Nicklisch, S.C.T., Rees, S.D., McGrath, A.P., Gökirmak, T., Bonito, L.T., Vermeer, L.M., Cregger, C., Loewen, G., Sandin, S., Chang, G., Hamdoun, A., 2016. Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure. Sci. Adv. 2, e1600001. Poustka, A.J., Kühn, A., Radosavljevic, V., Wellenreuther, R., Lehrach, H., Panopoulou, G., 2004. On the origin of the chordate central nervous system: expression of onecut in the sea urchin embryo. Evol. Dev. 6 (4), 227–236. Purdie, E.L., Samsudin, S., Eddy, F.B., Codd, G.A., 2009a. Effects of the cyanobacterial neurotoxin β-N-methylamino-L-alanine on the early-life stage development of zebrafish (Danio rerio). Aquat. Toxicol. 95 (4), 279–284. Purdie, E.L., Metcalf, J.S., Kashmiri, S., Codd, G.A., 2009b. Toxicity of the cyanobacterial neurotoxin β-N-methylamino-L-alanine to three aquatic animal species. Amyotroph. Lateral Sc. 10 (sup2), 67–70. Quiao, D., Nikitina, L.A., Buznikov, G.A., Lauder, J.M., Seidler, F.J., Slotkin, T.A., 2003.
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Inhibitory effects of neurotoxin β-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus
T
Aifeng Lia,b,*, Jose Espinozac, Amro Hamdounc a
College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao 266100, China c Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093–0202, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: β-N-methylamino-L-alanine (BMAA) Sea urchin Lytechinus pictus Embryonic development Malformation Efflux assay
Neurotoxin β-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 μg L−1. The EC50 values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 μg L−1 (1.4 μmol L-1) and 329 μg L−1 (2.8 μmol L1 ), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 μg L−1, even up to 500 μg L−1. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 μg L−1. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.
1. Introduction Neuroscientists have paid attention to one derivative of alanine, βN-methylamino-L-alanine (BMAA), due to its toxicity to motor neurons (Rao et al., 2006; Buenz and Howe, 2007; Lobner et al., 2007). BMAA was inferred as an environmental trigger for neurodegenerative disease such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the Chamorro people in Guam (Spencer et al., 1987; Cox et al., 2003). It was a further evidence for this hypothesis that researchers detected BMAA in the brain tissues of two Canadians died from progressive neurodegenerative disease (Murch et al., 2004). So far the neurotoxin was reported in diverse cyanobacterial samples in freshwater or seawater environments (Cox et al., 2003; Johnson et al., 2008; Brand et al., 2010; Violi et al., 2019) and marine eukaryotic diatoms (Jiang et al., 2014; Réveillon et al., 2016a), which hints a risk of BMAA exposure to wild organisms in aquatic ecosystems. Additionally, BMAA has also been found in marine invertebrates, such as mussels, from different sea area (Beach et al., 2015; Réveillon et al., 2016b; Li et al., 2018). Experiments carried out on the arthropod
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Daphnia magna, and the mussels Mytilus galloprovincialis and Dreissena polymorpha, also demonstrated the accumulation of BMAA along food chains under laboratory conditions (Lürling et al., 2011; Baptista et al., 2015; Esterhuizen-Londt et al., 2015; Lepoutre et al., 2018). Negative effects of BMAA were tested on organisms in diverse trophic levels, including the macrophyte (Ceratophyllum demersum), the zooplankter (D. magna), and the brine shrimp (Artemia salina) (Lürling et al., 2011; Esterhuizen-Londt et al., 2011, 2015; Brooke-Jones et al., 2018; Purdie et al., 2009a). Therefore, the risk of BMAA exposure to marine animals should be considered and assessed to understand the ecological risk of BMAA to wild organisms. Developmental toxicity of BMAA to aquatic animals should be performed to the potential release of BMAA from its producers in water environments. For example, the neuromuscular abnormality occurred in zebrafish when the exposure concentration of BMAA was above 50 μg L−1, while the heart rate of zebrafish reduced when the exposure of BMAA increased above 500 μg L−1 (Purdie et al., 2009b). According to the animal model of developmental toxicity test, sea urchins have been accepted internationally as a standardized marine pollutant assay
Corresponding author at: College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (A. Li).
https://doi.org/10.1016/j.aquatox.2020.105425 Received 14 December 2019; Received in revised form 27 January 2020; Accepted 27 January 2020 Available online 30 January 2020 0166-445X/ © 2020 Elsevier B.V. All rights reserved.
