The diffuse use of lithium in a number of industrial processes has produced a significant contamination of groundwater and surface water with it. The increased use of lithium has generated only scarce studies on its concentrations in ambient waters and on its effects on aquatic organisms. Only few contributions have focused on the toxicity of lithium in marine organisms (such as marine animals, algae and vegetables), showing that the toxic effect depends on the animal species. In the present study we describe the morphological and the molecular effects of lithium chloride (LiCl), using the sea urchin Paracentrotus lividus as a model organism. We show that LiCl, if added to the eggs before fertilization, induces malformations in the embryos in a dose-dependent manner. We have also followed by RT qPCR the expression levels of thirty seven genes (belonging to different classes of functional processes, such as stress, development, differentiation, skeletogenesis and detoxifications) to identify the molecular targets of LiCl. This study opens new perspectives for the understanding of the mechanism of action of lithium on marine organisms. The findings may also have relevance outside the world of marine organisms since lithium is widely prescribed for the treatment of human bipolar disorders.
Lithium is abundantly present in the earth’s crust1: it typically forms salts like phosphates, silicates and micas2,3. Mobilized by weathering processes, lithium is transported into soils where it can be potentially toxic to plants3. It is present in river’s water, in brine and in drinking waters. The lithium content ingested from food has been estimated at 2 milligrams per day, the primary source being grains and vegetables3.
The industrial use of lithium, e.g., for the production of metal alloys, ceramics, TV screens, color films, pool cleaning chemicals, contributes to environmental pollution4. Lithium is also involved in the production of synthetic rubber, pharmaceuticals, lubricants, coolant in nuclear reactors and batteries5,6,7. A source on environment contamination by lithium is the widespread habit of disposing spent batteries along with normal garbage6.
Only scarce information is available on the inhalation toxicity of lithium, although nasal irritations and coughing were reported in occupationally exposed workers4. Some data are available on rats exposed for 4 h to aerosols containing 80% lithium carbonate: they displayed ulcerative rhinitis often accompanied by squamous metaplasia, necrotic laryngitis and respiratory difficulties8. Moreover, signs of anorexia and dehydration were observed, showing that inhaled lithium crosses the lipid-rich layer lining the lung alveolar surface to reach the kidney via the blood9. Lithium can accumulate in marine animals, algae, vegetables. It is important to consider that grains and vegetables are the primary dietary sources of lithium, contributing from 66% to 90% of the total lithium intake3. To date, clear results on the concentration of lithium in marine environment are not available to evaluate its toxicity. A few data are available on the distribution and toxicity of lithium in the aquatic environment in the United States10.
Lithium toxicity has raised concerns, given its widespread use to treat bipolar disorders11,12. Doses resulting in serum levels from 0.8 to 1.2 mM are recommended and widely used, but they are associated with a higher incidence of side effects such as gastro intestinal, renal, neurological and endocrine disorders13,14,15,16,17,18. Despite its theraupeutic use, little is known about the way lithium can alter neurotransmission. Lithium has been shown to decrease the level of neuronal inositol through the inhibition of inositol monophosphatase (IMPase), which converts myo-inositol monophosphates to myo-inositol, to reconstitute the membrane phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2) pool. Eventually, the latter generates the Ca2+-mobilizing second messenger D-myo-Inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). The lithium-induced inositol depletion and the consequent disturbance of the Ca2+ signaling operation, affect the behavior of neurons in culture, impairing neurotransmission and altering growth cone and the cytoskeleton19,20.
A teratogenic effect of lithium on the development on Dictyostelium, zebrafish, Xenopus and sea urchin organisms has also been documented21. It has been shown that lithium perturbs pattern formation resulting in embryos that have a deformed dorso-ventral axis22,23,24,25,26. In line with a role of lithium in reducing cellular levels of myo-inositol and PIP2 concentrations, myo-inositol can protect Xenopus and sea urchin embryos by counteracting the teratogenic effect27. The administration of lithium, or of another IMPase inhibitor (L690,330), after fertilization of sea urchin, has been shown to block the cell cycle transitions in the first cleavage of embryos and to induce profound effects on sea urchin development28. These effects were reversed by myo-inositol29,30. However, it was also shown that the complete inhibition of IMPase had no effect on the morphogenesis of Xenopus embryos, thus, a new hypothesis for the molecular mechanism of lithium on development was proposed. I.e., it was claimed that lithium inhibits glycogen synthase kinase- kinase-3 (GSK-3), which regulates cell lineage determination in several embryos31. The issue is controversial, as more recent results of in vivo studies have revealed that lithium is not a specific, nor a potent inhibitors of GSK332.
Various studies suggest that some harmful effects of lithium could be related to oxidative stress33,34, whereas at therapeutic concentration lithium was found to confer protection against toxic stimuli inducing oxidative stress and apoptosis35,36,37,38. At the moment, the fine details of the pharmacological and toxicological mechanisms of the effects of lithium remain poorly understood39,40.
The aim of the present study was to explore the effects of lithium using Paracentrotus lividus embryos as model organisms. The sea urchin P. lividus, is a good model system to study the ecotoxicological response of marine invertebrates to environmental pollutants for several reasons: its ecological relevance, benthic and relatively sedimentary lifestyle, rapid response and high sensitivity to many types of contaminants, its transparent embryos that grow rapidly in the laboratory and its long reproductive period. We thus decided to adopt it for our study and treated embryos with increasing concentrations of lithium chloride to analyze the ensuing morphological changes and to define the mechanism of their production. We also monitored the response to the lithium exposure of thirty seven genes that have key roles in a broad range of functional responses, such as development, differentiation and detoxification processes.
