Toxicity of Vanadium during Development of Sea Urchin Embryos: Bioaccumulation, Calcium Depletion, ERK Modulation and Cell-Selective Apoptosis.

Vanadium toxicology is a topic of considerable importance as this metal is widely used in industrial and biomedical fields. However, it represents a potential emerging environmental pollutant because wastewater treatment plants do not adequately remove metal compounds that are subsequently released into the environment. Vanadium applications are limited due to its toxicity, so it is urgent to define this aspect. This metal is associated with sea urchin embryo toxicity as it perturbs embryogenesis and skeletogenesis, triggering several stress responses. Here we investigated its bioaccumulation and the correlation with cellular and molecular developmental pathways. We used cytotoxic concentrations of 1 mM and 500 µM to perform quantitative analyses, showing that vanadium accumulation interferes with calcium uptake during sea urchin development and provokes a disruption in the biomineralization process. At the end of the whole treatment, the accumulation of vanadium was about 14 and 8 µg for embryos treated respectively with 1 mM and 500 µM, showing a dose-dependent response. Then, we monitored the cell signaling perturbation, analyzing key molecular markers of cell survival/cell death mechanisms and the DNA fragmentation associated with apoptosis. This paper clarifies vanadium's trend to accumulate directly into embryonic cells, interfering with calcium uptake. In addition, our results indicate that vanadium can modulate the ERK pathway and activate a cell-selective apoptosis. These results endorse the sea urchin embryo as an adequate experimental model to study metal-related cellular/molecular responses.

Toxicological Impact of Rare Earth Elements (REEs) on the Reproduction and Development of Aquatic Organisms Using Sea Urchins as Biological Models.

The growing presence of lanthanides in the environment has drawn the attention of the scientific community on their safety and toxicity. The sources of lanthanides in the environment include diagnostic medicine, electronic devices, permanent magnets, etc. Their exponential use and the poor management of waste disposal raise serious concerns about the quality and safety of the ecosystems at a global level. This review focused on the impact of lanthanides in marine organisms on reproductive fitness, fertilization and embryonic development, using the sea urchin as a biological model system. Scientific evidence shows that exposure to lanthanides triggers a wide variety of toxic insults, including reproductive performance, fertilization, redox metabolism, embryogenesis, and regulation of embryonic gene expression. This was thoroughly demonstrated for gadolinium, the most widely used lanthanide in diagnostic medicine, whose uptake in sea urchin embryos occurs in a time- and concentration-dependent manner, correlates with decreased calcium absorption and primarily affects skeletal growth, with incorrect regulation of the skeletal gene regulatory network. The results collected on sea urchin embryos demonstrate a variable sensitivity of the early life stages of different species, highlighting the importance of testing the effects of pollution in different species. The accumulation of lanthanides and their emerging negative effects make risk assessment and consequent legislative intervention on their disposal mandatory.

Vanadium Toxicity Monitored by Fertilization Outcomes and Metal Related Proteolytic Activities in Paracentrotus lividus Embryos.

Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and a protease detection assay after 36 h of exposure. V affected the fertilization percentage and increased morphological abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected, and their proteolytic activity depended on the V concentration. Biochemical characterization shows that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+ requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs).

Toxic effects induced by vanadium on sea urchin embryos.

Vanadium, a naturally occurring element widely distributed in soil, water and air, has received considerable interest because its compounds are often used in different applications, from industry to medicine. While the possible medical use of vanadium compounds is promising, its potential harmful effects on living organisms are still unclear. Here, for the first time, we provide a toxicological profile induced by vanadium on Paracentrotus lividus sea urchin embryos, reporting an integrated and comparative analysis of the detected effects reflecting vanadium-toxicity. At the morphological level we found a dose-dependent induction of altered phenotypes and of skeletal malformations. At the molecular levels, vanadium-exposed embryos showed the activation of the cellular stress response, in particular, autophagy and a high degree of cell-selective apoptosis in a dose-dependent manner. The stress response mediated by heat shock proteins seems to counteract the damage induced by low and intermediate concentrations of vanadium while the high cytotoxic concentrations induce more marked cell death mechanisms. Our findings, reporting different mechanisms of toxicity induced by vanadium, contribute to increase the knowledge on the possible threat of vanadium for marine organisms and for both environmental and human health.

Interactive effects of increased temperature and gadolinium pollution in Paracentrotus lividus sea urchin embryos: a climate change perspective.

