Expert Opinion on Environmental BiologyISSN: 2325-9655

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Review Article, Expert Opin Environ Biol Vol: 6 Issue: 1

Sea Urchin Bioassays in Toxicity Testing: I. Inorganics, Organics, Complex Mixtures and Natural Products

Giovanni Pagano1,2*, Marco Guida1, Marco Trifuoggi1, Philippe Thomas3, Anna Palumbo2, Giovanna Romano2 and Rahime Oral4
1Federico II Naples University, I-80126 Naples, Italy
2Stazione Zoologica Anton Dohrn, I-80121 Naples, Italy
3Environment and Climate Change Canada, Science and Technology Branch, National Wildlife Research Center Carleton University, Ottawa, Ontario K1A 0H3, Canada
4Ege University, Faculty of Fisheries, TR-35100 Bornova, İzmir, Turkey
Corresponding author : Giovanni Pagano
Federico II Naples University, via Cinthia, I-80126 Naples, Italy
Tel: +39-335-7907261
Fax: +39-825-1911712
E-mail: [email protected]
Received: December 09, 2016 Accepted: January 18, 2017 Published: January 23, 2017
Citation: Pagano G, Guida M, Trifuoggi M, Thomas P, Palumbo A, et al. (2017) Sea Urchin Bioassays in Toxicity Testing: I. Inorganics, Organics, Complex Mixtures and Natural Products. Expert Opin Environ Biol 6:1. doi: 10.4172/2325-9655.1000142

Abstract

Sea Urchin Bioassays in Toxicity Testing: I. Inorganics, Organics, Complex Mixtures and Natural Products

Based on established knowledge in physiology and embryology, sea urchins have proven to be excellent bioassay models since the early 1970’s when evaluating the impacts to early life stages and cellular functions after exposure to a number of xenobiotics. This review attempts to build a comprehensive survey of reports on sea urchin gametes and embryos used in toxicity testing of different classes of xenobiotics including inorganics, organics, different complex mixtures, and natural products. Our results provide support for common endpoints used when evaluating adverse effects on sea urchin early life stages, including frequency of developmental defects and/or cytogenetic abnormalities, changes in sperm fertilization success and offspring damage, and other endpoints related to redox alterations, DNA damage and other molecular biomarkers. Current studies using sea urchin bioassays continue to provide support for this tool in toxicology including, amongst others, studies of ocean acidification, action mechanisms of candidate new drugs, and novel xenobiotic releases to the environment.

Keywords: Sea urchin; Fertilization; Embryogenesis; Cytogenetic abnormalities; Redox alterations

Keywords

Sea urchin; Fertilization; Embryogenesis; Cytogenetic abnormalities; Redox alterations

