Combined Effects of Cadmium and UVB Radiation on Sea Urchin

Article pubs.acs.org/crt

Combined Effects of Cadmium and UVB Radiation on Sea Urchin Embryos: Skeleton Impairment Parallels p38 MAPK Activation and Stress Genes Overexpression Rosa Bonaventura,* Roberta Russo, Francesca Zito, and Valeria Matranga* Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy ABSTRACT: Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-33epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.

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metals on marine/aquatic animals, including the water flea, the amphipods, and the fathead minnows.7−9 The sea urchin embryo has been successfully used as a model to evaluate the toxicity of many pollutants that generally cause morphological perturbations, mainly affecting skeleton formation (biomineralization) and patterning.10 These embryos are equipped with chemical defense genes to cope with chemical stressors.11 Moreover, they are able to protect themselves against physical stressors, such as excessive UVR, using mechanisms acting at different levels.12 In particular, Cd or UVB induced dose-dependent abnormalities affecting the elongation and patterning of the skeleton in Paracentrotus lividus embryos.13,14 In addition, UVB induced the activation of p38MAPK, one of the mitogen-activated protein kinases (MAPK).15 These are evolutionarily conserved mediators which are activated by a variety of environmental stresses, including ionizing radiation and Cd,16,17 and are subdivided into three major groups: (i) p38MAPK, (ii) extracellular

INTRODUCTION

Among metals, the cadmium (Cd) is identified as a priority hazardous substance within the Water Framework Directive1 because of its toxicity, persistence, and accumulation in the environment.2 Cd has no biological role inside the cells where it is accumulated causing several cytotoxic and metabolic effects; it interferes with essential metals through mechanism(s) that remain to be fully recognized.2 Ultraviolet radiation (UVR), particularly UVB (280−315 nm), is another important environmental stressor for marine ecosystems. Indeed, the reduction of the Earth’s ozone layer increases the penetration of UVB into the oceans mainly in the polar regions but also at temperate latitudes.3 UVB affects DNA, proteins, and lipids causing oxidative stress with severe consequences on the life of marine organisms.3 Many pollutants are present at the same time in the aquatic environment, even though most of them are present at very low concentrations.4 In addition, many pollutants are known to be more toxic in the presence of UVR, as, for example, polycyclic aromatic hydrocarbons (PAHs) in frog and sea urchin.5,6 Few studies have focused on UVR and on combined effects of heavy © XXXX American Chemical Society

Received: February 20, 2015

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DOI: 10.1021/acs.chemrestox.5b00080 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 1. Embryonic skeleton defects induced by Cd/UVB coexposure. (A) Schematic design of Cd/UVB coexposure experiments. Black line, hours postfertilization (hpf); red arrow, addition of CdCl2 at 0.5 hpf; yellow lightning bolt, irradiation with UVB at 3 hpf; red line, period of exposure to CdCl2; black arrows, timetable of withdrawals, 4 hpf for Western blots analyses; 27 hpf for IF, qPCR, and WMISH; 50 hpf for IF; and 72 hpf for microscopic inspection and IF. (B) The plot summarizes results from 3 independent experiments reporting the percentage of the embryos with a normal morphology scored at 72 hpf in controls, Cd-, UVB-, and Cd/UVB-embryos; the percentages are reported on the left of each box; boxes indicate the SD values; the lines in the middle of the boxes indicate the mean values; whiskers indicate the minimum and maximum values. (C) Normal embryo observed at 72 hpf. po, postoral rod; al, antero-lateral rod; vt, ventral-transverse rod; b, body rod. (D−H) Different morphologies of Cd/UVB embryos, observed 69 h after irradiation, i.e., 72 hpf.

adopted to cope with these toxic agents. The specific objectives were (1) the study of skeletal malformations induced during embryo development, using a mesodermic specific marker, msp130; (2) the identification of the signaling pathways involved by the analysis of the p38MAPK, ERK1/2, and JNK activation; (3) the analysis of the mRNAs levels of three stress markers, Pl-MT, Pl-14-3-3epsilon and Pl-jun by quantitative PCR, determining also their territorial localization by wholemount in situ hybridization.