Gradual ocean warming and marine heatwaves represent major threats for marine organisms already facing other anthropogenic-derived hazards, such as chemical contamination in coastal areas. In this study, the combined effects of thermal stress and exposure to gadolinium (Gd), a metal used as a contrasting agent in medical imaging which enters the aquatic environment, were investigated in the embryos and larvae of the sea urchin Paracentrotus lividus. Embryos were exposed to six treatments of three temperatures (18 °C, 21 °C, 24 °C) and two Gd concentrations (control: 0 µM; treated: 20 µM). With respect to developmental progression, increased temperature accelerated development and achievement of the larval stage, while Gd-exposed embryos at the control temperature (18 °C) showed a general delay in development at 24 h post-fertilization (hpf), and a stunting effect and impaired skeleton growth at 48 hpf. Elevated temperatures at near-future projections (+3 °C, 21 °C) reduced the negative effects of Gd on development with a lower percentage of abnormality and improved skeleton growth. Combined extreme warming at present-day marine heatwave conditions (+6 °C, 24 °C) and Gd treatment resulted in a lower proportion of embryos reaching the advanced larval stages compared to the 21 °C + Gd. At the molecular level, western blot analysis showed that Gd was the main driver for the induction of heat shock protein (HSP60, HSP70) expression. At 48 hpf, temperature increase was the main driver for activation of additional cellular stress response strategies such as autophagy and apoptosis. Combined treatments showed the induction of HSP60 at 24 hpf and autophagic and apoptotic processes at 48 hpf. Treatments having low levels of HSPs expression showed high levels of apoptosis, and vice versa, clearly demonstrating the antagonistic effects of HSPs expression and apoptosis. Detection of fragmented DNA in apoptotic nuclei showed selective apoptosis, likely in extremely damaged cells. Our results indicate that the negative effects of Gd-exposure on P. lividus larval development and biomineralization will be mitigated by a near-future ocean warming, up to a thermotolerance threshold when negative synergistic effects were evident. Our data highlight the use of biomarkers as sensitive tools to detect environmental impacts as well as the need for a better understanding of the interactions between the multiple stressors faced by marine species in coastal environments.

DNA fragmentation index, pAKT and pERK1/2 in cumulus cells are related to oocyte competence in patients undergoing in vitro fertilization programme.

Activated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus-oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the former and the intracellular accumulation of pAKT had a significant direct relationship. This relationship was not observed for the negative clinical outcome of the arrested embryos. Moreover, intracellular accumulation of pAKT and DFI value had a significant inverse relationship in all samples examined. The obtained data suggest that the intranuclear relocation of pERK1/2, along with an enhanced intracellular accumulation of pAKT, may exert a survival effect and increase cell viability, therefore providing a novel marker tool to choose the best oocyte to be fertilized and submitted to an intracytoplasmic sperm injection cycle.

Cadmium stress effects indicating marine pollution in different species of sea urchin employed as environmental bioindicators.

In recent years, researches about the defense strategies induced by cadmium stress have greatly increased, invading several fields of scientific research. Mechanisms of cadmium-induced toxicity continue to be of interest for researchers given its ubiquitous nature and environmental distribution, where it often plays the role of pollutant for numerous organisms. The presence in the environment of this heavy metal has been constantly increasing because of its large employment in several industrial and agricultural activities. Cadmium does not have any biological role and, since it cannot be degraded by living organisms, it is irreversibly accumulated into cells, interacting with cellular components and molecular targets. Cadmium is one of the most studied heavy metal inductors of stress and a potent modulator of several processes such as apoptosis, autophagy, reactive oxygen species, protein kinase and phosphatase, mitochondrial function, metallothioneins, and heat-shock proteins. Sea urchins (adults, gametes, embryos, and larvae) offer an optimal opportunity to investigate the possible adaptive response of cells exposed to cadmium, since these cells are known to accumulate contaminants. In this review, we will examine several responses to stress induced by cadmium in different sea urchin species, with a focus on Paracentrotus lividus embryos. The sea urchin embryo represents a suitable system, as it is not subjected to legislation on animal welfare and can be easily used for toxicological studies and as a bioindicator of environmental pollution. Recently, it has been included into the guidelines for the use and interpretation of assays to monitor autophagy.

Gadolinium perturbs expression of skeletogenic genes, calcium uptake and larval development in phylogenetically distant sea urchin species.