Paracentrotus lividus (Lamarck) sea urchins were collected from a location that is not privately-owned or protected in any way, according to Italian legislation of the Marina Mercantile (Decreto del Presidente dellaRepubblica DPR 1639/68, 09/19/1980 confirmed on 01/10/2000). The field studies did not involve endangered or protected species. All animal procedures were in compliance with the guidelines of the European Union (Directive 609/86).
Gamete collection, embryo culture, exposure to LiCl and morphological analysis
Adult sea urchins of the species, P. lividus, were collected during the breeding season by scuba-diving in the Gulf of Naples, transported in an insulated box to the laboratory within 1 hour after collection and maintained in tanks with circulating sea water until testing. Sea urchins were injected with 0.5 M KCl through the peribuccal membrane to obtain the emission of gametes. Eggs were washed with filtered sea water (FSW) and kept in FSW until use. Concentrated ‘dry’ sperm was collected and kept undiluted at +4 °C until use.
Lithium chloride was added to the eggs 10 minutes before fertilization (mpf) and 10 minutes post-fertilization (mpf) at the following concentrations: 1, 2, 3, 4, 5, 10 and 80 mM. Eggs were fertilized utilising sperm-to-egg ratios of 100:1 for both controls and treated embryos. Fertilized eggs were kept at 20 °C in a controlled temperature chamber on a 12 h:12 h light:dark cycle. Controls were also performed in FSW without LiCl.
After 48 hour of incubation and at 1 week post-fertilization, morphological malformations were determined for at least 200 plutei from each female (fixed in formaldehyde 4% in FSW) using a light microscope (Zeiss Axiovert 135TV, Carl Zeiss, Jena, Germany), in comparison to control embryos in FSW without LiCl.
For recovery experiments the procedure for the treatments with the LiCl was the same as reported above, adding LiCl before fertilization. The experiments have been performed at the concentration of 80 mM and embryos have been washed twice at different development: 10, 20 and 40 minutes post-fertilization (mpf). Embryos were grown at the pluteus stage. Controls were also performed, incubating embryos with LiCl, without washing. The number of abnormal embryos was evaluated by fixing embryos in formaldehyde (4% in FSW) and counting under the light microscope.
In the case of experiments to determine the teratogenic concentration for the molecular analysis, eggs were treated with LiCl at specific concentrations included in the range between 1.5–2 mM. Abnormal plutei were counted under the light microscope.
All experiments were conducted in triplicate using three egg groups collected from three different females.
Statistical analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego California USA).
RNA extraction and cDNA synthesis
About 8000 eggs in 50 mL of FSW were treated with 2 mM of LiCl for 10 minutes and then fertilized. Samples were then collected at 5, 21 and 48 hours post-fertilization (hpf) by centrifugation at 1800 relative centrifugal force for 10 minutes in a swing out rotor at 4 °C. The pellet was washed with phosphate buffered saline and then frozen in liquid nitrogen and kept at −80 °C. Experiments were conducted in triplicate using three egg groups collected from three different females.
Total RNA was extracted using GenEluteTM Mammalian Total RNA Miniprep Kit (SIGMA-ALDRICH®) according to the manufacter’s instructions. Contaminating DNA was degraded by treating each sample with a DNaseRNase-free kit (Roche, Milan, Italy) according to the manufacturer’s instructions. The amount of total RNA extracted was estimated by the absorbance at 260 nm and the purity by 260/280 and 260/230 nm ratios, using a NanoDrop spectrophotometer (ND-1000 UV-Vis Spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA). The integrity of RNA was evaluated by agarose gel electrophoresis. Intact rRNA subunits (28S and 18S) were observed on the gel indicating minimal degradation of the RNA. For each sample, 600 ng of total RNA extracted was retrotranscribed with an iScript™ cDNA Synthesis kit (Bio-Rad, Milan, Italy), following the manufacturer’s instructions. Synthetized cDNA was used in Real-Time qPCR experiments without dilution.
Gene expression by Real-Time qPCR
For all real-time qPCR experiments, the data from each cDNA sample were normalized using the zinc-finger transcription factor Pl-Z12–1 mRNA as endogenous control, because its expression is relatively constant in all developmental stages examined41. The expression level of thirty seven genes (see Table S1)42 were followed by Real Time qPCR.
Diluted cDNA was used as a template in a reaction containing a final concentration of 0.3 mM for each primer and 1 × FastStart SYBR Green master mix (total volume of 10 μL) (Applied Biosystems, Monza, Italy). PCR amplifications were performed in a ViiATM7 Real Time PCR System (Applied Biosystems, Monza, Italy) thermal cycler using the following thermal profile: 95 °C for 10 min, one cycle for cDNA denaturation; 95 °C for 15 s and 60 °C for 1 min, 40 cycles for amplification; 72 °C for 5 min, one cycle for final elongation; one cycle for melting curve analysis (from 60 °C to 95 °C) to verify the presence of a single product. Each assay included a no-template control for each primer pair. To capture intra-assay variability, all real-time qPCR reactions were carried out in triplicate. Fluorescence was measured using ViiATM7 software (Applied Biosystems, Monza, Italy). The expression of each gene was analysed and internally normalized against Pl-Z12-1 using REST software (Relative Expression Software Tool, Weihenstephan, Germany) based on the Pfaffl method43,44. Relative expression ratios above one cycles were considered significant. Experiments were repeated at least twice. Experiments were conducted in triplicate using three egg groups collected from three different females. Statistical analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego California USA).