Introduction

Sea urchins have proven to be extremely helpful in providing scientific insight in a number of biological disciplines, including physiology, embryology, biochemistry and genetics, in studies dating as far back as the late 19th century to the early 20th century [1-6]. Those studies provided a body of knowledge that became foundation in what we know in basic cellular events such as mitosis with the first reports on chromosomes [2], fertilization and embryogenesis [3-5], with implications well beyond echinoderm biology and thereafter translated into general biology and medicine. Relatively few reports on the exposures of sea urchin embryos to various chemicals were available, and focused primarily on the influence of some agents in modulating normal embryogenesis, rather than on investigating any adverse effects of chemicals, e.g. lithium-induced “animalization” [6,7].
In the wave of the early concern in radiobiology, drug toxicity, and environmental pollution, sea urchins were first utilized in pioneering studies evaluating the effects of sperm irradiation [8], marine pollution [9], pharmaceuticals and pesticides [10-12], and the action mechanisms of carcinogens [13,14], in bioassays viewed as complementary to the classical murine models. Those studies dating up to 1970’s set the grounds for continuing investigations up to present.
The body of literature of toxicity testing in the sea urchin model includes reports focused on a number of morphological and/or molecular endpoints assessing adverse effects. This prevents attempting extensive comparisons (e.g. scaling EC50’s) in-between several agents. Though with this limitation, the present review may both provide an unprecedented archive of sea urchin toxicity testing reports and a useful contribution in future study design.
Inorganic agents
Seawater acidification: As shown in Table 1, pH changes were initially studied in sea urchin embryos and sperm as a subject of bioassay standardization, or for their possible implications in freshwater systems, related to the recognized phenomenon termed acid rain [15]. Early reports found that developing embryos under slightly decreased pH conditions, in the order of 0.5 pH units, underwent damage to embryogenesis and mitotic aberrations, with stronger effects when treatment was performed before hatching than in later developmental stages (blastula/gastrula) [16,17]. When sea urchin sperm were exposed to pH changes, fertilization was inhibited by either pH decrease (<7) or increase (>8.5). Furthermore, transmissible damage to the offspring of low pH-exposed sperm was observed, both in terms of developmental defects and of decreased mitotic activity [18,19]. It should be noted that slightly decreased pH (such as 7.5) enhanced fertilization success, as confirmed in a more recent report [20]. A series of studies by Stumpp et al. [21-24] showed that elevated seawater pCO2, closely resembling the expected values by the end of this century, altered the expression of 26 representative genes important for metabolism, calcification and ion regulation, along with impacting growth and resulting in developmental delay of echinoid larvae. Adult sea urchins (P. lividus) were tested by Lewis et al. [25] for their sensitivity to micromolar copper levels in ambient vs. acidified seawater, resulting in DNA damage and oxidative stress responses, beyond the mere data of metal speciation.
Table 1: Reports on inorganics-associated effects on sea urchin early life stages.
Altogether, the available literature on the effects of pH decrease in sea urchin bioassays corroborates the of global concern about ocean acidification.
Other inorganics: A number of inorganics (Cd, As, Cr, Pb, Cl, Ag, Mn, La, Ce, Gd) were tested for their adverse effects on sea urchin development, fertilization success, mitotic activity, as well as other effects as induction of apoptosis, calcification defects, DNA damage and/or increasing production of reactive oxygen species (ROS) amongst others [26-59] (Table 1). Groups of inorganics were tested in an extensive body of studies providing comparative toxicity datasets for various agents [40,59]. This was the case for early studies [41,42] testing groups of inorganics as Ag, Cd, Cr, Hg and Pb; other studies reported on the effects of individual inorganics vs. their mixtures [43,54].
It has been reported that developmental impairment due to treatment of fertilized eggs with Cd and Mn [29,38] involved the action of the physiological messenger nitric oxide [47]. Moreover, maternal exposure to these metal ions impaired reproduction and progeny fitness [48].
We have reported on the comparative toxicities in P. lividus embryos and sperm of light rare earth elements (REEs) (Y, La, Ce, Nd, Sm and Gd) that induced developmental defects and mitotic abnormalities in exposed embryos, offspring damage following sperm treatment, and redox anomalies (ROS production, lipid peroxidation, and nitric oxide release) [58,59]. A current study of heavy REEs (Dy, Ho, Er, Yb and Lu) suggests more severe toxicity of heavy vs. light REEs.
