Overview of the molecular defense systems used by sea urchin embryos to cope with UV radiation

Marine Environmental Research xxx (2016) 1e11

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Overview of the molecular defense systems used by sea urchin embryos to cope with UV radiation Rosa Bonaventura*, Valeria Matranga Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 February 2016 Received in revised form 19 May 2016 Accepted 23 May 2016 Available online xxx This review is dedicated to the memory of Valeria Matranga who did not live enough to see this work published.

The sea urchin embryo is a well-recognized developmental biology model and its use in toxicological studies has been widely appreciated. Many studies have focused on the evaluation of the effects of chemical stressors and their mixture in marine ecosystems using sea urchin embryos. These are well equipped with defense genes used to cope with chemical stressors. Recently, ultraviolet radiation (UVR), particularly UVB (280
315 nm), received more attention as a physical stressor. Mainly in the Polar Regions, but also at temperate latitudes, the penetration of UVB into the oceans increases as a consequence of the reduction of the Earth’s ozone layer. In general, UVR induces oxidative stress in marine organisms affecting molecular targets such as DNA, proteins, and lipids. Depending on the UVR dose, developing sea urchin embryos show morphological perturbations affecting mainly the skeleton formation and patterning. Nevertheless, embryos are able to protect themselves against excessive UVR, using mechanisms acting at different levels: transcriptional, translational and post-translational. In this review, we recommend the sea urchin embryo as a suitable model for testing physical stressors such as UVR and summarize the mechanisms adopted to deal with UVR. Moreover, we review UV-induced apoptotic events and the combined effects of UVR and other stressors. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Echinoderm Sea urchin embryo Ionizing radiation Signaling pathways Stress response Developmental abnormalities

1. Introduction In the last decade, the interest on the evaluation of UV radiation (UVR) effects on aquatic and terrestrial ecosystems was high considering the environmental increase in UV irradiance (Andrady et al., 2012; The EEAP Report, 2014). Since the early 1900s, many studies used Echinoderms as model systems to evaluate the biological effects of UVR and understand how marine organisms protect themselves, as recently reviewed by Lamare et al. (2011). Among Echinoderms, the sea urchin embryo is beyond doubt an attractive model of study, which has played a key role in the fields of embryology, developmental and molecular biology, and has been unsurpassed for in vivo observations (Monroy, 1986; Ernst, 2011). Moreover, the sea urchin embryo is a widely used model in ecotoxicological studies to analyze the molecular defense systems to cope with: i) pollutants, as metals (Roccheri and Matranga, 2009; Pinsino et al., 2014); ii) climate change factors, as CO2-increase, acidification and ocean warming (Byrne, 2011); iii) nanoparticles

* Corresponding author. E-mail address: [email protected] (R. Bonaventura).

(NPs) (Corsi et al., 2014; Della Torre et al., 2014; Gambardella et al., 2015). 1.1. UVR in the marine environment The wavelengths “beyond violet”, i.e. the ultraviolet radiation (UVR), are a component of the electromagnetic radiation emitted by the Sun that are conventionally divided into UVC (200e280 nm), UVB (280e320 nm) and UVA (320e400 nm). The Earth’s atmosphere strongly absorbs UVC and provides some shielding to UV-A and UV-B that reach the biosphere (Maverakis et al., 2010). In the marine environment, the transmission of solar UVR depends on many variables, as for example the presence of dissolved materials and phytoplankton that in turn affect the amount and €der et al., 2011). UVA can wavelength distribution of UVR (Ha penetrate deeper than UVB, reaching depths between 40 and 60 m in clear ocean waters, affecting marine organisms differently distributed in the water column (Smith et al., 1992; Tedetti and  re , 2006). Generally, UVR can ionize molecules and thereby Sempe induce chemical reactions that in turn can be harmful to organisms by affecting their DNA, proteins, and lipids (Dahms and Lee, 2010). In particular, UVB has negative effects on primary producers, as

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cyanobacteria, phytoplankton, macroalgae and aquatic plants, as well as on many aquatic consumers, as zooplankton, crustaceans, €der et al., 2011). amphibians, fishes and corals (Ha The ecological importance of UVR led many studies towards the need to understand their biological effects. In addition, the discovery of the ozone-hole in Antarctica in the 1980s has strongly increased the attention of scientists on the hazardous effects of UVR. The ozone-hole is now slowly recovering since 1989, when the Montreal Protocol entered into force, although an episodic decrease of ozone was observed in the Artic region in spring 2011 (The EEAP Report, 2014). This apparent contradiction is due to changes in factors other than ozone depletion, as clouds, air pollution (including aerosols) and surface albedo, as well as the interactions between ozone depletion and climate change (increasing greenhouse gases) (McKenzie et al., 2011; The EEAP Report, 2014). All these factors cause a large variability in UVB radiation, also outside the Polar Regions, and have important implications for both ecosystems and human health (McKenzie et al., 2011; The EEAP Report, 2014). 1.2. Sea urchin embryo as a model to study UVR As many marine invertebrates, sea urchins release their gametes into the water column where fertilization occurs producing planktonic embryos and larvae that, depending on the species, need days or months to undergo metamorphosis and become juveniles (little sea urchins) (McEdward and Miner, 2001). During development, embryos have to cope with many adverse environmental conditions, including UVR, as well as many other pollutants that can affect their development, and consequently the success of their reproduction (Hamdoun and Epel, 2007). Starting from the late 1980s, the sea urchin embryos have been exploited not only in laboratory but also in field studies to evaluate the UVR response, although the available data concerning the molecular mechanisms activated following UVR exposure are still fragmented (Dahms and Lee, 2010; Lamare et al., 2011; Adams et al., 2012). Nevertheless, many protection strategies have been highlighted, including the avoidance, i.e. negative phototaxis to UVR exposure, and other non-protein defensive strategies, i.e. sunscreen compounds as Mycosporine-like amino acids (MAAs) and carotenoids (Lamare et al., 2011; Lamare and Barker, 2013). Here we gathered and elaborated all available information on the UVR molecular defense systems operating in the sea urchin embryo at: i) transcriptional, ii) translational and iii) posttranslational levels, both in laboratory and field experiments. In addition, we correlated the activated molecular defense systems to the alternative morphologies induced by UVR, which mainly affected the embryonic skeleton. Moreover, we analyzed the embryonic defense response, which had been activated after the combined exposure of UVR and other stressors. 2. UVR effects on embryonic development One of the advantages in the use of sea urchin embryos as a model is their transparency that easily allows the evaluations of the morphological effects caused by chemical and physical agents (Matranga et al., 2011). In many studies, gametes were irradiated before fertilization and the delay in cleavage analyzed afterwards. Table 1 summarizes data obtained in different studies, taking into account the i) emission properties of artificial lamps, ii) types of exposure, iii) stage chosen to irradiate gametes or embryos, both in laboratory and field experiments. For example, eggs from the European species Sphaerechinus granularis and Paracentrotus lividus have been irradiated with 65.6 kJ/m2 UVA and 7.6 kJ/m2 UVB, but following fertilization, only S. granularis embryos showed a delay or