Effects of LiCl on sea urchin development
Considering that 1 mM is the optimal range concentration of lithium plasma levels that is maximally beneficial for the treatment of bipolar disorder, we used this concentration as a starting point to study the effects of lithium on sea urchin development. In fact, increasing concentrations (1, 2, 3, 4, 5 and 10 mM) of LiCl were added separately to P. lividus eggs at 10 mbf and 10 mpf. Morphological analysis at the pluteus stage (48 hpf) revealed that the treatment at different concentrations tested induced the same malformations, which principally affected the arms, spicules and apex, in comparison with control embryos in FSW without LiCl (Fig. 1A). Such plutei had not-well-formed arms (Fig. 1B), or a poorly-formed apex with the spicules that appeared crossed at the apex (Fig. 1C) or disjoined at the tip (Fig. 1C), or arms that appeared broader (Fig. 1D) than in the controls (Fig. 1A).
Our results showed that the effects of LiCl were stronger before fertilization. At the concentration of 1 mM LiCl we observed an increase of about 30% malformed plutei in comparison with control embryos developed in FSW without LiCl. At 10 mbf a significant increasing percentage of abnormal plutei was observed from 1 mM up to 10 mM (Fig. 2). On the other hand, when LiCl was added after fertilization, a significant increase in the percentage of abnormal plutei was only detected at 5 and 10 mM. The times of exposure to 5 and 10 mM LiCl (added before fertilization) were extended to one week post-fertilization (wpf) to follow the fate of the plutei. After the pluteus stage, embryos began the retraction of the arms, firstly assuming a pyramid shape and then a characteristic “ampoule-like” shape (Fig. 3A)45. The treatment with LiCl (5 and 10 mM) induced malformations of the embryos at one wpf as reported in Fig. 3. In fact, microscopic observations showed that some embryos assumed the ampoule shape but the spicules appeared not-joined (Fig. 3B,C) or crossed at the tip (Fig. 3D). In these embryos the general body plan was definitely compromised and the entire body of the embryos was malformed (Fig. 3E–I). Several embryos had the pyramid shape and poorly-retracted and degraded arms (Fig. 3J–L). On the basis of these observations, the percentages of normal and abnormal embryos in samples treated with 5 and 10 mM LiCl and in the control were calculated after one week post-fertilization (Fig. 4): the percentage of abnormal plutei and ampoules increased at the concentration of 10 mM.
We also performed experiments adding LiCl at the concentration of 80 mM before fertilization. At this concentration LiCl blocked embryonic development at the blastula stage before the hatching (Fig. 5). Observations of embryos in 80 mM lithium after 48 hpf revealed that the blocked embryos then died. Recovery experiments were also performed to establish if sea urchin embryos exposed to 80 mM lithium were able to recover. Eggs were incubated with LiCl, fertilized and washed at three different times after fertilization: 10 mpf, 20 mpf and 40 mpf. After washing, embryos were grown to the pluteus stage to calculate the number of abnormal embryos. The results indicated that the embryos were able to recover. In fact, when washed, they were able to grow up to the pluteus stage (Fig. 6), even if some of them were malformed. In particular, we detected an increasing percentage of abnormal plutei with the increasing of the washing times. These results suggested that the time in which the embryos remained in contact with the litium was important: i.e., the number of malformed embryos increased with the time of exposure to lithium. The development of these embryos was followed until one week after the washing, showing that the percentage of abnormal embryos remained more or less the same (Supplementary Figure S1).
Effects of LiCl on gene expression
Embryos were treated with 2 mM LiCl, producing about 45% abnormal embryos (see Fig. 2). Samples were collected at different development times after fertilization, corresponding to the stages of early blastula (5 hpf), late gastrula (21 hpf) and pluteus (48 hpf). To detect potential gene targets of LiCl, the expression levels of thirty seven genes were followed by Real Time qPCR. These genes belong to different functional classes: stress-related genes, genes involved in development and differentiation processes, genes involved in detoxification and skeletogenesis processes (Supplementary Figure S2). The control gene for Real Time qPCR was Pl-Z12-1; variation of expression levels were calculated as relative expression ratios of the analyzed genes with respect to control embryos in sea water without LiCl. Only expression level greater than one-fold with respect to the controls were considered significant.
At early blastula stage (5hpf) three genes, HIF1A, p53 and p16 resulted up-regulated with an increase of 1.2-, 1.7-, 1-fold, respectively; δ-2-catenin was down-regulated with a 1.9-fold decrease in expression levels (Fig. 7). At the late gastrula stage, the GS and δ-2-catenin genes showed an increase of expression levels (1.3- and 1-fold, respectively), whereas two other genes were negatively affected: cytb (down-regulated by 1.2-fold) and sox-9 (down-regulated by 1.3-fold).
At the pluteus stage (48 hpf), only the up-regulation of the developmental gene hat was detected, with an increase of 1.1-fold with respect to the control.
The toxic effect of LiCl on embryonic development has been explored on different model organisms, such as squid, Xenopus, zebrafish and sea urchin46,47,48,49,50. The results have shown that LiCl inhibits the development along the animal vegetal axis and anterior midline of the squid embryos51. In Xenopus laevis it alters the axial patterning of the body, producing embryos with reduced posterior but exaggerated anterior structures and embryos with truncation of anterior structures52,53. LiCl also induces significant phenotypic abnormalities in zebrafish development, such as pericardial and yolk sac oedema, dispersed pigment cells54.