signal−regulated kinase (ERK1/2), and (iii) c-Jun N-terminal kinase (JNK). Concerning the gene level, we previously showed that Cdexposed embryos respond by increasing the expression levels of the Pl-Metallothionein (MT) gene, in a time- and dosedependent manner.13 Another study on P. lividus embryos reported the identification of five metallothionein genes (MT4MT8) and their induction following 10−4 M Cd exposure.18 Accordingly, the overexpression of MT genes after Cd exposure is well documented in many marine invertebrates, such as sponges, polychaetes, crabs, mussels, and echinoderms.19 In humans, it has been shown that MT genes are responsive to a wide spectrum of stressors, including ultraviolet radiation.20 In P. lividus embryos, we also showed that UVB caused the up-regulation of Pl-14-3-3epsilon mRNA, which was expressed ectopically in all embryonic territories.21 The 14-3-3 protein family, found from plants up to humans, is involved in the regulation of many cellular processes including the stress response to UV radiations.22 The use of 14-3-3 transcripts and proteins as markers of stress in marine organisms is well documented as they are induced by pesticides in sponges,23 salinity stress in shrimp,24 and treatment with UVR and toxic diatom aldehydes (PUAs) in sea urchin embryos.25,26 Recently, we extended the transcriptional analysis in UVB-exposed embryos to a group of selected genes, including Pl-14-33epsilon, Pl-MT, and Pl-jun.27 The jun proteins are a family of basic leucine zipper (bZIP) transcription factors present in a wide variety of organisms, including the sea urchin.28 They are involved in the response to physiological signals and environmental insults, such as Cd and UVB.29,30 As only a very small number of studies have used the sea urchin embryo to evaluate the effects of mixtures of pollutants, i.e., PAHs,31 heavy metals,32 and pesticides,33 the lack of information still persists and major gaps need to be filled. The aim of the present study was to investigate the effects of Cd and UVB applied in combination (Cd/UVB) to P. lividus sea urchin embryos, in order to evaluate the stress response

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EXPERIMENTAL PROCEDURES

Caution: CdCl2 is a hazardous chemical and should be handled caref ully. Embryo Culture. Gametes were collected from gonads of the sea urchin Paracentrotus lividus harvested along the Northwestern coast of Sicily. Eggs were fertilized and embryos reared at 18 °C in Millipore (Billerica, MA, USA) filtered seawater (MFSW) containing antibiotics (50 mg/L streptomycin sulfate and 30 mg/L penicillin). Cadmium Concentration and UVB Dose. To study the skeletal malformations induced by Cd/UVB, we took advantage of previous works on the effects caused by single stressors. We selected the sublethal doses of Cd and UVB that had mild consequence on the embryonic developmental program, i.e., between 60% and 90% of the embryos showing normal development, although with some delays. In particular, we selected the continuous exposure to the dose of 10−4 M CdCl2 (added just after fertilization) as it caused 64−90% of developmental delays and only 10−36% of abnormal development (after 48 h), i.e., abnormal blastulae with cells accumulating in a disorderly manner in the blastocoelic cavity.13 As for UVB, we decided to expose embryos at the cleavage stage at 200 J/m2 because we have previously shown that this dose caused 30% of developmental delays and 10% of abnormal development (abnormal blastulae) in 48 h-old embryos.14 In addition, for human skin, the dose of 200 J/m2 is assumed to be one MED (minimal erythema dose), i.e., the dose of UVR required to produce a barely perceptible erythema in people with skin type 1.34 Experimental Exposures to Cd, UVB, and Cd/UVB. Cadmiumexposure experiments were carried out as previously described by Russo et al.13 Briefly, embryos (4000/mL) were continuously cultured soon after fertilization in the presence of 10−4 M CdCl2 (C-2544, B