an inhibition in the first cell cleavage (Nahon et al., 2008). Later in development, abnormal plutei have been observed for both species with apically crossed body rods (percentage of normal plutei: 5.06% ± 1.07 SD for S. granularis and 38.46% ± 21.99 for P. lividus embryos) (Nahon et al., 2008). Exposure of Echinometra lucunter eggs to UVB with doses ranging from 0.9 to 7.2 kJ/m2 inhibited the first and second cleavage in a dose-dependent manner (Leite et al., 2014). The characteristic dose-dependent delay in cleavage has also been observed in Strongylocentrotus purpuratus embryos irradiated at 30 and 90 min post fertilization with a total dose of 41.31 kJ/m2 UVR (290e400 nm) and delivered over a period of 60 min (Campanale et al., 2011). P. lividus embryos irradiated during cleavage using UVB doses ranging from 0.01 to 0.8 kJ/m2 showed abnormal morphologies at 24 h post irradiation (hpi), starting from the dose of 0.15 kJ/m2. At the highest doses used, i.e. 0.4 and 0.8 kJ/m2 UVB, about 85.7% and 93.6% were abnormal embryos that lacked an organized epithelium and showed the blastocoelic cavity completely filled with cells (Bonaventura et al., 2006). This morphology was very similar to the so called “permanent” blastula, obtained irradiating Hemicentrotus pulcherrimus embryos with UVC at 0.038 and 0.45 kJ/m2 (Amemiya et al., 1986), and to the so called “packed” blastula, obtained irradiating S. purpuratus embryos 20 min after fertilization onwards with PAR þ UVA þ UVB (with cycles of 12 h light/dark) (Adams and Shick, 2001). Observations performed later in development (48hpi) showed that 100% of 0.8 kJ/m2 irradiated embryos were “packed blastula”, while 80% of the 0.4 kJ/m2 irradiated embryos were “packed blastula” with or without short skeleton elements (spicules), when control embryos were plutei (Bonaventura et al., 2006). Using the widely used skeletogenic cell marker, MSP130, in P. lividus embryos irradiated with 0.2 kJ/m2 and analyzed after 24 h, we found that some of the cells inside the blastocoel of the abnormal blastula (Fig. 1B) were skeletogenic cells (Fig. 1D), even though they lacked the typical organization (Fig. 1C) of the normal embryo (Fig. 1A), see also Table 2. P. lividus embryos exposed to UVB at the stage of mesenchyme blastula (Bonaventura et al., 2005) seemed to be more resistant to UVB if compared to embryos irradiated at early stages (Bonaventura et al., 2006), since they could tolerate the dose of 1.0 kJ/m2. In particular, when control embryos were at the pluteus stage, 0.3 kJ/ m2 irradiated embryos showed developmental delays and/or absence of skeleton and gut at 24hpi. At the same time, embryos irradiated with 1.0 kJ/m2 were nearly all blastulae- and early gastrulae-like embryos without skeleton and gut (Bonaventura et al., 2005). Whole mount in situ hybridization (WMIH) experiments using a Pl-SM30 DNA probe showed a dose-dependent reduced number of the skeletogenic cells expressing SM30 mRNA, a gene coding for one of the skeleton matrix proteins of the sea urchin embryo. In UVB-embryos exposed to 1.0 kJ/m2, Pl-SM30positive cells were absent indicating that the skeleton was a target structure damaged by UVB (Bonaventura et al., 2005), see also Table 2. Similarly, X-rays caused a reduced expression of the skeleton markers SM30 and msp130 in irradiated embryos, as assessed by RT-PCR and by WMISH respectively (Matranga et al., 2010). Using an appropriate floating devise for field experiments, Sterechinus neumayeri embryos have been suspended at 1, 3, and 5 m below the ice soon after fertilization and exposed for 5 days to ambient UVR (UVA and UVB) that was measured in situ (Lesser et al., 2004). Experiments carried out in spring 2002 and 2003 showed that UVR exposure determined significant mortality in embryos and caused abnormal development, i.e. “packed blastulae” among the survivor embryos (Lesser et al., 2004). Other field experiments showed different species-specific sensitivities, as indicated by the abnormal morphologies observed in exposed embryos from different geographic distributions: i) “packed blastulae” in

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Table 1 Emission properties of artificial lamps and types of exposure in laboratory and field experiments.

Laboratory exp.

Field exp.