Work on P. lividus sea urchin embryos, the species used in the present study, has demonstrated that lithium acts as a larval vegetalizing agent, i.e., it acts by enhancing the endoderm-mesoderm structures at the expense of the ectoderm55. Blastomeres isolated from the animal half of sea urchin embryos and treated with lithium manifested a morphology resembling that of blastomeres deriving from the vegetal half of embryos. At the molecular level, lithium induced the appearance of molecular markers specific for the differentiation of vegetal structures, such as a gut specific enzyme and a RNA transcript encoding a skeletal spicule protein of larvae24. Furthermore, an increase was found in the expression level of the vegetal plate marker Endo16, when whole Strongylocentrotus purpuratus sea urchin embryos were treated with different concentrations of lithium. The increased expression level of this specific cell lineage marker paralleled the increase in the number of endodermal cells as a result of vegetalization56. Berridge and colleagues57 first suggested that lithium affects the phosphoinositide (PI) metabolism, i.e., the pathway generating the second messengers InsP3 and diacylglycerol (DAG) which regulate Ca2+ signaling and protein kinase C (PKC) activation, respectively58. The InsP3-PKC signaling pathway has been suggested to play a crucial role in vegetalizing the ectodermal cells in experiments in which a brief exposure of 16-cell stage sea urchin embryos to the PKC activator 12-O-tetradecanoyl phorbol- 13-acetate (TPA) enhanced their endoderm-mesoderm structures59. Significantly, then, the vegetalization process could also be induced by lithium. The phosphoinositide pathway is blocked by lithium at the level of the myo-inositol 1-phosphomonoesterase: the supply of inositol necessary for the resynthesis of phosphatidyl inositol (PI) and the subsequent synthesis of the immediate precursor of InsP3 and DAG, PIP2 are thus inhibited60. In line with the suggestion that the vegetalizing effect of lithium was due to the blockade of the phosphoinositide cycle, the inhibition of the development of embryos could be reversed by the injection of myo-inositol into Xenopus and sea urchin embryos27,30,61. Considering that the PI cycle becomes activated immediately after fertilization in sea urchin eggs, the conclusion that the role for lithium in perturbing the PI metabolism and thus the mechanism of cell determination and differentiation is mediated by its effect on the PI cycle thus appears plausible.
Recent work has confirmed that the interplay of a Ca2+- PKC signaling pathway with actin cytoskeleton is essential for a variety of cell specification events in the early sea urchin embryos62. Studies on GSK-3, which phosphorylates β-catenin, a downstream effector of the Wnt signaling pathway that regulates fundamental aspects of development, including cell fate specification and increases the level of DAG and PKC activity63, have suggested that lithium exert its vegetalizing effect by inhibiting the activity of the kinase64,65.
Our results expand previous investigations on the negative effects of lithium on the embryogenesis of sea urchin embryos. Sea urchins have been widely used as sensitive indicators of biochemical, morphological and physiological changes linked to environmental stressors, such as pesticides, heavy metals, ionizing radiations, ocean warming and acidification, metal nanoparticles and natural toxins42,45,66,67,68,69,70,71,72,73,74,75. Our results have shown that lithium is able to induce malformations in sea P. lividus urchin embryos, exerting a strong effect if the treatment is made before fertilization at concentrations much lower than those applied after fertilization. This indicates that lithium affects the early stages of the fertilization processes that determine if the embryo development will proceed normally76,77.
Another interesting finding made in the present work was the strong effect of the pre-incubation of unfertilized eggs with 80 mM lithium on sea urchin embryogenesis. The pretreatment blocked the embryonic development at the blastula stage with the subsequent death of the embryos. The effect was reversible. After all the washing times tested after fertilization the embryos were able to develop until the pluteus stage, even if some of them resulted malformed.
Our study has also provided new information on genes as molecular targets of lithium. Interestingly, we demonstrated that LiCl, at the molecular level affected different classes of genes at specifically developmental stages. These results suggest that LiCl exerts its negative effect on physiological processes by affecting genes that play a key role in a broad range of functional responses, such as stress, development, differentiation, skeletogenesis and detoxification processes (Fig. 7). Our results have shown that at the early blastula stage (21 hpf) lithium preferentially affected the expression levels of two stress genes, HIF1A and p53, as well as of that of δ-2-catenin which is involved in development and differentiation processes. In previous work the effects of diatom-derived polyunsaturated aldehydes (PUAs) had been tested on sea urchin embryos and p53, HIF1A and δ-2-catenin have been shown by interactomic analysis to represent HUB genes74. HUB genes are viewed as important nodes in a network in which these three genes interact with many other genes. For instance, the p53 gene has a key role in regulating the cell cycle and functions as a tumor suppressor. It allows the repair or deletion of cells exposed to agents that cause DNA damage, like hypoxia, UVR, ROS or mutagens in the multicellular organism78,79,80,81. In sea urchins exposed to ultraviolet radiation the p53 gene product is down-regulation, leading to apoptosis82. HIF1A, a heterodimeric transcription factor, regulates cellular energy metabolism and angiogenesis in response to hypoxia83. In sea urchin the initial activation of aboral (situated at the opposite extremity from the mouth) genes depends directly on the redox sensitive transcription factor HIF1A84. The δ-2-catenin gene could also be a target of the toxic effect of lithium: its product is normally expressed in the brain and the catenin-presenilin interaction has implications for cadherin function and regulation of cell-to-cell adhesion85,86.
