Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models.

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

Copper oxide nanoparticles induce the transcriptional modulation of oxidative stress-related genes in Arbacia lixula embryos.

Copper oxide nanoparticles (CuO NPs) are widely used in various industrial applications, i.e. semiconductor devices, batteries, solar energy converter, gas sensor, microelectronics, heat transfer fluids, and have been recently recognized as emerging pollutants of increasing concern for human and marine environmental health. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this study, we evaluated the potential role of oxidative stress in CuO NP toxicity by exploring the molecular response of Arbacia lixula embryos to three CuO NP concentrations (0.7, 10, 20 ppb) by investigating the transcriptional patterns of oxidative stress-related genes (catalase and superoxide dismutase) and metallothionein, here cloned and characterized for the first time. Time- and concentration-dependent changes in gene expression were detected in A. lixula embryos exposed to CuO NPs, up to pluteus stage (72 h post-fertilization, hpf), indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs. These findings provide new insights into the comprehension of the molecular mechanisms underlying copper nanoparticle toxicity in A. lixula sea urchin and give new tools for monitoring of aquatic areas, thus corroborating the suitability of this embryotoxicity assay for future evaluation of impacted sites.

Expression and functional activity of neurotransmitter system components in sea urchins' early development.

Reverse-transcription polymerase chain reaction (RT-PCR) investigation of the expression of the components supposedly taking part in serotonin regulation of the early development of Paracentrotus lividus has shown the presence of transcripts of five receptors, one of which has conservative amino acid residues characteristic of monoaminergic receptors. At the early stages of embryogenesis the expressions of serotonin transporter (SERT) and noradrenaline transporter (NET) were also recognized. The activities of the enzymes of serotonin synthesis and serotonin transporter were shown using immunohistochemistry and incubation with para-chlorophenylalanine (PСРА) and 5-hydroxytryptophan (HTP). Pharmacological experiments have shown a preferential cytostatic activity of ligands characterized as mammalian 5-hydroxytryptamine (5-HT)1-antagonists. On the basis of the sum of the data from molecular biology and embryo physiological experiments, it is suggested that metabotropic serotonin receptors and membrane transporters take part in the regulatory processes of early sea urchin embryogenesis.

Effect of silver nanoparticles on Mediterranean sea urchin embryonal development is species specific and depends on moment of first exposure.

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use of silver nanoparticles (AgNP), their environmental fate and impact is therefore of particular concern. In this context we have investigated the species-specific effects of low concentrations of 60 nm AgNP on embryonal development in Mediterranean sea urchins Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. The sensitivity of urchin embryos was tested by exposing embryos to nanoparticle concentrations in the 1-100 µg L(-1) range, with times of exposure varying from 30 min to 24 h (1 h-48 h for S. granularis) post-fertilisation which corresponded with fertilized egg, 4 cell, blastula and gastrula development phases. The most sensitive species to AgNP was A. lixula with significant modulation of embryonal development at the lowest AgNP concentrations of 1-10 µg L(-1) with high numbers of malformed embryos or arrested development. The greatest impact on development was noted for those embryos first exposed to nanoparticles at 6 and 24 h post fertilisation. For P. lividus, similar effects were noted at higher concentrations of 50 µg L(-1) and 100 µg L(-1) for all times of first exposure. The S. granularis embryos indicated a moderate AgNP impact, and significant developmental abnormalities were recorded in the concentration range of 10-50 µg L(-1). As later post-fertilisation exposure times to AgNP caused greater developmental changes in spite of a shorter total exposure time led us to postulate on additional mechanisms of AgNP toxicity. The results herein indicate that toxic effects of AgNP are species-specific. The moment at which embryos first encounter AgNP is also shown to be an important factor in the development of abnormalities, and future applications of the sea urchin embryo development test for nanoparticle toxicity testing should carefully address the specific phase of development of embryos when nanoparticles are first introduced.

Developmental abnormalities and neurotoxicological effects of CuO NPs on the black sea urchin Arbacia lixula by embryotoxicity assay.

The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by using 24-well plates. Fertilized eggs were exposed to five doses of CuO NPs ranging from 0.07 to 20 ppb, until pluteus stage. CuO NPs suspensions in artificial seawater formed agglomerates of 80-200 nm size, and copper uptake was 2.5-fold up in larvae exposed to high NP concentrations in respect to control. Developmental delay and morphological alteration, including skeletal abnormalities, were observed, as well as impairment in cholinergic and serotonergic nervous systems. These findings suggest the potential of CuO NPs to interfere with the normal neurotransmission pathways, thus affecting larval morphogenesis. Overall, the embryotoxicity tests are effective for evaluation of nanoparticle effects on the health of aquatic biota. Furthermore, as the black sea urchin A. lixula demonstrated to be vulnerable to NP exposure, it may be a valid bioindicator in marine biomonitoring and ecotoxicological programmes.