Emitted λ

Type of UV exposure

Stage of irradiation

Species

Source

peak intensity: 340 nm continuous spectrum: 290e315 nm solar spectrum: 290e400 nm peak intensity: 312 nm peak intensity: 254 nm

UVA, UVB

Eggs

Nahon et al., 2008

UVB

gametes, zygotes

S. granularis P. lividus E. lucunter

Leite et al., 2014

UV-treated: 295e700 nm (PAR þ UVA þ UVB) UV-protected : 400e700 nm(PAR) UVB

first mitotic cleavage

S. purpuratus

Campanale et al., 2011

cleavage

P. lividus

Bonaventura et al., 2006

UVC

mesenchyme blastula

H. pulcherrimus

Amemiya et al., 1986

UVR treateda: UVA (UVA treatment) UVA þ UVB (UVT treatment) UV-protected: no UVR (UV0 treatment) UVR treatedb: PAR* þ UVA (UVA treatment) PAR* þ UVA þ UVB (UVT treatment) UV-protected: PAR* (UV0 treatment)

from fertilization onwards (5 days)

S. neumayeri

Lesser et al., 2004

from fertilization onwards (2, 4 and 5 days depending on the species)

S. neumayeri E. chloroticus T. gratilla D. savignyi

Lamare et al., 2007

Solar spectrum

Solar spectrum

λ: wavelength. PAR*: photosynthetically active radiation (from 400 to 700 nm). a Long-band pass filter Plexiglas were used to perform different UVR treatments. b Plexiglass filters that varied in their transmission of different wavelengths were used to perform different UVR treatments.

S. neumayeri (Antarctic species); ii) gastrula-delayed embryos and plutei with shortened or even absent arms in Evechinus chloroticus (temperate species), Tripneustus gratilla and Diadema savignyi (tropical species) (Lamare et al., 2007). In each sites, the weighted

UVR doses during field experiments have been calculated and the highest values, ranging from 0.26 to 0.10 kJ/m2, have been reported in the tropical site at the depths of 0.5, 1.5 and 4e5 m (Lamare et al., 2007).

Fig. 1. Localization of skeletogenic cells in normal and UVB exposes P. lividus embryos. Brigthfield images of normal (A) and 0.2 kJ/m2 UVB-exposed (B) embryos during cleavage observed at 27 h of development, i.e. 24hpi. Immunofluorescence (IF) with 1D5 mAb, recognizing the glycoprotein MSP130 specifically expressed on the skeletogenic cells of normal (C) and UVB-exposed (D) embryos. Indirect IF experiments on whole mount embryos have been performed as described by Bonaventura et al., 2015.

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Table 2 Skeletal markers used in P. lividus embryos exposed to UVB in lab experiments. Skeletal markers

Technique

Stage of irradiation

UVB doses

Source

MSP130 Pl-SM30

IF WMISH

during cleavage mesenchyme blastula

0.2 kJ/m2 0.05, 0.3, 0.5, 1.00 kJ/m2

see Fig. 1 Bonaventura et al., 2005

The abnormal morphologies of the survivor embryos indicate that UVR strongly affects development, in particular at the expenses of the skeleton, probably reflecting the energetic costs of the molecular mechanisms activated to prevent and repair damages. This relocation of the energetic resources from development to defense mechanisms might significantly influence larval fitness and⁄ or survivability as suggested by Lister et al. (2010b). 3. How sea urchin embryos protect themselves against excessive UVR: overview on the molecular defense systems Despite their fragile appearance, sea urchin embryos use many strategies to protect themselves against environmental changes that normally occur, including for example UVR and many other stressors/pollutants (Hamdoun and Epel, 2007). UVR causes damage on DNA, RNA, proteins and lipids and increases reactive oxygen species (ROS) (Herrlich et al., 2008). The UV-damage activates a number of “complex repair/defense reactions”, referred to as the “UV response” (Herrlich et al., 2008). Depending on the cell type, the UV response can arrest the cell cycle, repair damaged macromolecules, upregulate the levels of mRNAs coding for many stress, defense and repair proteins, modulate post-translational modification and protein turnover (Kültz, 2005; Herrlich et al., 2008). In the coming sections, we will review all these items. 3.1. UVR response at transcriptional level In the following sections, we will consider the types of DNA lesions caused by UVR exposure in sea urchin embryos and the genes involved on the DNA repair mechanisms as well as on the stress response, summarized in Table 3. 3.1.1. DNA repair genes Physical or chemical agents can damage DNA in many ways. In particular, the most common DNA lesions caused by UVR are cyclobutane pyrimidine dimers (CPDs) and (6e4) pyrimidinepyrimidone photoproducts [(6e4)PPs], usually simply referred to as photoproducts (Batista et al., 2009). The estimated number of

DNA lesions, both CPDs and 6e4 PP, is 105 per cell per day and it is due to solar UVR exposure at peak hours, i.e. the hours around solar noon (Ciccia and Elledge, 2010). These DNA lesions physically block both the replication and transcription machineries and need to be repaired, as reported (Batista et al., 2009). Both laboratory and field experiments on sea urchin embryos from different species showed that the CPDs are the main DNA lesions caused by UVR, repaired mainly by photolyase enzymes (Lamare et al., 2011). This type of DNA repair, known as “photoreactivation” (PER), is linked to the light-dependent activities of the CPD photolyases, typical of invertebrate organisms (Sancar, 2008), including also the sea urchin embryo (Oliveri et al., 2014). During evolution, mammals lost these repair genes, i.e. the photolyase genes, and they use the “nucleotide excision repair” (NER) as a mechanism to remove DNA photoproducts (Batista et al., 2009). In laboratory experiments using different sea urchins species (S. neumayeri, E. chloroticus, Diadema setosum), the CPDs concentration decreased exponentially in those UVR irradiated embryos that have been subsequently cultured under light conditions for 24 h, thus indicating a high rate of DNA photorepair (Lamare et al., 2006). Conversely, UVR embryos cultured in the dark showed a lower rate of DNA repair and a higher percentage of abnormally developed embryos (Lamare et al., 2006). In S. neumayeri, Strongylocentrotus franciscanus, E. chloroticus embryos a DNA sequence for photolyase has been identified (Isely et al., 2009), see Table 3. Using a floating device for field experiments, embryos of the Antarctic species S. neumayeri have been exposed to ambient solar radiation at 1, 2, 5 and 10 m below the sea ice surface or at the sea surface (without sea-ice), and the photolyase mRNA levels have been evaluated. Exposed embryos showed an increase in the photolyase mRNA levels as a result of increasing daily UVR dose, particularly at the shallowest depth of 0.5 m of a site without sea-ice (Isely et al., 2009). In addition to CPDs lesion caused by UVR, UVA wavelengths indirectly caused DNA damage by the formation/development of ROS, in the form of 8-OxoG (8-oxo-7,8-dihydro-2’-deoxyguanosin) or 8-OHdG (8-hydroxy-2’-deoxyguanosine), a well-known marker of DNA damage (Batista et al., 2009). Only a field study on

Table 3 UVR responsive genes in sea urchin embryos: DNA repair and stress genes studied.

