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Feb 2, 2011 - process in embryo development of marine organisms. Sea urchin embryos as a model system for studying autophagy induced by cadmium ...
Basic Research Paper

Autophagy 7:9, 1028-1034; September 2011; © 2011 Landes Bioscience

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress Roberto Chiarelli, Maria Agnello and Maria Carmela Roccheri* Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (sez. Biologia Cellulare); Università degli Studi di Palermo; Italy

Key words: autophagy, cadmium, stress, acidic vesicular organelles, bafilomycin A1, LC3, Paracentrotus lividus embryos Abbreviations: AO, acridine orange; AVOs, acidic vesicular organelles; Cd, cadmium chloride; CLSM, confocal laser scanning microscopy; HSP, heat shock protein; NR, neutral red

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.

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In this paper we utilized different techniques to detect autophagy in sea urchin embryos. Using neutral red (NR) and acridine orange (AO) vital staining, we found that embryos exposed to Cd display massive punctiform spots in the cytoplasm, indicative of acidic vesicular organelles (AVOs). These results have been confirmed using bafilomycin A1, a known inhibitor of this process. Furthermore these data were validated through both protein gel blotting and immunofluorescence in situ analysis of LC3-II, a specific marker of autophagy. We found that in P. lividus embryos autophagic processes occur in lesser amounts during physiological development and in greater amounts after Cd exposure. Introduction

Autophagy is a mechanism of self-eating described as the most important intracellular pathway responsible for degradation and recycling of long-term proteins and cytoplasmic organelles.1 The definition of autophagy often refers to the main subtype of this process, macroautophagy, which is the most common form of autophagic phenomenon. The execution and regulation of the autophagic program occurs by the expression of several autophagy-related (Atg) genes, having a high degree of conservation among species, from yeast to human. Similar to apoptotic cell death, autophagy is essential for development, growth and maintenance of homeostasis in multicellular organisms.2 Changes in the autophagic process are the cause of many pathological conditions, including vacuolar myopathies, neurodegenerative and liver diseases, as well as some types of cancer.3

Autophagy has recently been proposed as a crucial mechanism for the cellular remodeling that occurs during the development of multicellular organisms, such as Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and vertebrates.4 In addition, it has been demonstrated that autophagy is essential for pre-implantation development of mouse embryos.5 It is also known that, during mammalian embryonic development, autophagy often temporally precedes apoptosis, probably to promote the dismantling of some intracellular structures to reduce the cell volume, thus simplifying the removal of large quantities of apoptotic bodies.3 The autophagic process occurs constitutively at basal levels and appears to be activated as an adaptive response to a series of intracellular and extracellular stimuli, including nutrient deprivation (starvation), or hormonal treatments, bacterial or viral infections, accumulation of misfolded proteins and damaged organelles, toxic stimuli, radiation and many agents of stress.6 In general, it seems that autophagy is necessary for cell survival under stress conditions through the removal of damaged proteins and organelles. The chemicals of anthropogenic origin are of considerable interest for their ability to induce the activation of defense systems or interrupt the developmental program. Cd is a known stress agent and in some cases, as well as inducing apoptosis, is able to trigger autophagy.7-9 Furthermore, autophagy was identified as a biomarker of heavy metal toxicity in human stem cells.10 However, at present, there are few studies on the role of autophagy during development and on the putative protective function that it has in embryos exposed to stress.

*Correspondence to: Maria C. Roccheri; Email: [email protected] Submitted: 02/02/11; Revised: 05/06/11; Accepted: 05/11/11 http://dx.doi.org/10.4161/auto.7.9.16450 1028

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Figure 1. Quantification of AVOs by densitometric analysis, after neutral red (NR) vital staining and optical light microscopy observations. In the abscissa: hours (h) of development of the analyzed embryos, during physiological growth and after Cd-treatment; in the ordinate: intensity values obtained after quantification of the AVOs signal (arbitrary values). Data are presented as the mean of triplicate experiments (100 embryos for each sample).