Nanomaterials: As shown in Table 2, a number of studies have been focused on the effects of nanomaterials (NM) in sea urchin early life stages. The growing interest in the multi-fold applications of NM has led to concerns on their possible health effects in several biological models, including sea urchins [60-73]. To date, the reported effects of NM in sea urchin early life stages have included multiple responses in sea urchin embryos following exposures to carbon nanoparticles, including developmental defects, abnormal biomineralization, and altered gene expression [60,61]. Other adverse effects have been reported in terms of chemosensitization, increased multidrug resistance, and redox changes following exposures of sea urchin embryos to Zn and Co oxide nanoparticles [62-65]. Silica nanoparticles were found to increase developmental defects while inhibiting fertilization success, and led to the decrease of cholinesterase activity [67]. TiO2 nanoparticles were found to enhance phagocyte TLR/p38 MAPK signalling pathway by activating an internalization mechanism [72]. Altogether, sea urchin bioassays have been useful in revealing adverse effects after exposure to NM. It should be recognized, however, that a substantial body of literature, beyond the scope of this review, points to a number of beneficial effects of NM, especially in medical and agricultural applications. Ad hoc investigations are warranted to critically re-evaluate beneficial vs. harmful effects associated with NM exposures.
Table 2: Reports on nanoparticle-associated effects on sea urchin early life stages.
Organic agents
As shown in Table 3, an extensive body of literature is available regarding the effects on sea urchin early life stage after exposures to organic agents. Since early studies [13,14,74] and up to recent reports [75,76] polycyclic aromatic hydrocarbons (PAHs) and their hydroxylated derivatives have been investigated for their bioaccumulation in sea urchin embryos and/or for induction of developmental defects. Comparisons of the relative toxicities of select PAHs vs their model mixtures when subjected to fluorescent light exposure were also investigated in recent literature [76]. Other studies reported on the relative toxicities of low molecular-weight aromatic hydrocarbons (benzene and styrene) and their derivatives [77,78].
Table 3: Reports on the effects of organics, complex mixtures and natural products on sea urchin early life stages.
Extensive studies have focused on the effects of pharmaceuticals on sea urchin embryogenesis and fertilization since early reports [10,11,79-82] and up to recent studies [83-94]. The available database comprehends reports on several classes of agents including antibiotics and antiseptics [79-82], antineoplastic drugs [84-87,89], teratogens [10,11,86,88,93], anti-inflammatory drugs, hormones or hormone-like agents [88,92], anesthetics [94-96], cannabinoids [97-99], and antioxidants [86,100]. Altogether, the currently available literature on the adverse effects of several pharmaceuticals in sea urchin bioassays contributes to mechanistic information about druginduced toxicity to early life stages, warranting further investigations and risk assessment.
Pesticides and metal organics: Since a pioneering paper by Bresch and Arendt [12], several pesticides have been investigated in sea urchin early life stages. As shown in Table 3, a number of studies have reported on halogen, nitrogen, and pyrethroid derivative pesticides for their effects on sea urchin early development, fertilization, and offspring quality [101-120]. Several studies found embryo- and spermiotoxicity of chlorinated pesticides [101-109], showing differences in toxicity as a function of number of chlorine atoms per molecule [102,103]. Organophosphate pesticides were found to affect sea urchin metamorphosis [110], embryo- and spermiotoxicity, along with induction of offspring damage following sperm exposure [111].
In some cases pesticide preparations include organic and inorganic components, as in the case of R6 fungicide, a mixture of an acetamide pesticide, cymoxanil (CYM) and cupric oxychloride (Cu- OCl) [120]. The commercial mixture resulted in toxicity that failed to appear following exposure to the two mixture components alone. A mixture prepared from analytical grade CYM and Cu-OCl also failed to induce toxicity, a result that suggested the occurrence of more toxic components in the formulation of the commercial mixture [120]. Thus, a critical reappraisal may be suggested in studies where toxicity is commonly evaluated on pure chemicals, while disregarding the effects induced by the technical-grade counterpart.