DNA repair

Stress

a

Genes

Species

Type of experiment

Stage of irradiation

UV doses used

Source

Photolyase

S. neumayeri

Field

from fertilization

Isely et al.,2009

Pl-XPB-ERCC3 Pl-XPB-ERCC3 ercc1 xrcc4 xrcc5 xrcc6 pcna Pl-14-3-3ε Pl-NF-kB; Pl-jun Pl-FOXO Pl-MT Pl-14-3-3ε

P. lividus P. lividus L. variegatus L. variegatus

Lab Lab Lab Lab

cleavage cleavage pluteus pluteus

Environmental UVRa: 1266 kJ/m2 (0.5 m); 23.7 kJ/m2 (10 m) UVB 0.2 kJ/m2 UVB: 0.4 and 0.8 kJ/m2 UVC: 0.25, 0.5, 1.0, 3.0 kJ/m2 UVC: 0.25, 0.5, 1.0, 3.0 kJ/m2

L. variegatus P. lividus P. lividus

Lab Lab Lab

pluteus cleavage cleavage

UVC: 0.25, 0.5, 1.0, 3.0 kJ/m2 UVB: 0.4 and 0.8 kJ/m2 UVB: 0.4 and 0.8 kJ/m2

Reinardy and Bodnar, 2015 Russo et al., 2010 Russo et al., 2014a

€der et al., 2005 Schro Russo et al., 2014a Reinardy and Bodnar, 2015 Reinardy and Bodnar, 2015

Estimated daily UVR doses at the experimental depths of 0.5 and 10 m over the periods of embryo exposures below sea ice surface.

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Strongylocentrotus droebachiensis embryos quantified the concentration of 8-OHdG using an ELISA system, showing that exposed embryos had high levels of 8-OHdG at 1 m depth (Lesser, 2010). In the S. purpuratus genome, many highly conserved light independent genes have been revealed, which are associated with DNA repair functions, including: i) the base excision (BER), ii) the double-strand break, iii) the nucleotide excision (NER), and iv) the mismatch repair (MMR) (Fernandez-Guerra et al., 2006). Many proteins are implicated in the DNA repair pathways, for example in mammalian cells NER involves nearly 30 proteins to repair DNA lesions (Batista et al., 2009). Up to now, a few studies are available on the characterization of specific DNA repair genes and on the induction of pathways operating in the sea urchin embryos, as well as in the adults. The partial cDNA of the Pl-XPB-ERCC3, involved in the DNA repair by Homologous Recombination (HR), has been isolated from P. lividus embryos irradiated with UVB at the dose of €der et al., 2005). Furthermore, it 0.2 kJ/m2 during cleavage (Schro has been found that UVB induced the transcription of the Pl-XPBERCC3 gene in P. lividus embryos irradiated during cleavage with 0.4 and 0.8 kJ/m2 at 24hpi, while no induction was observed at 2hpi (Russo et al., 2014a), see Table 3. Recently, a panel of DNA repair genes has been analyzed by qRT-PCR on Lytechinus variegatus embryos and adult immune cells in response to selected genotoxicants, including also UVC, over a 24 h period of recovery (Reinardy and Bodnar, 2015). Although UVC (<280 nm) has no environmental relevance, (it is absorbed by ozone and does not reach the Earth), the study provided many information on the profiles of induced DNA-repair genes in exposed embryos: i) ercc1, involved in NER; ii) xrcc4, xrcc5 and xrcc6 involved in NHEJ (Non-homologous endjoining); iii) pcna involved both in NER and BER (Reinardy and Bodnar, 2015), see Table 3. BER and NHEJ DNA repair activities have been measured in vitro using embryonic extracts, and they appeared functional in embryos where DNA damage had been induced by MMS, a DNA alkylating agent (Le Bouffant et al., 2007). In addition, the study showed that early embryos could activate DNA damage checkpoint pathways, i.e. a cell cycle delay and no activation of the complex CDK1/cyclin B (Le Bouffant et al., 2007), that would explain the cleavage delay observed in early stage embryos after irradiation. As a further step, if the amount of DNA lesions were to exceed the efficiency of the repair systems, the unrepaired photoproducts could trigger apoptosis in sea urchin embryos, as reported for mammalian cells (Batista et al., 2009). Nevertheless, further studies are needed to deeply characterize the DNA repair tools and the mechanisms that regulate the recruitment of the several repair factors to the DNA sites damaged by UVR, in a fast mitotic cell system as the embryonic cells are. 3.1.2. Stress genes In man, mouse and derived cells in culture, the UV response determines the activation of a new program of gene expression (Blattner et al., 2000). Using DNA microarrays to study UV irradiated keratinocytes, three waves of induction can be identified considering the timing of induction and the genes analyzed early, intermediate and late (Mlakar and Glavac, 2007). For example in mammalian cells, c-fos, c-jun, and junB are early genes since their transcription is activated within 10 min after irradiation (Blattner et al., 2000). In general, a few studies have focused on stress genes specifically regulated by UVR in sea urchin embryos, see Table 3. UVB exposure of P. lividus embryos at cleavage caused the upregulation of Pl-14-3-3epsilon mRNA at 2hpi, together with its ectopic expression later in development (Russo et al., 2010). Thus, Pl-14-33epsilon could be implicated in the regulative cascade activated in response to UV-B irradiation, in addition to the role played in the regulation of many cellular processes in all organisms, from plants