or even stop it temporarily. The choice of an alternative pathway, however, can be a dangerous approach because it could lead to highly altered phenotypes.11 It was previously demonstrated that subacute/sublethal concentrations of Cd induce, during development of sea urchin embryos, morphological abnormalities, activation of specific stress proteins (heat shock proteins, HSPs), and expression of metallothioneins and apoptosis, in a dose/time-dependent manner.12-18 Essentially, Cd exerts its toxic action in the long-term, as it accumulates in cells. It has been suggested that survival systems adopted during embryonic development of sea urchins can operate in tandem through a putative crosstalk.17 The metallothioneins and HSPs are able to produce a detoxifying and antioxidant effect that is not always sufficient to block the action of the toxic metal, depending on the extent of cell damage.19,20 In such circumstances the mechanisms of programmed cell death, such as apoptosis, may be triggered.21 It is currently well accepted that autophagic cell death represents a separate route of programmed cell death significantly different from conventional apoptosis.22 However, autophagy and apoptosis should not be regarded as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in the cellular response to environmental changes, both physiological and pathological.23 The progress of studies about the role of autophagy in normal growth and development and the study of genes that encode the intermediate autophagy pathway represent a current area of research applied to various fields such as physiology, pathology and development. The marine invertebrate embryos are an appropriate model for this kind of research.

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Figure 2. NR vital staining on whole-mount embryos. The images, of representative embryos, were captured by light microscopy. (A) Control embryo, after 18 h of growth. (B) Cd-treated embryo for 18 h. (C) Embryo incubated with bafilomycin A1. (D) Cd-treated embryo for 18 h and then incubated with bafilomycin A1. (E and F) Enlargements of a section of (B and D), respectively. Bar = 50 μm.

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a suitable model system to investigate the adaptive response of cells exposed to stress during differentiation. The purpose of this study was to investigate whether autophagy occurs in the Paracentrotus lividus embryos, a species of sea urchin very common in the Mediterranean Sea, as a defense strategy after exposure to Cd stress, for the survival and maintenance of the developmental program. In extreme cases, the embryo may respond by changing the pathway of development,

In this work we highlight the activation of autophagy, as a further defense system against Cd stress, during sea urchin development. For this purpose we used different methods to detect the occurrence of autophagy in P. lividus embryos. Through incubation with the vital dye neutral red (NR), we studied the acidic vesicular organelles in whole embryos, just after fertilization, monitoring the control and those treated continuously with 1 mM Cd, from 12 to 24 h of development. NR is a lysosomotropic dye, changing from red to yellow as the pH shifts from acidic (pH 6.8) to basic (pH 8.0). This molecule, in its unprotonated form, is soluble in lipid membranes, and after entering an acid compartment it becomes protonated, staining the AVOs red. Therefore, we employed the NR vital stain as a new potential probe to identify these organelles in whole embryos. The Cd-treated embryos showed a progressive increase of AVOs, from 12 to 18 h, with a peak at 18 h, and a decrease of AVOs at 21 and 24 h. It should be noted that a basal autophagic signal was found in control embryos. The data, obtained measuring the intensity of pointed signal by densitometric analysis, have been summarized in the histogram of Figure 1. To confirm that the observed events were symptomatic of autophagy, we focused our attention on 18 h-treated embryos employing a specific inhibitor of autophagy, bafilomycin A1, an inhibitor of the vacuolar-type H+ ATPase of AVOs.6 As shown in Figure 2, after NR staining, the treated embryos displayed

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Figure 3. Quantification, by densitometric analysis, of AVOs after acridine orange (AO) vital staining and CLSM observations. In the abscissa: hours of development of analyzed embryos, during physiological growth and after Cd-treatment; in the ordinate: intensity values obtained after quantification of the AVOs signal (arbitrary values). Data are presented as the mean of triplicate experiments (100 embryos for each sample).

considerable perinuclear and cytosolic red dots (Fig. 2B and E), compared with controls (Fig. 2A). In addition, these embryos displayed not only a higher number, but also a larger size of AVOs, compared with the control, indicative of several cytosolic elements destined to be eliminated through autophagy. In contrast, the control and treated embryos, incubated for 30 min in 200 nM bafilomycin A1, a concentration that did not alter the embryos’ morphology, showed a drastic reduction in AVOs (Fig. 2C, D and F). These results were confirmed by an in vivo acridine orange (AO) fluorescence assay and confocal laser scanning microscopy (CLSM). AO is a metachromatic dye, emitting green fluorescence in monomeric form, and red fluorescence in bi/oligomeric form, related to the protonation of AVOs. The different optical sections captured by CLSM, and red stained, were grouped in stacks and analyzed through ImageJ software. In this manner it was possible to measure, in whole embryos, the amount of red AO-labeled organelles. These data have been summarized in the histogram reported in Figure 3. The external and equatorial optical sections of Figure 4 show that the embryos treated for 18 h, after AO vital staining, exhibited a considerable number of red dots located around nuclei (Fig. 4B1 and B2, E), much different than the controls (Fig. 4A1 and A2). The results obtained revealed important spatial features in the localization of AO granules: We found a great localization of AVOs in the ectoderm, probably due to the major exposure of this section of cells to Cd insult. By incubation with 200 nM bafilomycin A1 we observed a drastic reduction in AVOs (Fig. 4C1, C2, D1 and D2), confirming that the red granules were AVOs. In order to evaluate a specific marker of the autophagic process, we performed protein gel blot analysis to detect LC3. Although the antibody is able to recognize both the LC3-I and -II form, we found only LC3-II. In effect, using a vertebrate cell extract of the human breast cancer MDA-MB231 line, the antibody recognized both forms (even if the band of the inactive form is very faint) confirming the validity of the experimental procedure of protein extraction. In addition, employing a different polyclonal