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Chemical Research in Toxicology Sigma Chemical Co., St Louis, MO, USA), in the following referred to as Cd-embryos. We used the UVB exposure procedure previously described by Bonaventura et al.14 Briefly, embryos (4000/mL) at the 32 cell stage, i.e., about 3 h postfertilization (hpf), were irradiated with a UV−B lamp (model VL-6.M., Labortechnik GmbH & Co. KG, Wasserburg/ B, Deutschland) at the dose of 200 J/m2 corresponding to an exposure of 28 s, in the following referred to as UVB-embryos. After irradiation, UVB-embryos were cultured at 18 °C in the dark to prevent the activity of DNA repairing enzymes. In the experiments with Cd and UVB applied in combination (Cd/ UVB coexposure), sea urchin embryos (4000/mL) were cultured soon after fertilization in the presence of 10−4 M CdCl2 and were irradiated with UVB at the dose of 200 J/m2 at about 3 hpf, in the following referred to as Cd/UVB-embryos. The scheme of the experimental design used in this study is shown in Figure 1A. Morphological Analysis. Development was monitored at the pluteus stage, 69 h after irradiation (72 hpf). We used this experimental end-point, when control embryos (unexposed embryos) were well developed plutei, as used in recognized standard procedures for the assessment of embryo toxicity.35 The percentage of plutei with normal development in each treatment was determined by counting about 100 larvae using a Zeiss Axioscop 2 plus microscope (Zeiss, Jena, Germany), and images were recorded by a digital camera. Whole-Mount Indirect Immuno-Fluorescence. At 72 hpf, control and treated embryos were fixed in 4% paraformaldehyde in MFSW for 1 h at room temperature and stored in 100% MeOH at −20 °C until use.21 Indirect immuno-fluorescence (IF) on whole mount embryos was performed using the 1D5 monoclonal antibody (mAb), specific for the msp130 glycoprotein, which localizes only on skeletogenic cell membranes, diluted 1:10, and the Endo1 mAb, specific for an endoderm marker, which localizes in the hindgut and midgut, diluted 1:5. Embryos were incubated overnight at 4 °C with 1D5 or Endo1 mAbs. After washing, embryos were incubated for 1 h with fluorescein-conjugated goat antimouse IgM or IgG secondary antibodies (Sigma Chemical Co., St Louis, MO, USA), diluted 1:200. Embryos were observed under a Zeiss Axioscop 2 plus microscope (Zeiss) equipped with epifluorescence, and the images were recorded by a digital camera. Western Blot Analysis. Control, Cd-, UVB-, and Cd/UVBembryos were lysed as described previously, with minor modifications.28 Briefly, 1 h after irradiation, embryos were Douncehomogenized on ice in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton), supplemented with a cocktail of protease (Roche Applied Science, Penzberg, Germany) and phosphatase (Sigma, Chemical Co., St Louis, MO, USA) inhibitors. Protein amounts corresponding to 40 and 80 μg were separated by electrophoresis under reducing conditions on 4− 15% precast gels (Bio-Rad, Hercules, California, USA) and transferred to nitrocellulose membranes using a Semi-Dry electrophoretic transfer cell (Bio-Rad, Hercules, California, USA). Membranes were incubated with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) and then incubated with polyclonal antibodies directed against phospho-p38MAPK (Thr180/TyY182) (P-p38MAPK, dilution 1:250; #9211, Cell Signaling Technology, Danvers, MA, USA), phospho-p44/ 42 MAPK (Erk1/2) (Thr202/Tyr204) (P-ERK, dilution 1:250; #9101, Cell Signaling Technology), and phospho-SAPK/JNK (Thr183/Tyr185) (P-JNK, dilution 1:250; #9251, Cell Signaling Technology) with gentle agitation overnight at 4 °C. Protein levels were normalized using tubulin (Monoclonal anti-α-tubulin dilution 1:500, T5168, Sigma) as internal control, performing the immune reaction on the same membranes, for 1 h at room temperature with gentle agitation. The secondary antibodies, Alexa Fluor 680 goat antirabbit (dilution 1:5000, Molecular Probes, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 780 goat antimouse (dilution 1:4000, Molecular Probes, Life Technologies), were incubated with gentle agitation for 1 h at room temperature. Proteins were visualized using an Odyssey Infrared Imaging System (LI-COR, Biosciences, Lincoln, NE, USA) in accordance with the manufacturer’s instructions. Fluorescence quantification was performed with the application

software, version 3.0 (LI-COR, Biosciences). Protein quantification values were shown as Integrated Intensity (I.I., counts-mm2). Real-Time Quantitative Polymerase Chain Reaction (qPCR). Total RNA from control, Cd-, UVB-, and Cd/UVB-embryos was extracted according to Russo et al.13 One microgram of total RNAs was reverse transcribed according to the Applied Biosystems manufacturer’s instructions. An aliquot of the cDNA obtained was used to perform the qPCR. Quantification of gene expression was performed as described by the instruction manual of Applied Biosystems Step One Plus real time PCR, using a Comparative Threshold Cycle Method, with SYBR Green chemistry.36 Pl-Z12-1 was used as the endogenous control gene.37 The qPCR was run as described by Russo et al.21 The oligonucleotides used were described by Russo et al.27 Whole-Mount in Situ Hybridization (WMISH). WMISH was performed as previously described by Russo et al.38 for Pl-MT, Pl-jun,28 and Pl-14-3-3epsilon.21 All of the prehybridization and hybridization steps were carried out in 96-well plates, using 30−40 embryos per well. Antisense and sense RNA probes were synthesized by runoff transcription and labeled with digoxigenin (DIG) (Roche, Applied Science, Penzberg, Germany). DIG-labeled probes were detected by colorimetric staining using the NBT/BCIP substrate. After washings, embryos were mounted on glass slides and observed using a Zeiss Axioscop 2 plus microscope (Zeiss); images were recorded by a digital camera. Hybridization with sense probes showed no specific signal. An antisense msp130-DIG-labeled RNA probe was used as a positive control.28 Statistical Analysis. Values obtained for the morphological analysis, expressed as percentage (%) of embryos with normal morphology, were reported as the mean of three independent experiments ± standard deviation (SD). Two duplicates were carried out for the first two experiments and no replication in the third. QPCR values were reported as the mean of four independent qPCR analyses ± SD using two/three syntheses of cDNA. Results were compared using one way analysis of variance, ANOVA, with pairwise comparisons among treatments made using Tukey’s HSD test. The analyses were performed using the OriginPro 8.1 statistical program (OriginLab Corp., Northampton, MA, USA), and the level of significance was set to P ≤ 0.05.