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for more than 30 years due to some advantages such as simplicity, speed, sensitivity, unambiguity of results, uniformity and accuracy (Kobayashi, 1984). A standard ecotoxicology test procedure developed on the sea urchin embryos was adopted by Environmental Protection Agency, USA in 1995 (US EPA 600R95136). Sea urchin bioassays have been used to study diverse toxic chemicals, such as phosphate pollutants and mercuric chloride (Böttger and McClintock, 2001; Marc et al., 2002). The early development of sea urchins has also been used to study the effects of neurotoxins due to similarities between sea urchin and vertebrate development (Buznikov et al., 2001, 2007; Quiao et al., 2003). For example, the sea urchin embryos were used to test the effects of organophosphate pesticides on gene expression (Aluigi et al., 2008) and the effects of pesticide monocrotophos on serotonin metabolism (Xu et al., 2012). In addition to being a model for toxic exposure, the sea urchin embryo is also a model for cellular defense by ATP-binding cassette (ABC) transporters. More than half of the chemical defense genes that regulate ABC-transporters are expressed during embryonic or larval life stages (Goldstone et al., 2006). The apical transporters ABCB1a, ABCB4a, and ABCG2a overexpressed in the embryos of sea urchin (Stronglycentrotus purpuratus) can account for as much as 87 % of the observed efflux activity, providing a robust assay for their substrate selectivity (Gökirmak et al., 2012). The ABC transporters can be inhibited by ubiquitous pollutants found in fish and other foods due to specific binding of drug efflux transporter P-glycoprotein by pollutants, which argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals (Nicklisch et al., 2016). This study utilized the advantages of sea urchin (Lytechinus pictus) embryos to assess the effect of BMAA exposure to fertilization, development, and ABC transporter activity. Gametes and embryos were exposed to a series of BMAA concentrations and compared by microscopic observation. Possible effects of BMAA on ABC transporter activity were also assessed using a fluorescent substrate efflux assay.
albumin) were used to culture embryos. First, fifty healthy eggs of sea urchin were picked up and added to 9.5 mL FSW in each well. Then, BMAA stock solution was spiked to reach 10, 50, 100, 300 and 500 μg L−1 in 10 mL final volume. Meanwhile, a volume of 2 mmol L−1 HCl equivalent to the highest concentration of BMAA (500 μg L−1) was added to well as a solvent control treatment (denoted as 0 μg L−1 of BMAA). Finally, the diluted sperm was added to wells to start fertilization of eggs. Total larvae and active swimming larvae were counted under an inverted anatomical microscope after 96 h post fertilization (HPF). Every treatment was repeated in triplicate in one batch exposure test and total three batches of exposure tests were calculated in this experiment.
2. Materials and methods
2.6. Efflux assay for ABC transporter exposed to BMAA
2.1. Chemicals
Calcein-AM (CAM) is membrane permeable and non-fluorescent. After entry into the cell intercellular esterase activity cleaves the -AM moiety, leaving membrane impermeable calcein. Cells with active ABCB and ABC-C transporters efflux CAM, whereas cells with reduced ABC transporter activity accumulate fluorescent calcein. Embryos were incubated in CAM and intracellular calcein accumulation was measured using a Zeiss LSM-700 laser scanning confocal microscope (Jena, Germany) equipped with a Zeiss Plan APOChromat 20× air objective (numerical aperture, 0.8). At 5 HPF embryos were exposed to CAM and 50 μg L−1 BMAA or 2 mmol L−1 HCl for 90 min. The exposed embryos were washed 10 times with FSW to remove the background fluorescence and incubated for an additional 30 min before imaging. Two mmol L−1 HCl and 50 μg L-1 BMAA was added in treatment-1 and -2, respectively. Based on the results of treatment-1 and -2, additional ABCB transporter inhibitor 2 μmol L-1 PSC833 was added in the treatment-3 and -4, in order to calculate the efflux activity (discharge efficiency, DE, %). More than 15 embryos were imaged in each treatment. Micrographs were prepared and analyzed with ImageJ 1.47v (National Institutes of Health). The fluorescence intensity of micrographs of exposed embryos with and without PSC833 was calculated as FIEP and FIE, respectively. The DE value was calculated using the formula, DE = 100-(FIE/ FIEP)×100.