Temperature modulates the response of the thermophilous sea urchin Arbacia lixula early life stages to CO2-driven acidification.

The increasing abundances of the thermophilous black sea urchin Arbacia lixula in the Mediterranean Sea are attributed to the Western Mediterranean warming. However, few data are available on the potential impact of this warming on A. lixula in combination with other global stressors such as ocean acidification. The aim of this study is to investigate the interactive effects of increased temperature and of decreased pH on fertilization and early development of A. lixula. This was tested using a fully crossed design with four temperatures (20, 24, 26 and 27 °C) and two pH levels (pHNBS 8.2 and 7.9). Temperature and pH had no significant effect on fertilization and larval survival (2d) for temperature <27 °C. At 27 °C, the fertilization success was very low (<1%) and all larvae died within 2d. Both temperature and pH had effects on the developmental dynamics. Temperature appeared to modulate the impact of decreasing pH on the % of larvae reaching the pluteus stage leading to a positive effect (faster growth compared to pH 8.2) of low pH at 20 °C, a neutral effect at 24 °C and a negative effect (slower growth) at 26 °C. These results highlight the importance of considering a range of temperatures covering today and the future environmental variability in any experiment aiming at studying the impact of ocean acidification.

The effects of model polysiloxane and fouling-release coatings on embryonic development of a sea urchin (Arbacia punctulata) and a fish (Oryzias latipes).

In recent decades attention has focused on the development of non-toxic fouling-release coatings based on silicone polymers as an alternative to toxic antifouling coatings. As fouling-release coatings gain market share, they will contribute to environmental contamination by silicones. We report effects of eight model polysiloxane and three commercial foul-release coatings on embryonic development of sea urchins and fish, Japanese medaka. We used model coatings because they have known composition and commercially available components and molecules leaching from these coatings have been partially characterized. The commercial fouling-release coatings are purported to be non-toxic and components are proprietary. Our goal was to expose embryos of well studied model animals to the coatings to determine if the complex mixtures leaching from the coatings impact development. Urchins were chosen because development is rapid and embryos can enter the non-slip layer over surfaces. Medaka was chosen because the female deposits the sticky eggs onto the anal fin and then scrapes them off onto surfaces. Embryos were confined in water over coatings in 24 well plates. Fresh model coatings had no effect on urchin development while commercial fouling-release coatings inhibited development. Fish embryos had delayed hatching, increased mortality of hatchlings and dramatically decreased ability of hatchlings to inflate the swim bladder and reduced hatching success on all coatings. After one-month immersion of coatings in running seawater to simulate initial application in the marine environment, sea urchin embryos died when placed over model silicones. Effects of the commercial coatings were reduced but included retarded development. Effects on fish embryos over leached coating were reduced compared to those of fresh coating and included decreased hatching success, decreased hatchling survival and inability to inflate the swim bladder for commercial coatings. These findings suggest, similar to medical conclusions, compounds leaching from silicone coatings can impact development and the topic deserves study.

Parental dietary effect on embryological development response to toxicants with the sea urchin Arbacia punctulata.

The role of echinoid parental nutrition in early-life stage toxicity is not well understood. Arbacia punctulata were fed either a fresh diet consisting of organic lettuce and carrots or a dry feed. Embryos from parents fed the dry feed exhibited lower sensitivity to copper, whereas the opposite occurred with 1,3,5-trinitrobenzene and sodium dodecyl sulfate (SDS). EC(50) values for the dry and fresh feed treatments, respectively, were 41.0 and 29.9 microg/L for copper, 0.5 and 1.8 mg/L for 1,3,5-trinitrobenzene, and 3.5 and 5.6 mg/L for SDS. The data suggests that nutritional standardization for sea urchins in ecotoxicological laboratories needs to be addressed and further investigated.

Embryotoxic effects of nonylphenol and octylphenol in sea urchin Arbacia lixula.

Nonylphenol (NP) and octylphenol (OP), both of which are biodegradation products of alkylphenols, are widely used in industrial applications and in some domestic products. These chemicals are found widely in surface water and aquatic sediments. We have carried out a comparative embryotoxicity analysis of the effects of increasing concentrations of NP (seven concentrations ranging from 0.937 to 18.74 microg/l) and OP (six concentrations ranging from 5 to 160 microg/l) on embryos of the sea urchin Arbacia lixula. The indicators evaluated were larval malformations, developmental arrest and embryonic/larval mortality. The results revealed that low concentrations of these chemicals (NP, OP) generally caused malformations in the skeletal system. High concentrations (18.74 microg NP/l, 160 microg OP/l) were found to inhibit the growth of embryos in the early life stages by preventing mitosis. We conclude that NP and OP present a major risk to the normal development of A. lixula at the low concentrations that have been recorded in the environment. These chemicals are therefore most likely to represent an ecological hazard at the population level given the cumulative effects of other environmental pollutants.