Other genes could also be lithium targets. The p16 gene encodes for a small acidic protein involved in the formation of the biomineralized skeleton of sea urchin embryos and adults41. Recent studies have shown that this gene is also targeted by manganese and cadmium87 and by the diatom-derived hydroxyacid 5-HEPE45, confirming their important roles in skeletogenic processes. The finding that additional stress genes i.e., the GS gene which is responsible for the regulation of the glutamine synthetase activity in the metabolism of nitrogen are targeted by lithium supports the involvement of these genes in the stress response in sea urchin embryos. Marrone et al.88 have reported that at the prism stage (24 hpf) of sea urchin P. lividus embryos the treatment with the diatom-derived PUA decadienal up-regulated the GS gene. These findings were in line with studies in plants showing that salt stress affected glutamine synthetase activity and mRNA accumulation in potato plants in an organ-dependent manner89,90,91. The cyt b (cytochrome b) gene has also been suggested to intervene in the stress response in a single report in the literature which described its involvement in the response to the decadienal and 5-HEPE45,88. At prism stage (24 hpf) the expression of gene sox9, which is involved in left-right asymmetry processes during development and differentiation92, is affected by the treatment of sea urchin embryos with 15-HEPE45 and is also targeted by lithium. Finally, at the pluteus stage hat gene which is transiently expressed during the blastula stage as an early embryonic messengers93 is up-regulated. Its expression level has been found by us to increase in embryos treated with the three PUAs decadienal, heptadienal and octadienal42 and with the 5-HEPE45. In this contribution we have demonstrated how changes in gene expression levels may be considered early indicators of stressful conditions in the marine environment. As observed in most adaptive responses, the control of gene expression is tightly regulated in a fast response mode. This enables the cell to change the transcriptional pattern within minutes in the presence of stress and to return to the basal state after the stress is removed94. Our data suggest that LiCl is able to switch-on target genes at certain concentrations by a highly sophisticated mechanism. To be more detailed, we have probably detected effects on different genes at specifically developmental stages, as LiCl did not act directly on its target genes, but through the action of other genes belonging to the same functional pathway. Taking this into account, future efforts will focus on the analysis of the entire transcriptome and/or proteome to clarify the factors, e.g., mRNAs and/ or proteins, can be modulated by LiCl at specific developmental stages.
Furthermore, the aim of the present work was to contribute to the understanding of the morphological and molecular changes underlying the toxic effects of lithium on sea urchin embryogenesis. Lithium is widely used in patients with mood instability, but its mechanism of action still has obscure facets. The suggestion that its pharmacological effect involves the depletion of free myo-inositol appears plausible for the nervous tissue effects. In central neurons the activity of InsP3 and the Ca2+ release triggered by it are terminated by the dephosphorylation of the inositol monophosphate to inactive inositol. Since blood inositol is not available because of the blood-brain barrier, central neurons cannot replenish their plasma membrane with PIP2. This will prevent the formation of InsP3 and thus alter Ca2+ signals, somehow leading to the mood instability phenotype60. In line with this it should be mentioned that at fertilization of all animal species including humans, a sperm-induced signal transduction leads to the hydrolysis of PIP2 and to the formation of InsP3, which is suggested to be responsible for the release of Ca2+ in the fertilized eggs. A normal pattern and amount of Ca2+ release is a prerequisite necessary for the successful activation of the egg95,96. Lithium could cross the placental barrier of pregnant women treated with it for bipolar disorders and could be associated with the increased risk of neonatal malformations97.
An interesting and novel aspect of our work is that the lithium action on sea urchin embryo development is evident even after a short exposure (10 minutes) of unfertilized sea urchin eggs to it. This indicates that lithium exerts its effect on a process during which an increase in PIP2 takes place following fertilization98,99. Our previous work in starfish eggs100 and sea urchin (unpublished results) had shown that the PIP2 increase levels lasting for several minutes after sperm addition correlated with the dynamic changes of actin cytoskeleton at the egg surface: The latter are necessary for the structural reorganization of the cortex of the fertilized eggs that determine the incorporation of the sperm and direct the mitotic division of the zygote101. Thus, sea urchin eggs will be a useful model to clarify if the toxic effect action of lithium on sea urchin development is mediated by PIP2 depletion at the time of fertilization.
How to cite this article: Ruocco, N. et al. New insights into negative effects of lithium on sea urchin Paracentrotus lividus embryos. Sci. Rep. 6, 32157; doi: 10.1038/srep32157 (2016).
Habashi, F. Handbook of Extractive Metallurgy, vol. 4. Wiley-VCH, New York (1997).
Aral, H. & Vecchio-Sadus, A. Toxicity of lithium to humans and the environment- a literature review. Ecotoxicol Environ Saf 70, 349–356 (2008).
Schrauzer, G. N. Lithium: occurrence, dietary intakes, nutritional essentiality. J. Am. Coll. Nutr. 21, 14–21 (2002).
Beliles, R. P. Lithium, Li. In: Clayton G. D., Clayton F. E., eds. Patty’s Industrial hygiene and toxicology Vol 2. 4th ed. New York: John Wiley & Sons, 2087–2097 (1994).
Bleiwas, D. I. & Coffman, J. S. Lithium availability: Market economy countries: a minerals availability appraisal. Washington, D.C.: United States, Department of the Interior, Bureau of Mines (1986).
Ober, J. A. Lithium-2000: In Mineral Yearbook, vol. 1, Metal & Minerals, pp. 46.1–46.5 (2001).
NEMA. Spent consumer lithium batteries and the environment, National Electrical Manufacturers Association, March 2001.
Greenspan, B. J., Allen, M. D. & Rebar, A. H. Inhalation toxicity of lithium combustion aerosols in rats. J. Toxicol. Environ. Health 18, 627–637 (1986).
Allagui, M. S., Vincent, C., El feki, A., Gaubin, Y. & Croute, F. Lithium toxicity and expression of stress-related genes or proteins in A549 cells. Biochim Biophys Acta. 1773, 1107–1115 (2007).
Kszos, L. A. & Stewart, A. J. Review of lithium in the aquatic environment: distribution in the United States, toxicity and case example of groundwater contamination. Ecotoxicology 12, 439–447 (2003).
Schou, M. Lithium treatment at 52. J Affect Disord 67, 21–32 (2001).