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to humans (Morrison, 2009). Recently, Russo et al. (2014a) extended the transcriptional analysis of UVB-exposed embryos to a group of selected UVB-responder genes analyzed by QPCR at early (2hpi) and later (24hpi) stages. The Pl-14-3-3 epsilon mRNA levels increased at both 2 and 24hpi for the two UVB doses used (0.4 and 0.8 kJ/m2), while the mRNA levels of Pl-MT, a metal detoxification and stress gene, increased only with the dose of 0.8 kJ/m2 at 24hpi. This result agrees with the MT’s protective role against ionizing radiation previously observed in mammalian cells (McGee et al., 2010). Three transcription factors, Pl-NF-kB, Pl-jun and Pl-FOXO have been also analyzed. UVB irradiation seemed not to modulate the Pl-FOXO gene, one of the Forkhead genes involved in stress resistance, metabolism, apoptosis, also shown to be involved in S. purpuratus sea urchin embryos development (Tu et al., 2006). PlNF-kB appeared upregulated at 24hpi and in a dose-dependent manner. From studies on mammalian cells, it is known that NFkB is a key regulator of many signaling pathways, including survival, operating at both gene and post-translational levels. In addition, diverse external stimuli including UVR can activate NF-kB (Oeckinghaus and Ghosh, 2009). Similarly, Pl-jun is upregulated in a dose-dependent manner at 2hpi, while it is upregulated only at the dose of 0.8 kJ/m2 at 24hpi. Jun is a transcription factor of the AP-1 family that regulates UVB inducible genes in mouse tumorigenic cells, some of which promote cell survival (Cooper and Bowden, 2007). Recently, it has been showed that a Pl-jun transcript is specifically localized in the skeletogenic cells of the sea urchin embryo (Russo et al., 2014b). Depending on the stage of irradiation, the translation of maternal mRNA stored in the cytoplasm can be activated in embryos at early stages, as also suggested by Adams et al. (2012), while the transcription of zygotic genes (UVR-responder genes) can be activated in embryos at later stages. According to Lesser (2010), this could explain the greater tolerance towards damages induced by UVR of the late stage embryos compared to the early ones. One possible mechanism operating in UV-irradiated early embryos could be the stabilization of maternal mRNAs, as reported in murine fibroblasts where UVC irradiation induced the stabilization of several short-lived RNAs, such as c-fos. (Blattner et al., 2000). 3.2. UVR response at translational and post-translational levels The sea urchin embryos are equipped with an integrated network of genes and pathways involved in sensing and protecting cells from toxic chemicals termed “chemical defensome” (Goldstone et al., 2006). These chemicals can be natural compounds, as for example phytotoxins and microbial products, and/or anthropogenic pollutants, as for example heavy metals (Goldstone et al., 2006). The chemical defensome is a central part of a larger system, the “defensome” that includes the defense against many adverse environmental agents, as the UVR. In particular, in the following sections we will consider the proteins and signaling pathways involved in the UV-response in sea urchin embryos (see also Table 4). 3.2.1. HSP70 and anti-oxidative proteins A proteomic study on S. purpuratus embryos showed that during the first mitotic cleavage at least 176 proteins were involved in the UVR stress response (Campanale et al., 2011). Among the spots analyzed in lysates from embryos exposed to UVR, HSP70 underwent post-translational modification, becoming more acidic and thus reflecting a change in its function (Campanale et al., 2011). Studies on P. lividus species showed that embryos respond to UVB changing the levels of HSP70 protein, although with a different mode depending on the stage of irradiation. Indeed, HSP70 protein levels increased at doses as low as 0.15 kJ/m2 in embryos UVB-

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Table 4 Proteins and MAPK involved in the UVR stress response in sea urchin embryos. Proteins

Species

Type of exp.

Stage of irradiation

UV doses used

Source

HSP70; 14-3-3proteins; GPx

S. purpuratus

Lab

first mitotic cleavage

HSP70 (WBa)

P. lividus

Lab

cleavage

total dose of 41.31 kJ/m2 UVR (290e400 nm) delivered over a period of 60 min UVB 0.01, 0.05, 0.15, 0.2, 0.4 and 0.8 kJ/m2

HSP70 (WB )

P. lividus

Lab

mesenchyme blastula

UVB 0.05, 0.3, 0.5, 1 kJ/m2

SOD (WBa) SOD and CAT (enzymatic assays) SOD, GPx and GR (enzymatic assays) ABCs transporters

S. droebachiensis Lab S. neumayeri Field

different stages 3 days, starting within 12 h of fertilization within 12 h of fertilization

cycle of 12 h light/dark for 3 days Environmental UVR

Campanale et al., 2011 Bonaventura et al., 2006 Bonaventura et al., 2005 Lesser et al., 2003 Lister et al., 2010a

p53, p21 and cdc2 (WBa) P-p38MAPK (WBa)

S. droebachiensis Lab P. lividus Lab

different developmental stages mesenchyme blastula

UV doses ranged from 0.028 to 14.44 kJ/m (UVA, UVB) cycle of 12 h light/dark for 3 days UVB 0.05, 0.3, 0.5, 1.0 kJ/m2

P-p38MAPK (WBa)

P. lividus

Lab

cleavage

UVB 0.2 kJ/m2

P-JNK (WBa)

P. lividus

Lab

cleavage

UVB 0.8 kJ/m2

P-jun (WBa) P-jun localization

P. lividus P. lividus

Lab Lab

cleavage cleavage

UVB 0.8 kJ/m2 UVB 0.4 kJ/m2

a

a

T. gratilla E. lucunter

Field Lab

Environmental UVR

Lister et al., 2010b 2

Gametes, zygotes

Leite et al., 2014 Lesser et al., 2003 Bonaventura et al., 2005 Bonaventura et al., 2015 Bonaventura et al., 2015 Russo et al., 2014b see Fig. 2