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Figure 4. AO vital staining on whole-mount embryos. The images, of representative embryos, were captured by CLSM. Embryo after 18 h of growth: (A1–D1) external and (A2–D2) equatorial optical sections. (A1 and A2) Control. (B1 and B2) Cd-treated embryo for 18 h. (C1 and C2) Embryo incubated with bafilomycin A1. (D1 and D2) Cd-treated embryo for 18 h and then incubated with bafilomycin A1. (E and F) Enlargements of a section of (B1 and D1), respectively. Bar = 50 μm.

antibody against an isoform of LC3 (anti-LC3-B), we obtained a significant band of the inactive form for human cells, but not for embryos. Therefore, the results found in sea urchin could be explained considering that the employed antibodies are heterologous and/or that LC3-I is more labile and sensitive to the extraction conditions than LC3-II.24 Unfortunately, as described by Mizushima and Yoshimori,25 LC3-I protein is not always detectable using the currently available antibodies. This circumstance has been documented in other experimental models, for example in Hydra.26 Quantitative analysis showed an increase of LC3-II in embryos treated with Cd, from 12 to 24 h, with a peak at 18 h, followed by a decrease at 21 and 24 h (Fig. 5), different from that observed for the AVOs signal which remained at high levels for the same treatment times. The LC3-II reduction could be justified assuming that LC3-II, associated with the autophagosome membrane, is degraded by autophagy, when the cells are subjected to longlasting exposure to stress.27 Furthermore, the lower level of

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Figure 5. Representative immunoblotting detection against LC3. In the upper part, on the left: total lysates from control and Cd-treated embryos after 12, 15, 18, 21 and 24 h of development, reacted with anti-LC3 antibody. The same samples were incubated with anti-actin antibody. The figures show different parts of the same gel. In the lower part, on the left: densitometric analysis of bands visualized by immunoreaction. Results were obtained from the analysis of LC3-II band intensities normalized by comparison to actin and reported as arbitrary units. Data in the histogram are presented as the mean of triplicate experiments. In the upper part, on the right: embryos, control and Cd-treated (18 h), and MDA-MB231 human breast cancer cells, incubated with anti-LC3 antibody. In the lower part, on the right: embryos, control and Cd-treated (18 h), and MDA-MB231 human breast cancer cells, incubated with anti-LC3B antibody.

LC3-II could be due to apoptotic events that occur after a strong stress.15 In addition, we observed a marked increase of LC3-II in control embryos after 18 and 24 h of development. This evidence could be related to a stage-dependent metabolical requirement. Effectively, gastrulation is a critical developmental stage in which cell movements and migrations are consistent; therefore it is possible that autophagy could play a key role in this phase. Further analysis by immunofluorescence with the same antibody applying CLSM analysis, has shown, in situ, the recognition of both LC3-I and LC3-II, bypassing the extraction procedure of proteins for which the inactive form appears sensitive. Indeed, the signal of the cytosolic form (LC3-I) is widespread in the cytoplasm, while the signal for the active form of the protein (LC3-II), anchored in the membrane of autophagosomes is punctate. The analysis conducted by immunofluorescence confirmed a higher level of autophagosomes for embryos treated for 18 h with Cd (Fig. 6A and C), compared with the control (Fig. 6D and F). Through CLSM analysis, it was possible to confirm that the localization of autophagosomes is analogous to autolysosomes: cytoplasmic and perinuclear. Discussion Pioneering studies on eggs and embryos of invertebrates were performed in the 1960s. As regards eggs, the involvement of lysosomes in the digestion of yolk triggered after fertilization