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RESULTS Cd/UVB Induced Abnormal Branching in the Embryonic Skeleton. The plot in Figure 1B summarizes results from 3 independent experiments reporting the percentage of embryos with a normal morphology scored at 72 hpf. We found that 64.56% (±23.76 SD) of Cd-embryos and 70.18% (±13.12 SD) of UVB-embryos had a normal morphology. On the contrary, a marked decrease in the percentage of embryos with a normal morphology, namely 23.81% (±11.69 SD), was observed in Cd/UVB-embryos. Control embryos (unexposed embryos) developed normally (96.89% ± 1.67 SD) with a well organized skeleton, as shown in Figure 1C, where the different elements of the skeleton (rods) are indicated. Selected examples of developmental defects observed in 72 h-old Cd/ UVB-embryos are shown in Figure 1D−H. The Cd/UVB treatment caused a variety of abnormalities, mostly affecting skeleton morphogenesis. For example, the abnormal development of one of the two ventral-transverse rods produced the branching out of multiple supernumerary rods (Figure 1D). In other Cd/UVB embryos, the antero-lateral rods showed abnormal multiple small spines (Figure 1E). Other more severe abnormal morphologies were also observed with complex skeleton patterns showing a widespread (often radial) disorganization of the skeletal rods that resulted in the loss of a pluteus-like shape (Figure 1F−H). As the skeleton patterning was greatly affected by Cd/UVB exposure, we performed an IF analysis on the presence and C


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Figure 2. Cd/UVB coexposure affects the localization of the skeletogenic cells (PMCs). (A) Schematic drawing of a late gastrula embryo: PMCs are shown in green and are arranged in two ventrolateral (VL) clusters and ventral (V), dorsal (D), and longitudinal (L) chains; the triradiate spicules (skeleton) are shown in yellow; the primitive gut is depicted in plum/purple. (B−P) Whole mount immunofluorescence (IF) with 1D5 mAb, recognizing the glycoprotein MSP130 specifically expressed on PMCs, on normal and treated embryos at 27 hpf (B−H) and 50 hpf (J−P). (E) Unexposed embryos, (B−F) Cd-embryos, (C−G) UVB-embryos, and (D−H) Cd/UVB-embryos at 27 hpf. (I) Schematic drawing of a pluteus embryo: the postoral (PO), body (B), ventral-transverse (VT), and anterolateral (AL) rods constitute the larval skeleton showed in yellow; the three parts of the gut, hindgut (h), midgut (m), and foregut (f), are depicted in plum/purple. (M) Unexposed-embryo, (J−N) Cd-embryos, (K−O) UVBembryos, and (L−P) Cd/UVB-embryos at 50 hpf. (Q−T) Whole mount IF with Endo1 mAb, recognizing an endoderm marker localized in the hindgut (h) and midgut (m), on normal and treated embryos at 72 hpf. (Q) Unexposed-embryo, (R) Cd-embryo, (S) UVB-embryo, and (T) Cd/ UVB-embryos. The dotted white line bordered the hindgut.

compared to those present in unexposed late gastrula embryos (Figure 2E). In addition, a few PMCs moved away from their regular arrangement in both Cd-embryos (see the arrowheads in Figure 2B) and UVB-embryos (see the arrowheads in Figure 2C−G). In Cd/UVB embryos, the PMCs pattern is greatly affected, as cells were also found inside the blastocoelic cavity not positioned along the canonical chains (Figure 2D−H). At the pluteus stage, the skeleton rods develop in postoral (PO), antero-lateral (AL), body (B), and ventral-transverse (VT) rods (Figure 2I).10 In Cd-embryos at 50 h after exposure (Figure 2J−N), we found pluteus-like embryos with abnormal

organization of the skeletogenic cells, namely the primary mesenchyme cells (PMCs), using mAb for msp130, a PMCspecific cell surface marker.39 As outlined in Figure 2A, PMCs show a typical organization in two symmetric ventrolateral (VL) clusters at the late gastrula stage, with a ventral (V), a dorsal (D), and two longitudinal (L) chains.10 The deposition of a calcitic skeleton (composed of magnesium calcite plus occluded spicule matrix proteins) occurs at first within the two ventrolateral clusters in the form of triradiate spicules (in yellow). In Cd-embryos (Figure 2B−F), one of the two longitudinal chains was undersized (see asterisks), when D