WB: Western Blotting.

irradiated at the early stage of cleavage (Bonaventura et al., 2006) and at the doses of 0.5 and 1.0 kJ/m2 in embryos UVB-irradiated at the later stage of mesenchyme blastula (Bonaventura et al., 2005). These studies support a general defensive role for HSP70, but they also suggest a protective role against apoptosis in embryonic cells (see section 4). UVR exposure (with cycles of 12 h light/dark) of S. droebachiensis embryos at different developmental stages caused increased protein levels of the superoxide dismutase (SOD) enzyme, as detected using polyclonal antibodies against cytosolic SOD (Lesser et al., 2003). SOD is an indicator of increased ROS concentrations and thus of the oxidative stress caused by UVR, as reported in other marine organisms as copepod (Kim et al., 2015) and fish larvae (Lesser et al., 2001). Field experiments on S. neumayeri embryos exposed to UVR in marine sites without ice showed that SOD and CAT activities increased slightly, but in a significant way, even if this increase was not enough to counteract the oxidative damage (Lister et al., 2010a). Embryos of this Antarctic species, as many animals adapted to low temperatures, show moderate to high levels of both SOD and CAT enzymes when compared with other marine larvae (Lister et al., 2010a). Similar field experiments on the tropical T. gratilla embryos exposed to UVB, at the depth of 1 and 4 m in the water column, showed that these embryos had increased enzymatic activities of SOD, glutathione peroxidase (GPx) and glutathione reductase (GR), when compared with embryos protected from UVB and used as controls (Lister et al., 2010b). The proteomic analyses on early S. purpuratus embryos showed that UVB induced changes in a large number of proteins that are biomarkers of oxidative stress, i.e. not only classical markers, as for example the GPx, but also markers such as the cyclophilin D (Campanale et al., 2011; Adams et al., 2012). Interestingly, a recent study on the Atlantic species E. lucunter using inhibitors of the ABCs transporters (ATP-Binding Cassette) showed that these multi-drug efflux transporters are involved in the protection of early embryos (2 and 4 cells), after zygotes irradiation, against the harmful effects of UVB (Leite et al., 2014). Nevertheless, how ABCs transporters operate to protect these early embryos by UVB remains to be fully elucidated.

3.2.2. Pathways activated by UVR UVR can induce many signal transduction cascades, which can be triggered by DNA lesions, damaged ribosomal RNA, and inactivated protein tyrosine phosphatases (Herrlich et al., 2008). During development, sea urchin embryos express many signaling kinases, including the Mitogen-Activated Protein Kinases (MAPKs) (Bradham et al., 2006). Besides, a variety of environmental stresses, including UVR, might activate these evolutionarily conserved mediators (Munshi and Ramesh, 2013). Conventionally, three main groups characterize the MAPKs: (i) p38MAPK, (ii) extracellular signal
regulated kinase (ERK1/2), and (iii) c-Jun Nterminal kinase (JNK). UVB (0.05, 0.3, 0.5 and 1.00 kJ/m2) induced the activation of p38MAPK in P. lividus embryos irradiated at mesenchyme blastula stage, both at 1 and 24hpi (Bonaventura et al., 2005), see Table 4. One hour after irradiation, low UVB doses (0.2 kJ/m2) determined the activation of p38MAPK in P. lividus embryos irradiated during cleavage, while they did not affect the phosphorylation levels of ERK compared to control (Bonaventura et al., 2015). At the same UVB dose, no phosphorylated form of JNK was detectable, while a higher UVB dose (0.8 kJ/m2) activated JNK under the limits of detection in control embryos (Bonaventura et al., 2015). In addition, the 0.8 kJ/m2 UVB dose induced the activation of jun (phospho-jun) at 24hpi, which was detected as a faint band by immunoblot, while no band was detectable in the controls (Russo et al., 2014b). Thus, in sea urchin embryos UV irradiation activates JNK that in turn can activate jun, although a direct relationship between them needs further confirmation. The phosphorylated form of jun has been detected in the nuclei of some skeletogenic cells (Russo et al., 2014b), as shown in Fig. 2B, C. Indeed, p-jun co-localized with the skeletogenic cell surface marker WGA (Fig. 2B) and the DNA-binding dye Hoechst 33342 (Fig. 2C), in the cells lining the skeletal rods of the 52 h old normal pluteus (see also the magnifications in Fig. 2G, H). Embryos exposed to 0.8 kJ/m2 at cleavage, and observed at 49hpi, had a gastrula-like shape with an irregular organization of the skeletogenic cells that were p-jun positive (Fig. 2E, I). As showed by Russo et al. (2014b), few ectoderm and endoderm cells were p-jun-positive, as indicated by arrows and arrowhead both in control (Fig. 2B) and in irradiated

Please cite this article in press as: Bonaventura, R., Matranga, V., Overview of the molecular defense systems used by sea urchin embryos to cope with UV radiation, Marine Environmental Research (2016), http://dx.doi.org/10.1016/j.marenvres.2016.05.019

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Fig. 2. Phospho-jun localization in normal and UVB-exposed P. lividus embryos. Normal- (AeC) and 0.8 kJ/m2 UVB- embryos (DeF) exposed at cleavage and observed at 52 h of development, i.e. 49hpi. Merged images of the relative: Brightfield (BF) and immunofluorescence (IF) with anti phospho-jun (p-jun) (Fig A, D); IF with anti-p-jun and WGA, a skeletogenic-specific marker, (Fig. B, E); IF with anti-p-jun and Hoechst 33342, a DNA-binding dye, (Fig C, F). Images in G, H, I and J are magnifications of the rectangles in figure B, C, E and F, respectively. Each image shown in figures AeC is a composition of two captured images. Indirect IF experiments on whole mount embryos have been performed as described by Russo et al., 2014b.