has been suggested (interestingly, multivesicular bodies appeared prominently in this process);28 as regards embryos, a role for autophagy in insect metamorphosis was proposed that represents a dramatic developmental change associated with widespread cell death and complete disappearance of whole tissues.29 In the past ten years it has been shown that autophagy plays a key role during embryo development, differentiation and tissue remodeling of some invertebrates and vertebrates, contributing to the recycling of cytoplasmic components.4 At present, autophagy is not described in embryos of marine organisms. Sea urchins have been used as a model organism in developmental biology for many years and are considered the most primitive deuterostome with calcified skeletons and are related to vertebrates and protochordates. Therefore, knowledge of the autophagic process in this organism could be significant. In this paper we study the autophagic process, investigating whether this event can be activated as defense strategy in P. lividus sea urchin embryos under Cd stress, a heavy metal recognized as an environmental contaminant.30 It is important to emphasize that in this work we employed Cd as a stressor for the induction of autophagy purely as a toxic insult and it in no way constitutes an environmental stressor, as the concentration of Cd is many orders of magnitude higher than would be found in a polluted situation. On the other hand, we have previously demonstrated that long-lasting exposure to Cd concentrations, similar to those found in moderately or highly polluted seawaters, causes severe

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developmental delays and abnormalities during time in culture, showing that even very small amounts of Cd, if accumulated in cells, produce significant cytotoxic effects as well as apoptosis.16 Our study demonstrated that autophagy is a molecular process present in sea urchin embryos at a higher level after Cd treatment and at a basal level during physiological development. Our previous studies showed that Cd promotes different defense strategies such as the synthesis of HSPs and the activation of apoptosis, in a dose-time dependent manner.18 This work provides, through the use of different methods31 including NR and AO vital staining on whole mount and LC3 detection, both through protein gel blot and in situ immunofluorescence, new evidence concerning the autophagy mechanism induced by Cd during embryogenesis. Interestingly, the application of the techniques on whole organisms allows us to investigate the autophagic phenomenon in multipotent cells, which interact among themselves, in their natural position, bypassing the disadvantages of isolated cells, deprived of their normal network. Furthermore, autophagy seems to anticipate apoptosis temporally, suggesting a possible relationship between the two phenomena.32 Figure 6. Detection by immunofluorescence of LC3 protein on whole-mount embryos. On the one hand, we have previously shown that Equatorial optical sections captured by CLSM. (A, D and G) LC3 protein detection; (B, E if Cd insult is less than 15 h, P. lividus embryos and H) nuclei stained with propidium iodide; (C, F and I) merge of green and red. (A–C) Cd-treated embryo for 18 h. (D–F) Control embryos, after 18 h of growth. (G, H and I) restore normal development,13 probably because Negative control. Bar = 50 μm. only a few cells are damaged and removed through apoptosis, allowing the recovery of the morphology. On the other hand, if the Cd insult is much higher (i.e., for embryo activates the mechanism of apoptosis. In this case, the 24 h at the same concentration) the cells killed are more numer- second hypothesis, autophagy, could provide the ATP necessary ous, and the normal development cannot be restored.15 for apoptosis during development, by recycling of damaged celThe outcome from autophagy—a maximum peak after 18 lular components.33 On the basis of the third hypothesis, since h Cd exposure at the time in which apoptosis is just begin- the sea urchin embryo does not have specialized phagocytes, it ning—suggests that it could act as a cell survival process, when could begin the clearance of apoptotic bodies through autophagy. HSPs and metallothioneins induction is not able to preserve the In conclusion, although sea urchin embryos have a high resisdevelopmental program. Alternatively autophagy, like apoptosis tance to chemical pollutants,11 we suggest that chronic exposure and necrosis, can contribute to cell death in response to stress to Cd, which produces cellular accumulation of this metal, causes conditions (such as Cd accumulation), but the choice of aberrations in development, autophagy and, at last, apoptosis. In these mechanisms depends on cell type and the triggering fac- this context, autophagy could play a crucial role in stress response tors.7 The homeostatic relationship between apoptosis and of this suitable model system. autophagy during sea urchin development represents a very interesting chapter and further studies are in progress in our Materials and Methods laboratory. On the basis of actual data, we propose three different hypoth- Embryo cultures and Cd treatment. Gametes were collected eses about the role of autophagy: (1) hierarchical choice of defense from gonads of the sea urchin P. lividus harvested from the west mechanisms; (2) energetic hypothesis; (3) clearance of apoptotic coast of Sicily. Eggs were fertilized at a concentration of 5,000/ml bodies. According to the first hypothesis, we suggest that the and grown as previously described by Roccheri and colleagues.13 temporal choice of the two mechanisms depends on the fact that Just after fertilization, part of the embryo culture was continuthe embryo tries to oppose the stress conditions using, initially, a ously grown in 1 mM Cd (treated) for different times (12, 15, defense strategy that is less deleterious, namely autophagy, in an 18, 21 and 24 h), and part was grown as control without treatattempt to preserve the developmental program. However, if this ment. Embryo development was monitored by light microscopy process is not sufficient to offset the damage caused by stress, the (Olympus BX50).