Chelates of Gadolinium (Gd), a lanthanide metal, are employed as contrast agents for magnetic resonance imaging and are released into the aquatic environment where they are an emerging contaminant. We studied the effects of environmentally relevant Gd concentrations on the development of two phylogenetically and geographically distant sea urchin species: the Mediterranean Paracentrotus lividus and the Australian Heliocidaris tuberculata. We found a general delay of embryo development at 24h post-fertilization, and a strong inhibition of skeleton growth at 48h. Total Gd and Ca content in the larvae showed a time- and concentration-dependent increase in Gd, in parallel with a reduction in Ca. To investigate the impact of Gd on the expression of genes involved in the regulation of skeletogenesis, we performed comparative RT-PCR analysis and found a misregulation of several genes involved in the skeletogenic and left-right axis specification gene regulatory networks. Species-specific differences in the biomineralization response were evident, likely due to differences in the skeletal framework of the larvae and the amount of biomineral produced. Our results highlight the hazard of Gd for marine organisms.

Gene Expression and Apoptosis Levels in Cumulus Cells of Patients with Polymorphisms of FSHR and LHB Undergoing in Vitro Fertilization Program.

BACKGROUND/AIMS: FSH receptor (FSHR) Ala307Thr and Asn680Ser and LHß chain (LHB) Trp28Arg and Ile35Thr polymorphisms affect the response to pharmacological ovarian stimulation with r-FSH in women undergoing assisted reproductive treatment (ART). Here, we evaluated the expression level of selected genes involved in follicle maturation and the possible onset of apoptosis in cumulus cells of patients with single and double FSHR and LHB polymorphisms, as potential markers of oocyte competence. METHODS: Cumulus cells from 36 stimulated patients were collected and SNP genotyping performed by PCR. Gene expression was evaluated through real-time PCR, and apoptosis estimated via TUNEL assay, and cleaved caspase-3 and pAKT immunostaining. RESULTS: The cumulative data show significant correlations indicating that the genetic alteration of FSHR and/or LHB genes may lead to perturbations of the signaling network programmed to granulosa cell survival and follicle development. Notably, when double heterozygotes were compared to the rest of the patients, a higher level of apoptosis in terms of both DNA fragmentation index and amount of active caspase-3 was observed in cumulus cells. CONCLUSIONS: These results may help to define personalized stimulation protocols in ART programs, to increase the success rate of ICSI procedures in accordance with the polymorphic condition of the individual patient.

Relationship between apoptosis and survival molecules in human cumulus cells as markers of oocyte competence.

To select from a single patient the best oocytes able to reach the blastocyst stage, we searched for valuable markers for oocytes competence. We evaluated the DNA fragmentation index (DFI) and the level of some survival molecules, such as AKT, pAKT and pERK1/2, in individual cumulus cell-oocyte complexes (COC). The study included normo-responder women. The average age of the patients was 34.3. DFI in cumulus cells was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay in situ. AKT, pAKT and pERK1/2 were measured by immunological assay and densitometric analysis of fluorescent signals using NIS-Elements BR 3.10 image software. Statistical analysis was performed using STATA SE/14.1. The study focused on 53 patients involved after informed consent. Out of 255 MII oocytes, 197 were fertilized and the derived embryos had the following evolution: 117 completed the development to blastocyst and were transferred to uterus; 57 were vitrified at the blastocyst stage; and 23 were arrested during in vitro culture at different stages of cleavage. We found a significant statistical difference between the DFI of cumulus cells of the arrested embryos and the transferred blastocysts (P = 0.004), confirming that DFI could be considered as a valuable marker of oocyte competence. In addition, the pAKT/DFI ratio was higher in cumulus cells of oocytes able to produce blastocysts, indicating that DFI is significantly lower when pAKT is higher (P = 0.043). This study demonstrates for the first time that the relationship between apoptosis and survival molecules can be used as a marker to select the best oocytes.

Induction of skeletal abnormalities and autophagy in Paracentrotus lividus sea urchin embryos exposed to gadolinium.

Gadolinium (Gd) concentration is constantly increasing in the aquatic environment, becoming an emergent environmental pollutant. We investigated the effects of Gd on Paracentrotus lividus sea urchin embryos, focusing on skeletogenesis and autophagy. We observed a delay of biomineral deposition at 24 hours post fertilization (hpf), and a strong impairment of skeleton growth at 48 hpf, frequently displayed by an asymmetrical pattern. Skeleton growth was found partially resumed in recovery experiments. The mesodermal cells designated to biomineralization were found correctly migrated at 24 hpf, but not at 48 hpf. Western blot analysis showed an increase of the LC3-II autophagic marker at 24 and 48 hpf. Confocal microscopy studies confirmed the increased number of autophagolysosomes and autophagosomes. Results show the hazard of Gd in the marine environment, indicating that Gd is able to affect different aspects of sea urchin development: morphogenesis, biomineralization, and stress response through autophagy.