Either as antifouling agents or as pesticides, a number of metallorganic compounds have been evaluated for their toxicity to sea urchin early life stages (Table 3). Most of these reports have focused on organic tin derivatives used as antifouling agents, while some studies were aimed at comparing toxicities of individual agents vs. their mixtures [121-131].
Complex mixtures: An extensive body of literature refers to the effects of complex mixtures in sea urchin early life stages, since the 1970’s [132,133]. As shown in Table 3, a number of studies have been published on the effects of complex mixtures in sea urchin bioassays [132-160], including crude oil and oil fractions; spill-treating agents; refinery wastewater effluent; pulp mill effluent; bauxite manufacturing by-products; storm water and parking lot runoff; leather tanning effluents; landfill leachate; pisciculture effluents, and alum-coagulated municipal wastewater. This body of literature is not confined to studies of mixtures directly involved in marine pollution, as in the cases for industrial sludge and effluents [141-146,148-151,153,158], or landfill leachate [155]. Thus, one may recognize multiple applications of sea urchin bioassays in toxicity evaluation of complex mixtures both impacting on marine and on freshwater or terrestrial environments.
Another major application of sea urchin bioassays in testing complex mixture toxicity refers to sediment toxicity evaluations. This relevant subject is omitted in the present review and will be exposed in a subsequent paper.
Natural products: An established body of literature in sea urchin bioassays has focused on the effects of a number of natural products (NP) on sea urchin early life stages. The major goals of this research line have been devoted to either characterizing some natural products for their mitotic activity and their potential applications as pharmaceuticals, or as potential stressors to marine ecosystems.
Sea urchin bioassays were successfully used for assay-guided isolation of natural products from marine diatoms [161]. A body of literature has been devoted to these molecules, characterized as polyunsaturated aldehydes (PUAs), showing antimitotic and proapoptotic activity inducing malformations in developing embryos [162-168]. Interestingly, the effect was recorded when PUAs were added before or soon after fertilization, while they were almost ineffective when added at 40 minutes post fertilization onwards [167]. Molecular studies of differential gene expression induced by PUA treatments revealed that sea urchins activated an orchestrated defense system involving HSP70 as key stress response mediator [166] and showed that this effect is modulated by the messenger nitric oxide [165]. Analogous to PUA, diatom-derived oxylipins and hydroxyacids were reported recently to affect sea urchin development by antimitotic and pro-apoptotic mechanisms [169,170].
Other studies have reported on mitotoxic or other effects of natural products on sea urchin early development. Extracts from several biota, including algae, plants, and sponges were found to exert mitotic arrest in sea urchin cell division [171-175]. Terpenic compounds were obtained from sponges and corals providing evidence for mitotoxicity and embryotoxicity [176,177] and other effects, such as inhibition of DNA polymerases [178] and nucleic acid biosynthesis [179]. Two reports focused on the action mechanisms of β-amyloid in sea urchin early development [180,181]. A subtle reproductive impairment through nitric oxide-mediated mechanisms was reported in sea urchins from a site in the Gulf of Naples affected by (Ostreopsis cf. ovata) [182].
Sea urchin bioassays: Background and perspectives: After the early laboratory studies of sea urchin bioassays in 1970’s, some methodology reviews were published in testing developmental damage, spermiotoxicity and transmissible damage to sperm offspring, by either treating early life stages or adult sea urchins [183- 185].
A summarized scenario of assay timing and outcomes that are tested for in embryo exposure is depicted in Figure 1. The commonly used embryotoxicity protocol starts at zygote stage (10 min postfertilization) ending with the observation of developmental defects at pluteus larval stage, 48 to 72 hrs after fertilization, according to species and culture temperature. In order to elucidate the most sensitive developmental stage to the action of a given xenobiotic, embryo exposure may be confined to early, pre-hatching stages, with prevailing mitotic activity, or to post-hatching stages (blastula and gastrula), leading to larval differentiation. Thus, for example, allopurinol, some antioxidants (L-methionine and N-acetylcysteine) and diatom aldehydes [86,100,168] only induced developmental defects following early (pre-hatching) exposures. By contrast, cadmium and chlorobenzenes were only effective when administered to post-hatching embryos [26,77], while lead exerted significantly stronger effects following post-hatching compared to pre-hatching exposure [34].