2.4. Effect of BMAA on the fertility rate of sea urchin sperm Healthy eggs from one female and healthy sperm from three different individuals were used to test the effect of BMAA on the fertility rate of sea urchins. Fifty healthy eggs were added to 10 mL FSW. Sperm was diluted as follows: 5 μL concentrated sperm was added to 15 mL FSW, and 5 mL of this dilution was added to 25 μL FSW, 2 mM HCl or 0.1 mg mL−1 BMAA, respectively. The sperm dilution was kept 5 and 10 min in room temperature before it was used to start fertilization. Fertilization rates of eggs were calculated using the numbers of twocells embryos for each treatment. 2.5. Observation for embryo development of sea urchins exposed to BMAA Sea urchin embryos exposed to 500 μg L−1 BMAA at different developmental stages were compared with embryos in the solvent control group using a laser scanning confocal microscope (LSM 700, Zeiss). Normal development morphology for sea urchin embryos at early cleavage division, blastula, gastrula, and prism larvae and pluteus larvae stages were recorded in this study. Abnormal development of sea urchin embryos exposed to BMAA were also carefully observed.
Hydrochloric acid (HCl) and L-BMAA hydrochloride (B107, C4H10N2O2·HCl, 10 mg) were purchased from Sigma-Aldrich (Oakville, ON, Canada). The solution of 2 mmol L−1 HCl was used to dissolve BMAA at 0.1 mg mL−1 as a stock solution. Calcein-AM (CAM), plasma membrane permeable substrate of ABCB/P-gp and ABCC/MRP-type ABC transporters of echinoderm eggs and embryos, was obtained from Biotium (Hayward, CA). ABC-B inhibitor PSC833 was purchased from Millipore Sigma (St. Loius, MO). Stock solutions of CAM and PSC833 were prepared in dimethyl sulfoxide (DMSO) and diluted to the final concentrations in filtered seawater (0.22 μm, FSW). The final DMSO concentration in the assays did not exceed 0.5 %. Pure water (18.2 MΩ cm resistivity or better) was purified by a Milli-Q ultrapure water system (Millipore Corp., Billerica, MA, USA). 2.2. Echinoderm collection, gamete gathering and embryo culture Adults of sea urchin (Lytechinus pictus) collected from La Jolla (CA, USA) were held between 10 °C and 12 °C in flow-through seawater aquaria. Selected animals were stimulated to spawn by intra-coelomic injection of 0.55 mol L−1 KCl. Eggs obtained from females were washed twice with 0.22 μm FSW and diluted to either a 1 % (v/v) suspension, or to 500 eggs per mL in FSW. They were fertilized in a final sperm dilution of 1:250,000.
2.7. Statistical analysis The experiments of embryos to BMAA were conducted and repeated in three batches with each batch test containing three repetitions of each treatment. The exposure experiments for sperm were carried out in six repetitions, and the efflux assay for ABC transporter was tested in
2.3. Exposure experiments for sea urchin embryos to BMAA Six-well plates previously washed with 1 % BSA (bovine serum 2
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Fig. 2. Fertility rate (%) of sea urchin (Lytechinus pictus) eggs caused by sperms exposed to solvent HCl (∼10 μmol L−1) or BMAA (∼500 μg L−1) for 5 and 10 min. Note: Five μL of original sperms was diluted by 15 mL of filtered seawater, and part of 5 mL of the diluent sperms was added by 25 μL of 2 mmol L−1 HCl and 0.1 mg mL−1 BMAA used as solvent control and BMAA exposure, respectively. Data of each treatment were obtained from six repetitions, and * means significantly different from the control at p < 0.05 (one-way ANOVA).
300 μg L−1, except for the highest concentration 500 μg L−1 (Fig. 1B). The EC50 values of active swimming larvae and total larvae indexes were roughly inferred by an exponential equation as 165 μg L−1 (1.4 μmol L-1) and 329 μg L−1 (2.8 μmol L-1), respectively. The fertilization ability of sea urchin sperm exposed to BMAA is shown in Fig. 2. No significant difference occurred between blank control and solvent control treatments, which indicates that the additional low concentration of HCl did not affect fertilization. However, the fertilization ability of sea urchin sperm was significantly decreased (ANOVA, p < 0.05) after exposure to BMAA at 500 μg L−1 for 10 min. Fig. 1. Mortality (%) of sea urchin (Lytechinus pictus) embryos (A) and the percent of active swimming larvae in the surviving embryos (B) exposed to different concentrations of BMAA at 96 HPF (hours post fertilization). Note: An equivalent volume of 2 mmol L−1 HCl to the exposure group of 500 μg L−1 was added into the culture well as solvent control group marked as the concentration of BMAA at zero. Data of each treatment were obtained from the average of three batches of experiments and each batch contained different treatments in triplicate. Different letter shows the significantly different from each treatment at p < 0.05 (one-way ANOVA).