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Treatment with bafilomycin A1. At 18 h of development, the embryos, control and treated, were incubated with bafilomycin A1 (ALEXIS Biochemical, ALX-380-030), at 200 nM final concentration, for 30 min. Neutral red vital staining. Whole-mount embryos were stained with NR, to detect the AVOs’ number and volume. Control and treated embryos, at different development times, were incubated with NR (Molecular Probes, N3246) at 0.1 M final concentration, for 15 min. Excess dye was removed by repeated washings with Millipore filtered sea water. Embryos were examined by light microscopy under 40x magnification, and images were captured with a Nikon camera. Quantification of AVOs spots was performed by optical densitometry using ImageJ software. Acridine orange vital staining. AO staining was performed to detect the number and the volume of AVOs on whole-mount embryos. Control and treated embryos, at different development times, were incubated with AO (SIGMA-ALDRICH, A-6014) at 5 μM final concentration, for 1 min. Then, the embryos were washed twice with Millipore filtered sea water to remove the excess dye and observed using a CLSM (Olympus FV-300 equipped with argon, 488 nm and helium/neon, 543 nm, lasers) under a 60X oil immersion lens. Several optical sections (10-μm thick) of embryos were acquired using a Nikon camera. Image acquisition and analysis were performed as described by Morici and colleagues.34 Electrophoretic analysis and protein gel blot. Pellets of control and Cd-treated embryos were homogenized in lysis buffer (7 M urea, 2% CHAPS and 10 mM DTT) containing protease inhibitors cocktail tablets (Roche, 1836170). Protein concentration was evaluated using the Bradford method and 40 μg of samples were analyzed by SDS-PAGE (13% gel). The molecular masses were evaluated by comparison with a set of standard proteins (Fermentas, SM0671). After electrophoretic separation, the proteins were transferred and immunoreacted as described by Roccheri and colleagues.13 Specifically, the antibodies adopted were: rabbit polyclonal anti-LC3 (Sigma-Aldrich, L8918) and anti-actin (Sigma-Aldrich, A5060) diluted respectively 1:1,000 (overnight incubation) and 1:500 (1 h incubation) in 5% nonfat dried milk powder (EuroClone, EMR180500)/ TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween 20).

As secondary antibody we used an anti-rabbit IgG (Fc) alkaline phosphatase-conjugated (Promega, S373B), diluted 1:7,500 in TBST (1 h incubation). Immunoreaction signals were indicated by the reporter BCIP/NBT (Sigma-Aldrich, B1911). To test the specificity of the anti-LC3 antibody, we employed protein extracts (processed in the same manner) of the MDA-MB231 human breast cancer cell line, as a positive control. In addition, we employed anti-LC3-B (Cell Signaling, 2775) antibody, diluted 1:1,000 (overnight incubation). Bands were analyzed by ImageJ software and normalized with respect to the corresponding values obtained with the anti-actin antibody reaction. Data obtained by protein gel blot analysis are presented as mean ± SEM of triplicate experiments. Immunofluorescence. Immunofluorescence was performed on whole-mount control and treated embryos fixed, as previously described by Kiyomoto and colleagues.35 Subsequently, each sample was incubated for 1 h at room temperature in blocking solution: 0.5% albumin from bovine serum (Sigma-Aldrich, A7906) and 5% heat inactivated goat serum (Sigma-Aldrich, G9023) in PBS-T (phosphate-buffered saline, 0.1% Tween 20) and overnight at 4°C with anti-LC3 antibody, 1:125 diluted in blocking solution. In the negative controls, the primary antibody was omitted. After rinsing with PBS-T, embryos were incubated with a fluorescein conjugated secondary antibody anti-rabbit IgG (whole molecule)-FITC, developed in goat (Sigma-Aldrich, F0382), 1:50 diluted in blocking solution. The nuclei were stained with propidium iodide for 5 min. Samples were mounted on glass in 80% glycerol/PBS-T. The observations were performed by CLSM as described for AO detection. Image acquisition was performed measuring the intensity of autofluorescence in the negative control; this value becomes the threshold level for the capture of other samples.