Figure 1: Schematic view of timing and developmental stages in sea urchin embryos cultured at 18°C (e.g. P. lividus) showing sensitive stages to mitotoxic agents vs. embryo-selective agents (i.e. affecting later stages in embryogenesis). Abbreviations: N: Normally developed plutei; P1: Malformed pluteus; P2: Developmental arrest at abnormal blastula/gastrula stage.
The major embryological endpoints evaluated in larval cultures are the frequencies of pluteus malformations (P1), pre-larval arrest (P2), as shown in Figure 1. The most commonly scored endpoint, as a function of xenobiotic concentration, is the frequency of developmental defects (%DD)=(P1+P2). Another scored endpoint consists of the observation of dead plutei and dead pre-larval (or prehatching) embryos that point to acute effects; however, these effects are not usually observed in testing sub-acute developmental toxicity, confined to %DD.
In order to perform cytogenetic analysis, evaluating inhibition of mitotic activity and/or induction of mitotic aberrations in Carnoy’s fluid, cleaving embryos (approx. 5 hrs post-fertilization) are fixed and subsequently stained by acetic carmine allowing for observation of chromosomes, then scoring the frequencies of active mitoses per embryo, embryos lacking mitotic figures, and the frequencies of mitotic aberrations. The most commonly observed mitotic aberrations include anaphase bridges, lagging chromosomes, multipolar spindles, and scattered chromosomes (Figure 2).
Figure 2: Main cytogenetic endpoints include decreased mitotic activity (mitoses per embryo) and mitotic aberrations. A. Anaphase bridge; B.Lagging chromosomes; C. Multipolar spindle; D. Scattered chromosomes.
Aside embryological and cytogenetic endpoints, a number of biochemical or other molecular alterations may be detected in xenobiotic-exposed sea urchin embryos and larvae. This was the case for the induction of redox anomalies, with changes in ROS production, oxidative and nitrosative stress endpoints, and oxidative DNA damage [39,48,49,59,89,90]. Other reported endpoints, evaluated in testing developmental toxicity in sea urchin embryogenesis, included calcification defects [23,37-39], induction of multi-drug resistance [64] and MAPK-mediated signalling pathway [72], and production of the endogenous antioxidant, ovothiol [186].
Sea urchin sperm are exposed to agents for assigned time intervals varying upon species, e.g. 60 min for Paracentrotus lividus or 10 min for Sphaerechinus granularis. The endpoints measured following sperm exposures to xenobiotics include fertilization success and offspring damage, expressed as developmental defects and/or altered redox endpoints (Figure 3). Changes in fertilization success are mostly evaluated in zygotes by formalin fixation shortly after fertilization by a number of authors [49,95,96,114,183,184]. We follow - and recommend - a different procedure, by reading fertilization rate starting from 1 hr post-fertilization on live and cleaving embryos, which are then allowed to undergo further development up to pluteus stage. Indeed, reading fertilization rate in fixed zygotes is confined to the appearance of fertilization membrane, which may be more or less evident and lead to imprecise evaluation, or to fixation artifacts. Otherwise, reading fertilization rate in cleaving embryos is both supported by evidence for fertilization membrane and by on-going cleavage. Thus, the observation of any transmissible damage from sperm to offspring is warranted. The induction of developmental defects in the offspring of sperm exposed to xenobiotics has been reported in a number of studies [19,31,33,43,44,54,58,59,89,90,111,143-145,148-151]. The finding of developmental defects or embryonic larval mortality following sperm exposures may be attributed to induction of dominant lethals, an effect referred to since early genetics studies up to date [187,188].
Figure 3: Sperm pretreatment experiments, timing and observation endpoints: a) Changes in fertilization success; b) Offspring damage, detected as: b1) Developmental defects; b2) Altered redox endpoints.

Conclusions

The body of literature on the use of sea urchin in toxicity testing encompasses an extensive number of xenobiotics and mixtures that are relevant both in environmental science and in general pharmacology and toxicology. Well beyond the current database and the on-going research, the appearance of new pollutants, pharmaceuticals and occupational agents, along with the need for characterizing widespread complex mixtures, open the field for further research efforts and future regulation utilizing sea urchin bioassays in current and forthcoming work.
Beyond their half-century historical background, sea urchin bioassays represent a thriving tool in future investigations and health risk assessment, unconfined to marine environment and in prospect evaluation of novel xenobiotics.

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