3.2. No effect of BMAA on the ABC transporter activity of sea urchin embryos Accumulation of calcein dye and discharge efficiency of ABC transporter in sea urchin embryos are shown in Fig. 3. Fluorescence intensity, an indicator of calcein accumulation and low transport activity, extremely significantly (ANOVA, p < 0.01) increased when the ABC-B specific inhibitor PSC833 was present in treatment-3 and -4. But no significant difference was observed between the solvent control group (adding 2 mmol L−1 HCl) and BMAA exposure group (50 μg L−1) in three tests. A higher concentration of BMAA exposure, 500 μg L−1, was also analyzed in one test, but still no obvious difference for the accumulation of calcein in embryos (data not shown).
triplicate. Statistical analysis was performed using the IMB SPSS Statistics V25.0 software (IBM Corporation, New York, USA). If assumptions of homogeneity of variances were satisfied, one-way analysis of variance (ANOVA) followed by LSD’s test was used. A P-value < 0.05 was considered to be statistically significant. 3. Results
3.3. Abnormal division and malformation development of embryos exposed to BMAA
3.1. Inhibitory effects of BMAA on embryonic development and sperm fertilization
Normal morphologic images of sea urchin in the early 96 HPF development stage in the solvent control (10 μmol L−1 HCl) are shown in Fig. 4. The embryo started the first cleavage after about 2 HPF at room temperature 25 °C and finished the first three cleavage divisions in 3 HPF. Then the embryo developed to the blastula and gastrula stage at about 7 and 24 HPF, respectively. Finally, the embryo developed as a four-arm pluteus larva at 96 HPF. Selected images of sea urchin embryos in the solvent blank control group (10 μmol L−1 HCl) and BMAA exposure group (500 μg L-1) after 24 HPF and 31 HPF are shown in Fig. 5. Some selected abnormal division and malformation development of sea urchin embryos exposed to BMAA (500 μg L-1) are shown in Fig. 6.
The effect of chronic BMAA exposure on mortality and normal development was determined using a 96-h exposure to various BMAA concentrations (Fig. 1). The mortality was calculated by averaging three independent tests with each test containing triplicate treatments. No significant effects on mortality were found in the treatments of embryos exposed to BMAA at 100 μg L−1 or lower, while the mortality significantly (ANOVA, p < 0.05) increased when the concentration of BMAA increased above 300 μg L−1 (Fig. 1A). However, the percentage of swimming larvae in the survived embryos were not significantly (ANOVA, p > 0.05) reduced when BMAA concentrations increased to 3
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(Fig. 1). However, there was a significant effect on mortality in BMAA exposures at 300 and 500 μg L−1. Observation of embryos exposed to BMAA at the highest concentration 500 μg L−1 only found that the percent of actively swimming four-armed plutei in the surviving embryos significantly decreased, while the other positive treatments for BMAA (≤ 300 μg L−1) were not significantly different from the control group. This phenomenon hints that the primary motor nerves controlling ciliary beating were not obviously destroyed by BMAA at the blastula stage in the experiment. But the standard deviations of the percent of active swimming larvae amplified when the concentration of BMAA ≥ 50 μg L−1, which demonstrated that the swimming ability was slightly inhibited by higher concentrations of BMAA. The roughly calculated EC50 value of active swimming larvae (165 μg L−1) was much lower than that of larval mortality (329 μg L−1), which also indicates that BMAA reduced the development of active swimming larvae of sea urchin. Some larvae only in situ rotated like a peg-top in the culture cell with higher concentration of BMAA (1000 μg L−1) although they developed to the two-armed pluteus stage at 96 HPF. Wada et al. (1997) reported that the neurotransmitters dopamine decreased, but serotonin increased, the beat frequency averaged over the ciliated epithelium of sea urchins (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) at the four-armed pluteus stage, which suggests that both the stability and the direction of ciliary beating are under nervous control. The serotonergic nervous system was also suggested to regulate the ciliary movement in mollusks and pond snails due to the association of cilia and the end of axons from serotonergic cells (Uhler et al., 2000; Kuang and Goldberg, 2001; Kuang et al., 2002). However, the irreversible inhibitor of tryptophan 5-hydroxylase, p-chlorophenylalanine (CPA), exclusively