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changes in their levels, i.e., around 1.5-fold in both UVB- and Cd/UVB-embryos lysates compared to those of controls. Cd seems not to affect the activation of P-ERK, as we found only a 1.17-fold increase compared to that of controls. The anti-P-JNK failed to detect any band even after increasing the amount of total proteins loaded for each embryo lysate. To prove the cross-reactivity of the antibody, embryos irradiated with a higher dose of UVB (800 J/m2) were used (see box in Figure 3) as a positive control. In this case, a band of 54 kDa was recognized, suggesting that JNK might not be activated by the former conditions of exposure used or that the small amount of phosphorylated protein was undetectable by this method. Overexpression of Pl-MT, Pl-14-3-3epsilon, and Pl-jun Genes in Cd/UVB-Embryos. We recently analyzed the expression of specific genes encoding for DNA repair proteins, transcription factors, and adapter and structural proteins and reported their dose- and time-dependent response in UVBembryos (400 and 800 J/m2). On the basis of our analysis, genes were classified as early (2 h after irradiation) and late (24 h after irradiation) UVB responders, along with a number of genes that were either not responsive or both early and late UVB responders. Among them, in this study we selected three genes coding for (i) the stress protein metallothionein (Pl-MT), a late UVB-responder gene; (ii) the 14-3-3 (Pl-14-3-3 epsilon); and (iii) the jun protein (Pl-jun), both early and late UVBresponders.27 As Pl-MT is an exclusively late UVB-responder gene, we decided to analyze the expression of the three selected genes in embryos at 27 h of development. As shown in Figure 4, mRNAs levels of all genes tested increased considerably in embryos treated with both agents. In particular, the Pl-14-3-3epsilon transcription levels increased by approximately 15-fold in Cd/UVB-embryos, compared with control embryos (Figure 4A). This value is approximately 3fold higher than that observed in Cd- or UVB-embryos. We found very high levels of Pl-MT mRNA in Cd/UVB-embryos, that increased by approximately 70-fold, compared with that of control embryos (Figure 4B). This value is 3-fold higher than that observed in Cd-embryos and 8-fold higher than that found in UVB-embryos. As for Pl-jun mRNA levels, we found a 4-fold increase over control values, but only in Cd/UVB-embryos, with no overexpression detected in either Cd- or UVB-embryos (Figure 4C). Ectopic Expression of Pl-MT, Pl-14-3-3epsilon, and Pljun mRNAs in Cd/UVB-Embryos. Given the considerable increase in the expression of Pl-14-3-3epsilon, Pl-MT, and Pl-jun in Cd/UVB-embryos at 27 h of development, it was interesting to analyze their mRNAs localization by WMISH. As shown in Figure 5A, Pl-14-3-3epsilon was expressed in the presumptive gut at the gastrula stage in control embryos, in accordance with findings reported previously.21 In Cd/UVB-embryos, where the elongation of the gut was delayed, the transcripts were expressed not only in the invaginating gut but also in many ectopic cells inside the blastocoel (Figure 5B). Pl-MT mRNA was expressed in the gut of the control gastrula embryos (Figure 5C), as previously described.38 In Cd/UVB-embryos, Pl-MT mRNA was localized in the gut, as expected, as well as abnormally distributed in some cells inside the blastocoelic cavity (Figure 5D). Pl-jun mRNA was expressed in the skeletogenic cells (PMCs) of the control gastrula embryos (Figure 5E), as recently described.27 In Cd/UVB-embryos, Pljun mRNA remained localized in the PMCs (Figure 5F). A confirmation of the irregular positioning of the PMCs observed

rods (i.e., the antero-lateral rod; see arrowhead in Figure 2J) and a lower percentage of embryos with a gastrula-like shape and an abnormal arrangement of PMCs (Figure 2N). In UVBembryos, we observed pluteus-like embryos with undeveloped postoral rods (see arrows in Figure 2K) and gastrula-like embryos with a short lateral chain (see arrow in Figure 2O), lacking the typical subequatorial ring organization of the PMCs around the gut (Figure 2O). In Cd/UVB embryos, we observed an abnormal PMCs arrangement in pluteus-like embryos (see the arrowheads in Figure 2L), and a few cells moved away from the regular arrangement in gastrula-like embryos (see arrows in Figure 2P). We also noted that in all the treatments (Cd, UVB, and Cd/UVB) the number of PMCs was frequently different from the number normally present in the P. lividus species,40 both in excess or in shortage (not shown). We carried out an IF analysis to test gut differentiation on whole mount embryos at 72 hpf employing an antibody against an endoderm-specific marker (Endo1) that labels the hindgut and midgut but not the foregut of sea urchin embryos.41 In Cd-, UVB-, and Cd/UVB-embryos (Figure 2R−T), we observed a normal tripartite gut and the regular localization of Endo1, as found in control unexposed embryos (Figure 2Q). Sea Urchin Embryos Respond to Cd/UVB Exposure Activating p38MAPK 1 h after UVB Irradiation. To understand if one or more of the MAPKs known to be involved in the Cd- and UVB- stress response was eventually activated upon the combined and/or single exposure, we analyzed the activation of p38MAPK, ERK, and JNK in sea urchin embryos exposed to Cd, UVB, and Cd/UVB. Figure 3 shows

Figure 3. Cd/UVB coexposure activates p38MAPK. Western blot analysis of total proteins from normal, Cd-, UVB-, and Cd/UVBembryos harvested 1 h after irradiation, i.e., 4 hpf, and reacted with anti-P-p38MAPK (P-p38) (A), anti-P-ERK (B), and anti-P-JNK (C) antibodies. Protein levels were normalized using antitubulin antibodies (tub) on the same membranes. In the box: total proteins from embryos exposed to a high dose of UVB (800 J/m2) reacted with antiP-JNK antibody.

representative Western blots performed on lysates from embryos collected at 4 hpf (see experimental design, Figure 1A). The anti-P-p38MAPK antibody detected a band with an apparent molecular weight of approximately 40−42 kDa, whose levels were 2.3- and 7.7-fold higher in UVB- and Cd/UVBembryo lysates, if compared to that of controls. In Cd-embryo, the P-p38MAPK levels did not show significant changes compared to those of controls. The anti-P-ERK 1/2 antibody detected a band of about 42 kDa in lysates from all the exposed embryos but with slight E