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References 1.

2.

3.

4.

5.

Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000; 290:171721; PMID:11099404; http://dx.doi.org/10.1126/science.290.5497.1717. Penaloza C, Lin L, Lockshin RA, Zakeri Z. Cell death in development: shaping the embryo. Histochem Cell Biol 2006; 126:149-58; PMID:16816938; http:// dx.doi.org/10.1007/s00418-006-0214-1. Kelekar A. Autophagy. Ann NY Acad Sci 2005; 1066:259-71; PMID:16533930; http://dx.doi. org/10.1196/annals.1363.015. Di Bartolomeo S, Nazio F, Cecconi F. The role of autophagy during development in higher eukaryotes. Traffic 2010; 11:1280-9; PMID:20633243; http:// dx.doi.org/10.1111/j.1600-0854.2010.01103.x. Tsukamoto S, Kuma A, Mizushima N. The role of autophagy during the oocyte-to-embryo transition. Autophagy 2008; 4:1076-8; PMID:18849666.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgements

We thank MD Joe Louis Scilabra for language revision and Mr. Giovanni Morici for technical assistance. This work was supported by 60% MIUR to Prof. Maria Carmela Roccheri.

6. Tasdemir E, Galluzzi L, Maiuri MC, Criollo A, Vitale I, Hangen E, et al. Methods for assessing autophagy and autophagic cell death. Methods Mol Biol 2008; 445:29-76; PMID:18425442; http://dx.doi. org/10.1007/978-1-59745-157-4_3. 7. Dong Z, Wang L, Xu J, Li Y, Zhang Y, Zhang S, et al. Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells. Toxicol In Vitro 2009; 23:10510; PMID:19061949; http://dx.doi.org/10.1016/j. tiv.2008.11.003. 8. Wang SH, Shih YL, Kuo TC, Ko WC, Shih CM. Cadmium toxicity toward autophagy through ROSactivated GSK-3beta in mesangial cells. Toxicol Sci 2009; 108:124-31; PMID:19126599; http://dx.doi. org/10.1093/toxsci/kfn266. 9. Templeton DM, Liu Y. Multiple roles of cadmium in cell death and survival. Chem Biol Interact 2010; 188:267-75; PMID:20347726; http://dx.doi. org/10.1016/j.cbi.2010.03.040.

10. Di Gioacchino M, Petrarca C, Perrone A, Farina M, Sabbioni E, Hartung T, et al. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells. Sci Total Environ 2008; 392:50-8; PMID:18166216; http://dx.doi. org/10.1016/j.scitotenv.2007.11.009. 11. Hamdoun A, Epel D. Embryo stability and vulnerability in an always changing world. Proc Natl Acad Sci USA 2007; 104:1745-50; PMID:17264211; http:// dx.doi.org/10.1073/pnas.0610108104. 12. Russo R, Bonaventura R, Zito F, Schröder HC, Müller I, Müller WE, et al. Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos. Cell Stress Chaperones 2003; 8:232-41; PMID:14984056; http://dx.doi.org/10.1379/14661268(2003)0082.0.CO;2. 13. Roccheri MC, Agnello M, Bonaventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004; 321:80-7; PMID:15358218; http:// dx.doi.org/10.1016/j.bbrc.2004.06.108.