perturbed synthesis of serotonin but not nervous system organization, and CPA-treated sea urchin (H. pulcherrimus) larvae did not swim, despite the occurrence of ciliary beating in culture chamber (Yaguchi and Katow, 2003). Thus serotonin is not essential for ciliary beating but is necessary for swimming behavior. It is possible that catecholaminergic neurons, such as dopaminergic neurons or peptidergic neurons, may be involved in ciliary beating (Yaguchi and Katow, 2003). The SH-SY5Y neuroblastoma cells exposed to BMAA showed that BMAA perturbed alanine, aspartate and glutamate metabolism pathway, arginine and proline metabolism pathway, as well as in specific intermediary metabolites such as GABA and taurine which were involving several neurotransmitters/neuromodulators (Engskog et al., 2017). Rats treated with BMAA displayed alterations in the levels of glutamate, GABA and taurine in the motor cortex, which hints the excitotoxic mechanism due to loss of GABAergic inhibition involved in ALS in humans (de Munck et al., 2015). While BMAA-fed flies demonstrated the neurotoxin prolonged the NMDAR open time, which would allow excessive Ca2+ entry into the postsynaptic cell and lead to neurodegenerative effects (Koenig et al., 2015). Therefore, we hypothesized that the neurotoxin BMAA possibly disrupted the synthesis of dopamine and serotonin in the early embryo of sea urchin in this study. Effect of BMAA on the activity of these synthesis pathways should be explored in future studies. Fertilization percentage was significantly decreased when sperm was exposed to BMAA at 500 μg L−1 for 10 min (Fig. 2). The sulfated polysaccharides in the jelly coat surrounding the egg play a role in an initial step to induce the acrosome reaction of sea urchins (Alves et al., 1997). A high level of internal ATP needs to be maintained causing the stable sperm binding to induce a successful fertilization event (Hirohashi and Lennarz, 1998). Faster sperm of sea urchin (Lytechinus variegatus) demonstrated higher fertilization rates than slower sperm (Levitan, 2000). The mitochondrion swelled and shifted to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface after the occurrence of acrosome reaction (Kazama et al., 2006). A significant cytotoxic effect of BMAA on fish immune cell line (CLC) was also confirmed (Sieroslawska and Rymuszka, 2019), which was characterized by the reduction of cell membrane integrity and an increasement of ATP level in cells exposure to BMAA. Therefore, the
Fig. 3. Fluorescence intensity of Calcein-AM accumulated by embryos (A) and discharge efficiency (B) tested by the efflux assay of sea urchin (Lytechinus pictus) ABC-transporter with or without BMAA exposure. Data of each treatment were obtained from triplicate tests, and very significantly difference between Treat 1 - Treat 3, and Treat 2 - Treat 4, respectively (A). Average efflux assay of ABC-transporter exposed to BMAA was not significantly different from the control (B), p > 0.05 (one-way ANOVA).
4. Discussion The sea urchin has been adopted as a standard animal model for marine ecotoxicology (US EPA 600R95136). Just as in the mammalian central nervous system, sea urchin embryos can synthesize, store and release neurotransmitters like acetylcholine, serotonin (5 H T), dopamine and norepinephrine, and possess homologous populations of receptors and their downstream signaling cascades (Buznikov et al., 2007). The chemical reagents that target specific neurotransmitter mechanisms or signaling cascades in the mammalian brain have counterparts in “pre-nervous” morphogenetic anomalies in the developing sea urchin (Shmukier, 1993; Buznikov et al., 2001; Quiao et al., 2003). The effects of exogenous chemicals on the early development of sea urchin embryos could reflect drug-induced damage to mammalian neurons to some degree, and cell signaling during sea urchin development could be adopted to assess toxicity of environmental contaminants (Angelini et al., 2005). Considering that BMAA has adverse effects on motor neurons in some animal models (Rao et al., 2006; Buenz and Howe, 2007; Lobner et al., 2007), the early development of embryos of sea urchin (Lytechinus pictus) after exposure to the neurotoxin BMAA was observed in this study. No significant inhibitory effects were found on the fertilization and development of sea urchin embryos exposed to BMAA ≤ 100 μg L−1 4
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Fig. 4. Confocal microscopy observation for normal sea urchin (Lytechinus pictus) embryos in the early 96 h development stage, ‘HPF’ means hours post-fertilization, scale bar equals to 50 μm.