Geddes, J. R., Burgess, S., Hawton, K., Jamison, K. & Goodwin, G. M. Long-term lithium therapy for bipolar disorder: systematic review and meta-analysis of randomized controlled trials. Am J Psychiatry 161, 217–222 (2004).
Gelenberg, A. J. et al. Comparison of standard and low serum levels of lithium for maintenance treatment of bipolar disorder. N. Engl. J. Med. 321, 1489–1493 (1989).
Bocchetta, A. et al. Thyroid abnormalities during lithium treatment. Acta. Psychiatr. Scand. 83, 193–198 (1991).
Tam, V. K., Green, J., Schwieger, J. & Cohen, A. H. Nephrotic syndrome and renal insufficiency associated with lithium therapy. Am. J. Kidney Dis. 27, 715–720 (1996).
Lazarus, J. H. The effects of lithium therapy on thyroid and thyrotropin-releasing hormone. Thyroid 8, 909–913 (1998).
Turan, T. et al. Effects of short- and long-term lithium treatment on kidney functioning in patients with bipolar mood disorder. Prog. Neuropsychopharmacol Biol. Psychiatry 26, 561–565 (2002).
Adityanjee, Munshi K. R. & Thampy, A. The syndrome of irreversible lithium-effectuated neurotoxicity. Clinical Neuropharmacology 28, 38–49 (2005)
Berridge, M. J. Inositol trisphosphate, calcium, lithium and cell signalling. J. Am. Med. Asso. 262, 1834–1841 (1989).
Williams, R. et al. A molecular cell biology of lithium. Biochem. Soc. Trans. 32, 799–802 (2004).
Berridge, M. J., Downes, C. P. & Hanley, M. R. Neural and developmental actions of lithium: A unifying hypothesis. Cell 59, 411–419 (1989).
Maeda, Y. Influence of ionic conditions on cell differentiation and morphogenesis of the cellular slime molds. Dev. Growth Differ. 12, 217–227 (1970).
Van, Lookeren, Campagne, M. M., Wang, M., Spek, W., Peters, D. & Schaap, P. Lithium respecifiescyclic -AMP-lnduced cell-type specific gene expression in Dictyostelium. Dev. Gen. 9, 589–596 (1988).
Livingston, B. T. & Wilt, F. H. Lithium evokes expression of vegetal-specific molecules in the animal blastomeres of sea urchin embryos. Proc. Natl. Acad. Sci. USA 86, 3669–3673 (1989).
Maslanski, J. A., Leshko, L. A. & Busa, W. B. Lithium-sensitive production of inositol phosphates during amphibian embryonic mesoderm induction. Science 256, 243–245 (1992).
Stachel, S. E., Grunwald, D. J. & Myers, P. Z. Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development (Cambridge, UK) 117, 1261–1274 (1993).
Busa, W. B. & Gimlich, R. L. Lithium-induced teratogenesis in frog embryos prevented by a polyphosphoinositide cycle intermediate or a diacylglycerol analog. Dev. Biol. 132, 315–324 (1989).
Sconzo, G., Cascino, D., Amore, G., Geraci, F. & Giudice, G. Effect of the IMPase inhibitor L690,330 on sea urchin development. Cell. Biol. Int. 22, 91–94 (1998).
Giudice, G. et al. Myo-inositol counteracts the vegetalizing effect of lithium. Cell Biol. Int. Rep. 16, 47–52 (1992).
Becchetti, A. & Whitaker, M. Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol. Development 124, 1099–1107 (1997).
Klein, P. S. & Melton, D. A. A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. 93, 8455–8459 (1996).
Kremer, A., Louis, J. V., Jaworski, T. & Van Leuven F. GSK3 and Alzheimer’s Disease: Facts and Fiction. Front. Mol. Neurosci. 4, 17 (2011).
Kielczykowska, M., Pasternak, K., Musik, I. & Wroniska, J. The effect of lithium administration in a diet on the chosen parameters of the antioxidant barrier in rats. Ann. Univ. Mariae Curie Sklodowska 59, 140–145 (2004).
Efrati, S. et al. N-Acetylcysteine ameliorates lithium-induced renal failure in rats. Nephrol. Dial. Transplant. 20, 65–70 (2005).
Chalecka-Franaszek, E. & Chuang, D. M. Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc. Natl. Acad. Sci. USA 96, 8745–8750 (1999).
Lai, J. S., Zhao, C., Warsh, J. J. & Li, P. P. Cytoprotection by lithium and valproate varies between cell types and cellular stresses. Eur. J. Pharmacol. 539, 18–26 (2006).
Shao, L., Young, L. T. & Wang, J. F. Chronic treatment with mood stabilizers lithium and valproate prevents excitotoxicity by inhibiting oxidative stress in rat cerebral cortical cells. Biol. Psychiatry 58, 879–884 (2005).
Chen, C. L., Lin, C. F., Chiang, C. W., Jan, M. S. & Lin, Y. S. Lithium inhibits ceramide- and etoposide-induced protein phosphatase 2A methylation, Bcl-2 dephosphorylation, caspase-2 activation and apoptosis. Mol. Pharmacol. 70, 510–517 (2006).
Shaldubina, A., Agam, G. & Belmaker, R. H. The mechanism of lithium action: state of the art, ten years later. Prog. Neuropsychopharmacol. Biol. Psychiatry 25, 855–866 (2001).
Hill, E. J. et al. Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development. PLoS One 8, e58822 (2013).
Costa, C., Karakostis, K., Zito, F. & Matranga, V. Phylogenetic analysis and expression patterns of p16 and p19 in Paracentrotus lividus embryos. Dev. Genes Evol. 222, 245–251 (2012).