Mitochondria during sea urchin oogenesis.

Sea urchin represents an ideal model for studies on fertilization and early development, but the achievement of egg competence and mitochondrial behaviour during oogenesis remain to be enlightened. Oocytes of echinoid, such as sea urchin, unlike other echinoderms and other systems, complete meiotic maturation before fertilization. Mitochondria, the powerhouse of eukaryotic cells, contain a multi-copy of the maternally inherited genome, and are involved directly at several levels in the reproductive processes, as their functional status influences the quality of oocytes and contributes to fertilization and embryogenesis. In the present paper, we report our latest data on mitochondrial distribution, content and activity during Paracentrotus lividus oogenesis. The analyses were carried out using confocal microscopy, in vivo incubating oocytes at different maturation stages with specific probes for mitochondria and mtDNA, and by immunodetection of Hsp56, a well known mitochondrial marker. Results show a parallel rise of mitochondrial mass and activity, and, especially in the larger oocytes, close to germinal vesicle (GV) breakdown, a considerable increase in organelle activity around the GV, undoubtedly for an energetic aim. In the mature eggs, mitochondrial activity decreases, in agreement with their basal metabolism. Further and significant information was achieved by studying the mitochondrial chaperonin Hsp56 and mtDNA. Results show a high increase of both Hsp56 and mtDNA. Taken together these results demonstrate that during oogenesis a parallel rise of different mitochondrial parameters, such as mass, activity, Hsp56 and mtDNA occurs, highlighting important tools in the establishment of developmental competence.

Autophagy is required for sea urchin oogenesis and early development.

Autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium.

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.

Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

PURPOSE: An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. METHODS: In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. RESULTS: After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p<0.05), but we found a significant reduction in patients with high basal DFI values (>15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. CONCLUSIONS: Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction.

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 µM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 µM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 µM CdCl2). We found also that, in response to a lethal dose of cadmium (1 µM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Heavy metals and metalloids as autophagy inducing agents: focus on cadmium and arsenic.

In recent years, research on the autophagic process has greatly increased, invading the fields of biology and medicine. Several markers of the autophagic process have been discovered and various strategies have been reported studying this molecular process in different biological systems in both physiological and stress conditions. Furthermore, mechanisms of metalloid- or heavy metal-induced toxicity continue to be of interest given the ubiquitous nature and distribution of these contaminants in the environment where they often play the role of pollutants of numerous organisms. The aim of this review is a critical analysis and correlation of knowledge of autophagic mechanisms studied under stress for the most common arsenic (As) and cadmium (Cd) compounds. In this review we report data obtained in different experimental models for each compound, highlighting similarities and/or differences in the activation of autophagic processes. A more detailed discussion will concern the activation of autophagy in Cd-exposed sea urchin embryo since it is a suitable model system that is very sensitive to environmental stress, and Cd is one of the most studied heavy metal inductors of stress and modulator of different factors such as: protein kinase and phosphatase, caspases, mitochondria, heat shock proteins, metallothioneins, transcription factors, reactive oxygen species, apoptosis and autophagy.

Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton.

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrates.

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress.

It is well known that sea urchin embryos are able to activate different defense strategies against stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to Cd adopt autophagy as an additional stratagem to safeguard the developmental program. At present, there are no data focusing on the role of this process in embryo development of marine organisms.

Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects.

In the marine environment increasing concentrations of bio-available compounds often result from anthropogenic activities. Among metal ions, manganese represents a new emergent factor in environmental contamination. Here, we studied the effects of manganese on Paracentrotus lividus sea urchin embryos using biological and biochemical approaches for the analysis of impact on development, tissue accumulation and stress markers. Embryos were continuously exposed from fertilization to manganese at concentrations ranging from 1.0 to 61.6 mg l(-1), monitored for developmental abnormalities at 48 h after fertilization, and used for atomic spectrometric analysis at various times from 6 to 72 h. We found that concentration- and time-dependent increases in morphological abnormalities were directly correlated to manganese accumulation, with major defects in skeleton formation at 48 h. Concurrently, we found an upregulation of the hsc70 and hsc60 stress proteins detected by immunoblotting, whereas no induction of apoptosis or ROS production was observed by TUNEL and live tests, respectively. Taken together, our findings demonstrate that the observed manganese embryo-toxicity is related to both its intracellular accumulation and misregulated homeostasis, and confirm the importance of stress proteins as protective agents in the acquisition of tolerance and resistance to apoptosis.