embryos (Fig. 2E). Consequently, UVB irradiation does not affect the p-jun localization, probably in relation to its specialized function related to the embryonic skeletogenesis (Russo et al., 2014b). The signaling pathways potentially affected by UVR in sea urchin embryos need further analysis that should include other kinases, as the PI3K/Akt and ATR/AMT that are known to be involved in the UV response in mammalian cells (Herrlich et al., 2008). In addition, it should be considered the role of the phosphatases in UVR-exposed embryos, since these enzymes regulate the phosphorylated state of many proteins in numerous cellular activities, also during the sea urchin embryo development (Byrum et al., 2006). In addition, the stress sensing mechanisms need more attention. A common form of damage in response to stress is the lipid peroxidation occurring at the plasma membrane that generates H2O2, an upstream intermediate signal that can activate multiple signaling pathways (Kültz, 2005). Lipids oxidative damage occurred in T. gratilla embryos exposed to solar radiation at depths of 1 and 4 m in field experiments (Lister et al., 2010b). In parallel, the activities of antioxidant enzymes significantly increased (see Section 3.2.1), indicating that embryos upregulate their antioxidant defenses to provide protection to oxidative damages of proteins as well as lipids (Lister et al., 2010b). 4. Apoptosis induced by UVR A few studies have analyzed the effects caused by UVR in terms of induced apoptosis in sea urchin embryos. Following exposure to many stressors, sea urchin embryos can activate apoptosis, reviewed by Agnello and Roccheri (2010). The sea urchin genome contains many homologs of the vertebrate apoptotic pathways, but with some specificities (Robertson et al., 2006). In particular, five distinct subfamilies of caspases have

been identified, as for example the two classes of the signalresponsive initiator caspases-8/10 and 9, that underwent to an “echinoderm-specific” expansion, and a new group of caspases, named caspase-N (for “Novel”), different from any of the already known vertebrate caspases (Robertson et al., 2006). S. droebachiensis embryos showed increased p53 and p21 protein levels when exposed to UVR from low (400 nm) to high (280 nm) doses, with cycles of 12 h light/dark at different developmental stages (freshly fertilized, blastula and gastrula embryos) and analyzed after three days of development (Lesser et al., 2003). Interestingly, in P. lividus embryos, other ionizing radiation, such as X-Ray, determined increased levels of a putative p63 protein, detected using a commercially available antibody against a human p53 fusion protein (Bonaventura et al., 2011). In mammalian cells, p53 and the other family members are well known “genome guardians” with key roles as cycle checkpoint factors and apoptosis regulators (Vousden and Lane, 2007). In particular, p63 and p73 are mainly involved in the coordination of normal development €tsch et al., 2010). In almost all invertebrates, including the sea (Do urchin, a p63/p73 common ancestor gene has been found (Belyi et al., 2010), but in sea urchins its molecular characterization and functional studies are still lacking, both at physiological and stressful conditions (Costa et al. in preparation). As suggested by Lesser et al. (2003), p53 might act together with p21, an inhibitor of kinases like cdc2, which showed decreased protein levels after increasing exposure to UVR in freshly fertilized embryos. In addition, by the TUNEL assay, a significant increase in the number of apoptotic cells has been observed in embryos exposed to UVR at different developmental stages (see Table 5, Lesser et al., 2003). According to the physiological apoptosis occurring in sea urchin embryos (Voronina and Wessel, 2001; Roccheri et al., 2002), lower percentages of apoptotic cells were also found in the controls, i.e.

Please cite this article in press as: Bonaventura, R., Matranga, V., Overview of the molecular defense systems used by sea urchin embryos to cope with UV radiation, Marine Environmental Research (2016), http://dx.doi.org/10.1016/j.marenvres.2016.05.019

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embryos exposed to artificial visible radiation (400 nm), see Table 5 (Lesser et al., 2003). A Proteomic analysis of S. purpuratus embryos irradiated at early stages identified i) proteins indirectly involved in apoptosis, as the mitochondrial chaperone cyclophilin D which might sense changes in the cellular redox states following irradiation, ii) many other proteins involved in the cell cycle delays, and iii) the HSP70s (Campanale et al., 2011; Adams et al., 2012). Interestingly, many studies have shown that HSP members could regulate the apoptotic pathway at several levels (Kennedy et al., 2014). Thus, as previously suggested, HSP70 might promote cell survival counteracting apoptosis in sea urchin embryos exposed to UVR, as well as to others stress agents such as cadmium (Roccheri et al., 2004), manganese (Pinsino et al., 2010), and X-Ray (Bonaventura et al., 2011). Further studies are needed to explain how UVRs trigger apoptosis in embryonic cells and through which pathways, i.e. intrinsic or extrinsic signaling pathways (Robertson et al., 2006). Indeed, extracellular signals can activate apoptosis through the extrinsic pathway and intracellular signals can activated the apoptotic pathway where mitochondria have a pivotal role (Robertson et al., 2006). Concerning the balance among the mechanisms involved in the embryonic fate, it should also be taken into account the role of autophagy, known to be involved in promoting the survival of cells in P. lividus embryos exposed to cadmium (Chiarelli et al., 2011). In addition, the relationship between autophagy and apoptosis was studied in cadmium-exposed embryos (Chiarelli et al., 2014), suggesting this as another intriguing topic to be studied in UV exposed embryos.