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Figure 4. Overexpression of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes in Cd/UVB-embryos. Quantitative PCR analysis of Pl-14-3-3epsilon (A), Pl-MT (B), and Pl-jun mRNAs in normal, Cd-, UVB-, and Cd/ UVB-embryos at 27 hpf. Relative levels are expressed in arbitrary units as fold increase compared to control samples assumed as 1 in the histograms, using the endogenous gene Pl-Z12-1 for normalization. Each bar represents the mean of four independent qPCR experiments ± SD using two batches of cDNA. Asterisks (*) indicate significant difference between the control and treated embryos by the ANOVA test (P < 0.05) followed by Tukey’s test.

Figure 5. Ectopic expression of Pl-14-3-3epsilon, Pl-MT, and Pl-jun mRNAs in Cd/UVB-embryos. Whole-mount in situ hybridization of normal (A, C, E, and G) and Cd/UVB-embryos (B, D, F, and H) at 27 hpf with Pl-14-3-3epsilon (A-B), Pl-MT (C-D), Pl-jun (E-F), and msp130 (G-H) RNA probes.

by IF in Cd/UVB-embryos comes from the WMISH with the msp130 RNA probe which labels the PMCs irregularly positioned within the blastocoel (Figure 5H).

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short period of time, i.e., 0−48 h. Relevant ecological UVB doses ranged from 1.2 to 1.8 kJ m2 (mean hourly) as measured in situ at the depth of 1−5 m in Cook Islands.43 The decrease in the number of well developed plutei after Cd/UVB exposure seems to be caused by a synergistic effect. In particular, the effect of Cd/UVB on the skeleton patterning observed in 72 h-old embryos was never described in previous studies where embryos were exposed singly to Cd or UVB. Indeed, 10−4 M Cd or 200 J/m2 UVB caused developmental and skeleton growth delays in P. lividus embryos, but spicules with abnormal branching and irregular patterns were never observed.13,14 Cd/UVB exposure caused an atypical distribution of PMCs inside the blastocoel, often displaying a radial organization (compare Figure 2G−H with Figure 1G−H) determining the abnormal branching/patterning of spicules observed in this study. Interestingly, similar defects in skeleton morphology have been observed in sea urchin embryos overexpressing the growth factors FGFA or VEGF (VEGF-3 ligand).44,45 Indeed, microinjection of FGFA or VEGF transcripts strongly

DISCUSSION In this article, we investigated for the first time the combined exposure to Cd and UVB of early stage embryos of the sea urchin P. lividus to evaluate the defense strategies adopted to cope with these toxic agents. Analyzing the morphology of treated embryos, we found that skeleton patterning is greatly affected by Cd/UVB coexposure. Moreover, Cd/UVB induced the activation of the p38MAPK as well as increasing the transcription of Pl-14-3-3epsilon, Pl-MT, and Pl-jun genes at levels higher than those induced by Cd or UVB used singly. In addition, Pl-14-3-3epsilon and Pl-MT mRNAs were misexpressed, as they were found in ectopic embryonic territories, indicating a deregulation of gene expression. Concentrations of Cd, determined in the field, vary greatly, depending on different seawater latitudes and depths,42 and reach a very high level (12 μg/L, i.e., 0.65 × 10−4 M for CdCl2) in waters highly impacted as is the case of a disused mine in Kanayama.32 In this case study32 and in the laboratory study,13 acute Cd-exposure was evaluated on sea urchin embryos for a F