Varrella, S. et al. Molecular response to toxic diatom- derived aldehydes in the sea urchin Paracentrotus lividus. Mar. Drugs 12, 2089–2113 (2014).
Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29, e45 (2001).
Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST) for groupwise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30, e36 (2002).
Varrella, S. et al. First morphological and molecular evidence of the negative impact of diatom-derived hydroxyacids on the sea urchin Paracentrotus lividus. Toxicol. Sci. pii: kfw053. [Epub ahead of print], doi: 10.1093/toxsci/kfw053 (2016).
Anderson, B. G. The toxicity threshold of various sodium salts determined by the use of Daphnia magna. Sewage Works J. 18, 82–87 (1946).
Emery, R. D., Klopfer, C. & Skalski, J. R. The incipient toxicity of lithium to freshwater organisms representing a salmonid habitat. PNL-3640, UC-11 Pacific Northwest Laboratory: Richland, WA, 364 p. (1981).
Hamilton, S. J. Hazard assessment of inorganics to three endangered fish in the Green River, Utah. Ecotoxicol. Environ. Safety 30, 134–142 (1995).
Long, K. E., Brown, R. P. Jr. & Woodburn, K. B. Lithium chloride: a flow-through embryo-larval toxicity test with the fat-head minnow, Pimephales promelas Rafinesque. Bull. Environ. Contam. Toxicol. 60, 312–317 (1998).
Dwyer, F. J., Burch, S. A., Ingersoll, C. G. & Hunn, J. B. Toxicity of trace element and salinity mixtures to striped bass (Morone saxatilis) and Daphnia magna. Environ. Toxicol. Chem. 11, 513–520 (1992).
Crawford, K. Lithium chloride inhibits development along the animal vegetal axis and anterior midline of the squid embryo. Biol. Bull. 205, 181–182 (2003).
Fredieu, J. R., Cui, Y., Maier, D., Danilchik, M. V. & Christian, J. L. Xwnt-8 and lithium can act upon either dorsal mesodermal or neurectodermal cells to cause a loss of forebrain in Xenopus embryos. Dev. Biol. 186, 100–114 (1997).
Leroy, P. & DeRobertis, E. M. Effects of lithium chloride and retinoic acid on the expression of genes from the Xenopus laevis Hox 2 complex. Dev. Dynamics 194, 21–312 (1992).
Ali, S., Aalders, J. & Richardson, M. K. Teratological effects of a panel of sixty water-soluble toxicants on zebrafish development. Zebrafish 11, 129–141 (2014).
Giudice, G. The sea urchin embryo: a developmental biological system. Springer, Berlin (1986).
Nocente-McGrath, C., McIsaac, R. & Ernst, S. G. Altered cell fate in LiCl-treated sea urchin embryos. Dev. Biol. 147, 445–450 (1991).
Berridge, M. J., Downes, C. P. & Hanley, M. R. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206, 587–595 (1982).
Ciapa, B. & Whitaker, M. J. Two phases of inositol polyphosphate and diacylglycerol production at fertilization. FEBS Lett. 195, 347–351 (1986).
Livingston, B. T. & Wilt, F. H. Phorbol esters alter cell fate during development of sea urchin embryos. J. Cell Biol 119, 1641–1648 (1992).
Berridge, M. J. Unlocking the secrets of cell signaling. Annu. Rev. Physiol. 67, 1–21 (2005).
Ciapa, B. & Maggio, K. Effect of lithium on ionic balance and polyphosphoinositide metabolism during larval vegetalization of the sea urchin Paracentrotus lividus. Dev Biol. 159, 114–121 (1993).
Yazaki, I. et al. Ca2+ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signaling and the oral-aboral axis formation in early sea urchin embryos. Zygote 23, 426–446 (2015).
Drummond, A. H. & Raeburn, C. A. The interaction of lithium with thyrotropin-releasing hormone-stimulated lipid metabolism in GH3 pituitary tumour cells. Blochem. Y. 224, 129–136 (1984).
Wu, D. & Pan, W. GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem. Sci. 35, 161–168 (2010).
O’Brein, W. T. & Klein, P. S. Validating GSK3 as an in vivo target of lithium action. Biochem. Soc. Trans. 37, 1133–1138 (2009).
Aluigi, M. G., Angelini, C., Corte, G. & Falugi, C. 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 (2008).
Pinsino, A., Matranga, V., Trinchella, F. & Roccheri, M. C. Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: Developmental and stress response effects. Ecotoxicology 19, 555–562 (2010).
Russo, R. et al. Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos. Cell Stress Chaperones 8, 232–241 (2003).
Bonaventura, R., Poma, V., Costa, C. & Matranga, V. UVB radiation prevents skeleton growth and stimulates the expression of stress markers in sea urchin embryos. Biochem. Biophys. Res. Commun. 328, 150–157 (2005).
Bonaventura, R. et al. Stress response gene activation protects sea urchin embryos exposed to X-rays. Cell Stress Chaperones 16, 681–687 (2011).
Lister, K. N., Lamare, M. D. & Burritt, D. J. Sea ice protects the embryos of the Antarctic sea urchin Sterechinus neumayeri from oxidative damage due to naturally enhanced levels of UV-B radiation. J. Exp. Biol. 213, 1967–1975 (2010).
Byrne, M. Global change ecotoxicology: identification of early life history bottlenecks in marine invertebrates, variable species responses and variable experimental approaches. Mar. Environ. Res. 76, 3–15 (2012).
Falugi, C. et al. Toxicity of metal oxide nanoparticles in immune cells of the sea urchin. Mar. Environ. Res. 76, 114–121 (2012).