www.landesbioscience.com Autophagy

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14. Agnello M, Filosto S, Scudiero R, Rinaldi AM, Roccheri MC. Cadmium accumulation induces apoptosis in P. lividus embryos. Caryologia 2006; 59:403-8. 15. Agnello M, Filosto S, Scudiero R, Rinaldi AM, Roccheri MC. Cadmium induces an apoptotic response in sea urchin embryos. Cell Stress Chaperones 2007; 12:4450; PMID:17441506; http://dx.doi.org/10.1379/ CSC-229R.1. 16. Filosto S, Roccheri MC, Bonaventura R, Matranga V. Environmentally relevant cadmium concentrations affect development and induce apoptosis of Paracentrotus lividus larvae cultured in vitro. Cell Biol Toxicol 2008; 24:603-10; PMID:18322810; http:// dx.doi.org/10.1007/s10565-008-9066-x. 17. Agnello M, Roccheri MC. Apoptosis: Focus on sea urchin development. Apoptosis 2010; 15:322-30; PMID:19876739; http://dx.doi.org/10.1007/s10495009-0420-0. 18. Roccheri MC, Matranga V. Cellular, biochemical and molecular effects of cadmium on marine invertebrates: focus on Paracentrotus lividus sea urchin development. In: Parvau RG, Ed. Cadmium in the Environment. New York, NY: Nova Science Publishers 2010; 337-66. 19. Alhama J, Romero-Ruiz A, Jebali J, López-Barea J. Total metallothionein quantification by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization. In: Parvau RG, ed. Cadmium in the Environment. New York, NY: Nova Science Publishers 2010; 389-405. 20. Samali A, Cotter TG. Heat shock proteins increase resistance to apoptosis. Exp Cell Res 1996; 223:16370; PMID:8635489; http://dx.doi.org/10.1006/ excr.1996.0070. 21. Hamada T, Tanimoto A, Sasaguri Y. Apoptosis induced by cadmium. Apoptosis 1997; 2:359-67; PMID:14646532; http://dx.doi. org/10.1023/A:1026401506914.

22. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009; 16:3-11; PMID:18846107; http://dx.doi. org/10.1038/cdd.2008.150. 23. Bursch W, Ellinger A, Gerner C, Fröhwein U, Schulte-Hermann R. Programmed cell death (PCD). Apoptosis, autophagic PCD or others? Ann NY Acad Sci 2000; 926:1-12; PMID:11193023; http://dx.doi. org/10.1111/j.1749-6632.2000.tb05594.x. 24. Settembre C, Fraldi A, Jahreiss L, Spampanato C, Venturi C, Medina D, et al. A block of autophagy in lysosomal storage disorders. Hum Mol Genet 2008; 17:119-29; PMID:17913701; http://dx.doi. org/10.1093/hmg/ddm289. 25. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007; 3:542-5; PMID:17611390. 26. Buzgariu W, Chera S, Galliot B. Methods to investigate autophagy during starvation and regeneration in hydra. Methods Enzymol 2008; 451:409-37; PMID:19185734; http://dx.doi.org/10.1016/S00766879(08)03226-6. 27. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 2005; 1:84-91; PMID:16874052; http://dx.doi. org/10.4161/auto.1.2.1697. 28. Dalcq AM. The relation to lysosomes of the in vivo metachromatic granules. In: de Reuck AVS, Cameron MP, eds., Ciba Foundation Symposium on Lysosomes. Boston, MA: Little, Brown and Company, 1963:226-63. 29. de Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol 1966; 28:435-92; PMID:5322983; http:// dx.doi.org/10.1146/annurev.ph.28.030166.002251.

30. Wang SH, Shih YL, Ko WC, Wei YH, Shih CM. Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway. Cell Mol Life Sci 2008; 65:3640-52; PMID:18850067; http://dx.doi. org/10.1007/s00018-008-8383-9. 31. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4:151-75; PMID:18188003. 32. Fimia GM, Piacentini M. Regulation of autophagy in mammals and its interplay with apoptosis. Cell Mol Life Sci 2010; 67:1581-8; PMID:20165902; http:// dx.doi.org/10.1007/s00018-010-0284-z. 33. Mellén MA, de la Rosa EJ, Boya P. The autophagic machinery is necessary for removal of cell corpses from the developing retinal neuroepithelium. Cell Death Differ 2008; 15:1279-90; PMID:18369370; http:// dx.doi.org/10.1038/cdd.2008.40. 34. Morici G, Agnello M, Spagnolo F, Roccheri MC, Di Liegro CM, Rinaldi AM. Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development. J Microsc 2007; 228:165-73; PMID:17970916; http://dx.doi. org/10.1111/j.1365-2818.2007.01860.x. 35. Kiyomoto M, Zito F, Costa C, Poma V, Sciarrino S, Matranga V. Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrixectoderm interactions in Paracentrotus lividus sea urchin embryos. Dev Growth Differ 2007; 49:731-41; PMID:17983367; http://dx.doi.org/10.1111/j.1440169X.2007.00967.x.

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Volume 7 Issue 9