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they induced p38 MAPK activation. We already demonstrated that UVB induced a dose-dependent p38MAPK activation in P. lividus embryos irradiated at a later stage of development (mesenchyme blastula) probably to protect embryos.15 At the transcriptional level, the up-regulation of the p38MAPK gene was reported in P. lividus embryos exposed to toxic diatom aldehydes,26 suggesting its involvement in promoting embryo survival. The same role is suggested by another recent study on a benthic copepod where UVB induced p38MAPK activation.55 In conclusion, our result confirms the involvement of p38MAPK signaling in the stress response of sea urchin embryos. Concerning the ERK kinase, it was not activated by Cd/ UVB, nor by Cd or UVB used singly. The presence of its activated form in all the exposed embryos, as well as in control embryos, is in agreement with its physiological role during sea urchin embryo development starting from early stages.56 A similar finding was reported in a study on cultured human keratinocytes where a weak ERK activation was induced by UVB.57 Conversely to ERK, activated JNK was not detectable in Cd/UVB-, Cd-, and UVB-exposed as well as in control embryos 1 h after irradiation. Here, for the first time in sea urchin embryos, we showed that only a high dose of UVB (800 J/m2) activated JNK, whose signaling pathway has been poorly addressed in sea urchin embryo development to date. To summarize, ERK and JNK are not involved in the stress response to Cd/UVB, nor to Cd or UVB, at least at the doses and stages examined in this study. We cannot exclude that Cd/ UVB could activate other stress signaling pathways, as it occurs, for example, in HK-2 human renal proximal tubular cells, where Cd activates the PI3K/Akt to promote cell survival.58 At 24 h post irradiation, Cd/UVB exposed embryos showed a considerable up-regulation of Pl-14-3-3epsilon, Pl-MT, and Pljun genes. We first demonstrated that the Pl-14-3-3epsilon gene is an UVB responsive gene in P. lividus embryos both at early and late stages (2 and 24 h after irradiation).21,27 In agreement, a proteomic study showed that 14-3-3 proteins in the sea urchin embryo undergo changes in their phosphorylation states following UVR, suggesting a role in UV-induced cell cycle arrest as in mammalian cells.25 Furthermore, here we showed for the first time that Cd induced Pl-14-3-3epsilon transcription at levels similar to those induced by UVB. Similarly, in a small arthropod living in the soil, Cd exposure increases the 14-33zeta protein levels.59 Another study investigated the role of the human 14-3-3 (β/α isoform) protein in promoting cell survival following Cd exposure.60 Although the increase of transcripts found in our studies is only an indication of high protein levels, we suggest that Pl-14-3-3epsilon is involved in promoting cell survival in sea urchin embryos. Here, we found that Cd/UVB exposure induces Pl-MT expression, with an increase over control values that is very high if compared to that found in Cd- or UVB-exposed embryos. A similar trend was reported for the level of the MT protein in bone marrow cells of rats coexposed to Cd [low dose = 0.1 mg CdCl2/(kg·d)] and γ-radiation. The authors found high MT protein levels in the coexposure condition greater than the MT protein levels found in cells exposed to Cd (low dose) or γradiation singly.61 A recent study on P. lividus embryos showed that 10−4 M Cd induced the activation of three inducible MT genes, PlMT4, PlMT5, and PlMT6, and the up-regulation of two constitutive MT genes, PlMT7 and PlMT8.18 Authors suggested that PlMT8 could correspond to the Pl-MT that we first described in P. lividus embryos.13 Here, we found that the

perturbed morphogenesis, causing an irregular PMC organization and frequent examples of abnormal branching of the spicules.44,45 The increase in the number of PMCs reported by Rottinger et al.44 correlates with the observed higher number of PMCs we observed in some embryos. Further investigation on the involvement of FGF and/or VEGF signaling pathways in the abnormal skeletal branching induced by Cd/UVB are needed. The Cd ion has a charge and radius similar to those of zinc (Zn) and calcium (Ca) ions, and thus it can substitute them in the binding sites of many proteins that use Zn or Ca as cofactors.2 In fact, it has been shown that Cd disturbed or abolished the functions of many enzymes, transcription factors, and signaling proteins.2 For example, the carbonic anhydrases (CAs), widely distributed Zn-metalloenzymes, could be a potential target of Cd. CAs occur in all the kingdoms of life and are involved in diverse physiological functions. In various metazoans, CAs catalyze the reversible hydration of carbon dioxide to form bicarbonate and protons, playing a pivotal role in the biomineralization process.46 The CA sensitivity to Cd has been investigated in Mytilus galloprovincialis where it was shown that the protein activity and expression significantly increased after in vivo Cd exposure.47 Accordingly, since this enzyme is involved in the biomineralization processes occurring in sea urchin embryos,48 it is reasonable to hypothesize that its activity and/or expression increases in Cd/UVB embryos and that this probably accounts for the branching/patterning of spicules. A study on frog larvae demonstrated that UVB enhanced Cd uptake, although the mechanism should be elucidated.49 Similarly, UVB could enhance Cd uptake in sea urchin embryos where it was demonstrated that Cd is accumulated during development.50 DNA is directly damaged by Cd or UVB, but it could also be indirectly damaged by the production of the reactive oxygen species (ROS) and the inhibition of DNA repairing enzymes causing many cellular harmful effects, including the impairment of sea urchin embryo development.51 Adult sea urchin immune cells were also used to evaluate the effects of Cd/UVB coexposure analyzing the DNA integrity.51 Another study on sea urchin embryos (L. variegatus) exposed to PAH and UVB suggested that the coexposure could increase oxidative damage (ROS production).6 A field study using sea urchin embryos confirmed that UVB determined ROS production and that embryos protected themselves inducing antioxidant enzymes.42 Nevertheless, proteins were damaged by ROS, and embryos showed developmental abnormalities.42 Therefore, Cd/UVB coexposure could enhance DNA damage as well as ROS production affecting the functions of critical proteins needed for embryo development. The vast majority of the signaling kinases, including MAPKs, are expressed in the developing embryo.52 For the first time in sea urchin embryos at an early stage of development (4 h), we showed that p38MAPK is activated by Cd/UVB and also by UVB. Strong p38MAPK activation occurred at the mesenchyme blastula stage (15 h) when the skeletogenic cells entered into the blastocoel.53 This is in agreement with its physiological role already shown in sea urchin embryo skeletogenesis.53,54 Starting from 15 h of exposure, an overload of the essential metal manganese (MnCl21.12 mM) induced p38MAPK activation in P. lividus embryo.53 Differently to our finding, embryos exposed to Mn showed inhibition of skeleton elongation and patterning. Thus, Mn and Cd/UVB affected in different ways the embryonic skeleton despite the fact that G