Varrella, S. et al. Toxic diatom aldehydes affect defence gene networks in sea urchins. PLoS One, 11, e0149734 (2016).
Ruocco, N. et al. Activation of caspase-8 and caspase3/7 by diatom-derived oxilipins in sea urchin Paracentrotus lividus. Aquatic Toxicology 176,128–140 (2016).
Santella, L., Vasilev, F. & Chun, J. T. Fertilization in echinoderms. Biochem. Biophys. Res. Commun. 425, 588–594 (2012).
Santella, L., Limatola, N. & Chun, J. T. Calcium and actin in the saga of awakening oocytes. Biochem Biophys Res Commun 460, 104–113 (2015).
Graeber, T. G. et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379, 88–91.
Renzing, J., Hansen, S. & Lane, D. P. Oxidative stress is involved in the UV activation of p53. J. Cell Sci. 109, 1105–1112 (1996).
Clarke, A. R., Howard, L. A., Harrison, D. J. & Winton, D. J. p53 mutation frequency and apoptosis in the murine small intestine. Oncogene 14, 2015–2018 (1997).
Evans, M. D., Griffiths, H. R. & Lunec, J. Reactive oxygen species and their cytotoxic mechanisms. Chipman, J. K. eds. Mechanisms of Cell Toxicity 20, 25–73 (JAI Press Inc. London, 1997).
Lesser, M. P., Kruse, V. A. & Barry, T. M. Exposure to ultraviolet radiation causes apoptosis in developing sea urchin embryos. J. Exp. Biol. 206, 4097–4103 (2003).
Van Uden, P., Kenneth, N. S. & Rocha, S. Regulation of hypoxia-inducible factor-1α by NF-κB. Biochem. J. 412, 477–484 (2008).
Ben-Tabou de-Leon, S., Su, Y. H., Lin, K. T., Li, E. & Davidson, E. H. Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs and cell fate lockdown Dev. Biol. 374, 245–254 (2013).
Israely, I. et al. Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction. Curr. Biol. 14, 1657–1663 (2004).
Teo, J. L., Ma, H., Nguyen, C., Lam, C. & Kahn, M. Specific inhibition of CBP/beta-catenin interaction rescues defects in neuronal differentiation caused by a presenilin-1 mutation. Proc. Natl. Acad. Sci. USA 102, 12171–12176 (2005).
Migliaccio, O., Castellano, I., Romano, G. & Palumbo, A. Stress response to cadmium and manganese in Paracentrotus lividus developing embryos is mediated by nitric oxide. Aquatic Toxicology 156, 125–134 (2014).
Marrone, V. et al. Defensome against toxic diatom aldehydes in the sea urchin Paracentrotus lividus. PLoS One 7, e31750 (2012).
Teixeira, J. & Fidalgo, F. Salt stress affects glutamine synthetase activity and mRNA accumulation on potato plants in an organ-dependent manner. Plant Physiol. Biochem. 47, 807–813 (2009).
Cai, H. et al. Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Rep. 28, 527–537 (2009).
Lee, H. J. et al. Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress. Plant Cell Rep. 32, 1521–1529 (2013).
Duboc, V., Rottinger, E., Lapraz, F., Besnardeau, L. & Lepage, T. Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev. Cell 9, 147–158 (2005).
Lepage, T., Sardet, C. & Gache, C. Spatial expression of the hatching enzyme gene in the sea urchin embryo. Dev. Biol. 150, 23–32 (1992).
Nadal, E. D., Ammerer, G. & Posas, F. Controlling gene expression in response to stress. Nat. Rev. Genet. 12, 833–845 (2011).
Santella, L., Lim, D. & Moccia, F. Calcium and fertilization: the beginning of life. Trends Biochem Sci. 29, 400–408 (2004).
Chun, J. T., Limatola, N., Vasilev, F. & Santella, L. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules. Biochem. Biophys. Res. Commun. 450, 1166–1174 (2014).
Thiels, C. Pharmacotherapy of psychiatric disorder in pregnancy and during breastfeeding: a review. Pharmacopsycology 20, 133–146 (1987).
Turner, P. R., Sheetz, M. P. & Jaffe, L. A. Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature (London) 310, 414–415 (1984).
Halet, G., Tunwell, R., Balla, T., Swann, K. & Carroll, J. The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs. J. Cell Sci. 115, 2139–2149 (2002).
Chun, J. T., Puppo, A., Vasilev, F., Gragnaniello, G., Garante, E. & Santella L. The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling. PLoS One. 5(11), e14100 (2010).
Vasilev, F., Chun, J. T., Gragnaniello, G., Garante, E. & Santella, L. Effects of ionomycin on egg activation and early development in starfish. PLoS One 7, e39231 (2012).
Nadia Ruocco (Ph.D. in Biology, University of Naples Federico II) has been supported by a Ph.D fellowship co-funded by the Stazione Zoologica Anton Dohrn and Bio-Organic Chemistry Unit of the Institute of Biomolecular Chemistry-CNR, Pozzuoli, Naples (Italy). We thank the Molecular Biology and Bioinformatics Unit of the SZN for providing primers for PCR experiments and the Fishing Service of the SZN for providing sea urchins. Many thanks are also due to Davide Caramiello of the Marine Resources for Research Unit for his technical support in sea urchin maintenance and gamete collection. We also thank Massimo Perna of the Department of Integrative Marine Ecology for providing formaldehyde to fix embryos.
The authors declare no competing financial interests.
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Ruocco, N., Costantini, M. & Santella, L. New insights into negative effects of lithium on sea urchin Paracentrotus lividus embryos. Sci Rep 6, 32157 (2016). https://doi.org/10.1038/srep32157
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