5. UVR in combination with other stressors In the environment, UVR acts in concert with other factors both environmental, as food availability and climate changes, and anthropogenic, as metals, polycyclic aromatic hydrocarbons (PAHs) and nanoparticles (NPs). These interactions with UVR could be independent or interactive, either in an additive, synergistic or antagonistic manner. Few studies have analyzed the combined effects of UVR and other stressors on sea urchin embryos. For example, L. variegatus gametes have been exposed to PAHs (benzo [a]pyrene and phenanthrene) for 30 min before fertilization and then UVB irradiated for 1 or 4 h. PAHs/UVB had an additive toxicological effect on embryos evaluated as growth rate, i.e. counting how many embryos reached the 4- and 32-cell stages (Steevens et al., 1999). The Combined exposure of P. lividus embryos to cadmium, from fertilization, and UVB, at cleavage stage, (Cd/UVB exposure) induced the activation of p38MAPK at 1hpi, while ERK and JNK seemed not to be activated (Bonaventura et al., 2015). Furthermore, Cd/UVB exposure induced the upregulation of Pl-143-3 epsilon, Pl-MT and Pl-jun mRNAs later in development, i.e. at 24hpi. Interestingly, Cd/UVB caused abnormal branching and irregular skeletal patterns occurring in 72 h-old embryos (Bonaventura et al., 2015). Such abnormal morphologies have never

been described in previous studies exposing embryos singly to Cd or UVB (Bonaventura et al., 2015). How Cd/UVB operate together in sea urchin embryos remains to be fully elucidated. One of the possibilities is an enhanced production of ROS, as suggested by Steevens et al. (1999) for the co-exposure of L. variegatus embryos to PAHs/UVB, and that might similarly occur in P. lividus embryos after Cd/UVB exposure. Moreover, the increased Pl-MT mRNA levels support this possibility, in agreement with the MT role as radicalscavenger and/or its ROS-quenching activity (McGee et al., 2010). Emerging technologies are increasingly using engineered nanoparticles (NPs) or nanomaterials (ENMs) to produce many different products, as for example sunscreens and cosmetics. Possible health risks of NPs is a topic of growing concerns (The EEAP Report, 2014) and, thus, further studies are needed to assess the health as well as the “ecosafe” production and application of ENMs. Until now, it seems that NPs (Ti or Zn oxides) in sunscreens do not cause toxicity in skin cells (Therapeutic Goods Administration, 2013). Nevertheless, UV radiation could enhance the penetration of engineered NPs in solar exposed skin (Jatana and DeLouise, 2014). Sea urchin immune cells and embryos are used as model to evaluate nanomaterials effect on immunity and development (Corsi et al., 2014; Pinsino et al., 2015). Thus, sea urchins can be used to evaluate the putative interaction between nanomaterials and UVR.

6. Conclusions We have summarized the molecular defense systems adopted by sea urchin embryos to counteract UVR effects, as showed in Fig. 3. The UVR response involves the activation of DNA repair mechanisms, the induction of stress genes, as well as many defensive proteins and signaling pathways. The activation of these defense-systems have an energetic cost that affects the embryonic development contributing in the determination of alternative morphotypes, i.e. abnormal embryos as the “packed blastula” or embryos with skeleton malformations. In addition, some of the defensive gene and protein markers have a dual role, i.e. protection against environmental hazards, as the UVR, and regulation of the developmental program, and thus their recruitment in protective mechanisms can affect embryonic development. The fate determination of UV-irradiated embryos is also linked to the balance between survival and apoptotic signaling. In the UV-defense systems operating at several levels in sea urchin embryos, other aspects should be taken into consideration. For example, the geographic distribution of the species used, as Antarctic and Tropical species have different sensitivity to UVR. The developmental stages chosen for the UV-irradiation, i.e. early/late stage or continuous exposure, could determine different molecular and morphological responses. Field and/or laboratory exposure should have different effects, also in the same species. Indeed, it is not easy to compare data obtained in different laboratories, which use different types of UV lamps, with different emission properties and irradiation times. Until now, only a study used a proteomics approach in early

Table 5 Percentage of apoptotic cells in irradiated S. droebachiensis embryos (Lesser et al., 2003). Irradiation stagea

FFE blastula gastrula a

UVB

UVA

Vis. radiation

280 nm

305 nm

320 nm

375 nm

400 nm

36% 78% 100%

32% 57% 61%

18% 53% 54%

17% 37% 43%

10% 34% 29%

TUNEL assay performed after three days of development.

Please cite this article in press as: Bonaventura, R., Matranga, V., Overview of the molecular defense systems used by sea urchin embryos to cope with UV radiation, Marine Environmental Research (2016), http://dx.doi.org/10.1016/j.marenvres.2016.05.019

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Fig. 3. Defensive-response mechanisms and pathways involved in the UVR response in sea urchin embryos. DNA repair/stress genes, defensive/anti-oxidative proteins and MAPKs involved in the UVR-response in sea urchin embryos from published data. Dashed arrows indicate hypothetical connections. Arrows indicate the molecular targets of UVR, connections and relationships among the UVR-response mechanisms that act at several levels and that can influence the balance between apoptosis and survival of the embryonic cells.

stage cleavage embryos (Campanale et al., 2011) and further studies using omics strategies are needed. The use of the microarray technology is encouraged to expand the information both on up and downregulated genes in sea urchin embryos exposed to UVR, taking also into account that these transcriptional responses can vary with the doses and the time elapsed after irradiation, as reported for human cells (de la Fuente et al., 2009). Sea urchin embryos are a useful in vivo-model to study the UVresponse, which allows the expansion of knowledge on the defense systems operating in the cells exposed to UVR, both at physiological and stressful conditions, and also in combination with other stress factors. Indeed, in addition to UV radiation alone, the interactive effects of UV and other environmental and/or anthropogenic factors also need major attention.

Acknowledgement We are grateful to the members of the group for their encouragement, support and helpful discussions during the writing of this review. Authors wish to thank Francesca Zito and Annalisa Pinsino for the immunofluorescence experiments showed in Figs. 1 and 2, and Professor David McClay for the kind gift of the monoclonal antibody 1D5. The EU program UV-TOX to V. Matranga, Contract No. EVK3-CT1999e00005 greatly supported this work. CNR Flagship Project POM-FBdQ 2011e2013 partially supported this work.

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