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Chemical Research in Toxicology Pl-MT gene is up-regulated by 10−4 M Cd in agreement with MT’s role in metal detoxification that also occurred in the cells of marine invertebrates.19 Furthermore, we recently showed that Pl-MT mRNA is up-regulated in embryos irradiated during cleavage at a high UVB dose (800 J/m2) and analyzed at 24 h after irradiation.27 This is in agreement with MT’s protective role against ionizing radiation related to its radical-scavenging and/or ROS-quenching activities.20 Cd/UVB exposure induces Pl-jun mRNA expression with a fold increase over control value of the transcript that is lower than that found for Pl-14-3-3epsilon and Pl-MT. During sea urchin embryo development, Pl-jun mRNA is specifically expressed at very low levels in the skeletogenic cells suggesting its involvement in skeletogenesis.28 We also showed that the Pljun gene is up-regulated by 800 J/m2 UVB at 24 h after irradiation.27 At the protein level, we found that 800J/m2 UVB induced Jun phosphorylation.28 It is known that UVB induces Jun, both at the gene and protein levels. In turn, Jun, which belongs to the activator protein-1 (AP-1) transcription factor family, regulates UVB inducible genes, some of which promote cell survival in tumorigenic cells.30 In conclusion, we suggest that, Pl-jun, together with Pl-14-3-3epsilon and Pl-MT, is involved in promoting cell survival after Cd/UVB exposure. The ectopic expression of Pl-14-3-3epsilon and Pl-MT mRNAs induced by Cd/UVB seems in agreement with their roles in promoting cell survival.60 Conversely, the finding that Pl-jun mRNA remains localized in the skeletogenic cells probably reflects its highly specialized function in skeletogenesis.28 A study on mouse embryonic fibroblasts showed that UVB induced the up-regulation of VEGF expression through the AP-1(c-Jun/c-Fos) transcription factor.62 Similarly, in sea urchin embryos the Pl-jun up-regulation induced by Cd/UVB could in turn induce the overexpression of VEGF that caused an abnormal skeleton branching.45 Further studies are needed in this direction. Nevertheless, studies on Pl-14-3-3epsilon, Pl-MT, and Pl-jun mRNA expression do not necessarily reflect protein expression and function, indicating that further studies on the proteins they code for are needed to better understand their role in Cd/ UVB stress response. Moreover, the proteins and genes analyzed in this study, as well as those analyzed in other studies performed on sea urchin embryos displaying experimentally induced skeleton malformations, often have a dual function, i.e., a protection/survival role against environmental hazards and a regulative/structural role in the developmental program. In the EU as in other parts of the world, the chemicals’ legislation is built predominantly on the assessment of single substances. A communication from the European Commission called for a stronger commitment to ensure that the risks associated with chemical mixtures are properly understood to ensure a high level of protection of human health and the environment.63 In this direction, for example, a recent work carried out experiments testing two complex mixtures on 11 organisms from different trophic levels, microcosm and cell lines.4 However, the interactions between chemical and physical agents such as UV radiation should not be underestimated. In conclusion, we propose once again the sea urchin embryo as a suitable model for toxicity testing mimicking conditions occurring in the field to better understand the molecular mechanisms that operate in cellular protection against a mixture of chemical and physical stressors. Further studies using

“omics” approaches are needed to deeply characterize the cell stress response.

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AUTHOR INFORMATION

Corresponding Authors

*(R.B.) Phone: +39-091-6809592. Fax: +39 091 6809122. Email: [email protected]. *(V.M.) Phone: +39 091 6809551. Fax: +39 091 6809557. Email: [email protected]. Funding

This research was supported in part by the EU-ITN Biomintec Project (Contract No. PITN-GA-2008-215507) and CNR Flagship Project POM-FBdQ 2013-2014 (Screening of Bioactive Molecules and Toxicological Studies) to V.M. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Caterina Costa for the msp130 RNA probe, Mr. Mauro Biondo for assistance in the maintenance of sea urchins in aquaria, and all of the other members of the group for helpful discussions and suggestions. We are also indebted to Professor David McClay for the kind gift of the monoclonal antibodies 1D5 and Endo1.

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ABBREVIATIONS Cd, cadmium; MAPK, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MT, metallothionein; bZIP, basic leucine zipper; IF, immunofluorescence; mAb, monoclonal antibody; qPCR, realtime quantitative polymerase chain reaction; WMISH, wholemount in situ hybridization; PMC, primary mesenchyme cell; VL, ventrolateral; V, ventral; D, dorsal; L, longitudinal; VT, ventral-transverse; Zn, zinc; Ca, calcium; CA, carbonic anhydrase

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REFERENCES

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Combined Effects of Cadmium and UVB Radiation on Sea Urchin

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