sea urchin blastulas at the early development stage. The sea urchin (L. pictus) embryos quickly developed in the control group at room temperature, 25 °C, in this study. The first three cleavage divisions started at 2 HPF and finished at 3 HPF, then the embryo entered into blastula stage at about 7 HPF (Fig. 4). The two-armed pluteus and four-armed pluteus could be clearly observed at 48 and 96 HPF, respectively (Fig. 4). Selected gastrulas at 24 HPF and early two-armed pluteus were compared in the control group and exposure group at 500 μg L−1 (Fig. 5). Under the observation of confocal microscopy, many primary mesenchyme cells (PMCs) could be seen to organize the skeletal rods in the gastrula stage (Fig. 5B) and two arms normally developed with the organization of skeletal rods (Fig. 5C and D) of sea urchin in the control group. However, fewer PMCs entered the blastocoel the skeletal rods did not form in the BMAA exposure group (Fig. 5E-H). In the control group the pigment granules were dispersed throughout the larva (Fig. 5C and D), but only a few black granules were distributed along the ring of the edge of sea urchin gastrula
ability of the mitochondrion to maintain high ATP levels is very important for high fertilization ability of sea urchin sperm. One possible mechanism for the reduction in fertilization percentage observed in the present study could be that BMAA inhibited the synthesis of ATP in sperm mitochondria. This mechanism should be investigated in future studies. CAM is a commonly used, membrane permeable substrate for ABC-B and ABC-C subfamily transporters in sea urchin research (Gökirmak et al., 2012, 2014). Cells with high ABC-B and ABC-C transporter activity would accumulate less calcein and be less fluorescent (Gökirmak et al., 2014). Without the ABC-B specific inhibitor PSC833 (treatments 1 and 2) sea urchin embryos accumulated very little calcein (Fig. 3A), however there was no significant difference between solvent control and BMAA exposed embryos. No significant difference in discharge efficiency was found between the sea urchin blastulas exposed to control solvent and BMAA (Fig. 3B). Results showed that BMAA did not affect the ABC-B and ABC-C transporter activity in the cytomembrane of 5
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Fig. 5. Confocal microscopy observation for sea urchin (Lytechinus pictus) embryos exposed to 10 μmol L−1 dissolvent HCl (A–D) and BMAA toxin at 500 μg L-1 (E–H) after 24 h (A, B, E, F) and 31 h (C, D, G, H) post-fertilization, scale bar equals to 50 μm.
first cleavage division (Fig. 6A), and some embryos underwent the-third cleavage division with unnormal arrangement of cells (Fig. 6B). Some eggs leaked cytoplasm and did not successfully fertilize in the BMAA exposure group (Fig. E). Some tenacious embryos slowly developed to blastula and two-armed pluteus at 24 HPF (Fig. 6F) and 48 HPF
exposed to BMAA (Fig. 5G and H). Some embryos treated with BMAA at 500 μg L−1 developed to the gastrula stage by 24 HPF, but few embryos had become two-armed plutei by 31 HPF. Most embryos exposed to 500 μg L−1 BMAA were arrested at different developmental stage with various malformation (Fig. 6). Some embryos could not complete the 6
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Fig. 6. Confocal microscopy observation for abnormal sea urchin (Lytechinus pictus) embryos exposed to BMAA of 500 μg L−1 in the early 48 h development stage, ‘HPF’ means hours post-fertilization, scale bar equals to 50 μm.
hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices, which demonstrated a significant decrease of PP2A activity and a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites such as Tyr307. BMAA has been directly proven to be transferred into astrocytes and neurons cells through cystine/glutamate antiporters (system xc−), which resulted in the imbalance of cystine uptake and a reduction of glutathione levels (Albano and Lobner, 2018). Therefore, one explanation for our data is that BMAA possibly affected the stability of microtubules in the mitotic spindle. This toxicological mechanism should be confirmed in future studies. It is unclear whether BMAA exposure would affect the differentiation on the neurogenic ectoderm. The Onecut gene is exclusively expressed in cells of the emerging cilliary band from late blastula stage. In later development Onecut is predominantly expressed in the cilliary band, but is also expressed in the apical organ and the elongating arms in the early pluteus stage (Poustka et al., 2004). Prominent, beating cilia were still observed in the abnormal gastrulas or pluteus exposed to BMAA, which suggested that the differentiation of the neurogenic ectoderm and ciliary band were not affected by BMAA in the early
(Fig. 6G-I), respectively. However, these tenacious embryos did not develop healthy organs such as mouth, stomach, and anus due to BMAA exposure. BMAA has been reported to affect all the main glutamate receptors, i.e., NMDA, AMPA/kainate, and metabotropic receptors (Lobner, 2009). In this study, BMAA had the following adverse effects on sea urchin development: (1) the fertilization process was arrested, (2) the first three cleavage divisions could not be normally arranged for embryos, and (3) late development of sea urchin blastulas was obviously inhibited by the neurotoxin BMAA. Exposure of sperm to BMAA decreased the fertilization rate, possibly by inhibiting intercellular ATP synthesis (Kazama et al., 2006). Our observations suggested that mitosis could not occur normally in many of the embryos exposed to BMAA. The mitotic spindle, the machinery for the separation of chromosomes during mitosis, mainly consists of microtubules (MTs) (Collins and Vallee, 1986). It has been well documented that MT disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed (Larkin and Danilchik, 1999). Arif et al. (2014) studied the effect of BMAA on PP2A activity and tau 7
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development of sea urchin. Sea urchin embryos can synthesize, store and release neurotransmitters like acetylcholine, serotonin, dopamine and norepinephrine, which take part in cell differentiation and morphological assembly (Buznikov et al., 2007). The initial cleavage divisions of the sea urchin embryo are controlled in part by serotonin responsive mechanisms (Shmukler, 1993). The toxic effects of BMAA that we have observed in the early development of sea urchin embryos possibly occur by disrupting the synthesis or function of neurotransmitters, especially of serotonin. This mechanism will be confirmed in a future study.
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5. Conclusions A toxic effect of BMAA on the early development of sea urchin embryos was found and discussed for the first time in this study. Fertilization and development of sea urchin embryos were significantly inhibited by BMAA above 300 μg L−1. The EC50 values calculated for active swimming larvae and larval mortality at 96 HPF was 165 μg L−1 (1.4 μmol L−1) and 329 μg L−1 (2.8 μmol L-1), respectively. Fertilization ratio also significantly decreased resulting from sperm exposed to BMAA for 10 min. Abnormal divisions and development arrest occurred at different developmental stages in the treatment of BMAA at 500 μg L−1. Results suggested that the intercellular ATP synthesis of sperm, cell division, and the function of neurotransmitters such as serotonin of embryos were possibly affected and disrupted by BMAA. Multiple mechanisms may underlie the effects of BMAA on the early development of sea urchin embryos. CRediT authorship contribution statement Aifeng Li: Conceptualization, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Jose Espinoza: Methodology, Writing - review & editing. Amro Hamdoun: Methodology, Resources, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant # 41676093) and the National Study Abroad Fund Sponsored by China Scholarship Council (Grant # 201706335006). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquatox.2020.105425. References Albano, R., Lobner, D., 2018. Transport of BMAA into neurons and astrocytes by system xc−. Neurotox. Res. 33, 1–5. Aluigi, M.G., Angelini, C., Corte, G., Falugi, C., 2008. The sea urchin, Paracentrotus lividus, embryo as a “bioethical” model for neurodevelopmental toxicity testing, Effects of diazinon on the intracellular distribution of OTX2-like proteins. Cell Biol. Toxicol. 24, 587–601. Alves, A.P., Mulloy, B., Diniz, J.A., Mourao, P.A.S., 1997. Sulfated polysaccharides from the jelly layer are species-specific inducers of acrosome reaction in sperms of sea urchins. J. Biol. Chem. 272, 6965–6971. Angelini, C., Aluigi, M.G., Sgro, M., Trombino, S., Thielecke, H., Falugi, C., 2005. Cell signalling during sea urchin development: a model for assessing toxicity of environmental contaminants (book chapter). In: Matranga, V. (Ed.), Progress in Molecular and Subcellular Biology, pp. 45–70 39. Arif, M., Kazim, S.F., Grundke-Iqbal, I., Garruto, R.M., Iqbal, K., 2014. Tau pathology
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