Effects of three zinc-containing sunscreens on development of purple sea urchin (Strongylocentrotus purpuratus) embryos.

The growing popularity of physical sunscreens will lead to an increased release of ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations may be released into marine environments by use of sunscreens. The extent of the consequences of this addition of Zn to the ocean are not fully understood. We investigated the effects of materials released by ZnO- sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos incubated in various concentrations of Zn (0.01, 0.05, 0.1, 0.5, and 1 mg/L), the sources of which included zinc-containing compounds: ZnO and zinc sulfate (ZnSO4); and ZnO sunscreens: All Good, Badger, and Raw Elements brands. Based on EC50 values, ZnO-containing sunscreens were slightly, but not significantly, more toxic than ZnO and ZnSO4, suggesting that sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn-exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescence confocal microscopy. The concentration of Zn2+ internalized by the developing embryos correlated positively with the concentration of Zn in seawater. Additionally, exposure to both ZnO sunscreens and ZnO and ZnSO4 at 1 mg/L Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is one of the first studies documenting ZnO-containing sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms.

Methods for toxicology studies in echinoderm embryos and larvae.

Sea urchin embryos have been used in toxicological studies for many decades as they are an accepted model system for investigations of chemicals that impact development. Here we describe methods for using pulse-chase experiments to study the impacts of environmental chemicals on early development as well as development of larvae. This includes the application of fluorescence plate assays with living embryos and fluorescent probes to assess cell functions (mitochondrial membrane potential, lysosome abundance, reactive oxygen species, and esterase activity) based on total cell numbers. We also describe how to use some of these fluorescent probes in embryos/larvae with confocal microscopy for the localization of cellular damage in response to toxics exposure. Finally, we assess skeleton formation in sea urchin larvae and present methods for using polarized light microscopy to examine spicule morphology.

Effects of soluble copper and copper oxide nanoparticle exposure on the immune system of mussels, Mytilus galloprovincialis.

Copper and copper oxide nanomaterials (nCuO) can enter the marine environment negatively impacting mussels, an environmental and commercially relevant organism. We analyzed the effects on the immune system of adult mussels exposed to soluble copper (CuSO4 , 20-50 µg/L) or nCuO (100-450 µg/L). CuSO4 caused significant copper accumulation in gills and cell-free hemolymph, while nCuO caused cell damage to gills and significant copper accumulation in hemocytes, the most abundant cells in the hemolymph. Both sources of copper caused cellular toxicity in hemocytes by increasing reactive oxygen species production and lysosome abundance, and decreasing multi-drug resistance transporter activity. Though hemocyte abundance was not affected, their in-vitro phagocytic activity decreased, explaining the slight (but not statistically significant) increase in bacterial proliferation in mussels exposed to the pathogenic bacteria Vibrio tubiashii following copper exposure. Thus, exposure to non-lethal concentrations of CuSO4 or nCuO can potentially increase mussel susceptibility to bacterial infections.

Facilitation of trace metal uptake in cells by inulin coating of metallic nanoparticles.

Trace elements such as zinc and iron are essential for the proper function of biochemical processes, and their uptake and bioavailability are dependent on their chemical form. Supplementation of trace metals through nanostructured materials is a new field, but its application raises concerns regarding their toxicity. Here, we compared the intracellular zinc uptake of different sources of zinc: zinc sulfate, and ZnO and core-shell α-Fe2O3@ZnO nanoparticles, coated or uncoated with inulin, an edible and biocompatible polysaccharide. Using mussel haemocytes, a well-known model system to assess nanomaterial toxicity, we simultaneously assessed zinc accumulation and multiple cellular response endpoints. We found that intracellular zinc uptake was strongly enhanced by inulin coating, in comparison to the uncoated nanoparticles, while no significant effects on cell death, cell viability, mitochondrial membrane integrity, production of reactive oxygen species or lysosome abundance were observed at concentrations up to 20 ppm. Since no significant increments in toxicity were observed, the coated nanomaterials may be useful to increase in vivo zinc uptake for nutritional applications.

Stage specific effects of soluble copper and copper oxide nanoparticles during sea urchin embryo development and their relation to intracellular copper uptake.

The effects of exposure to either soluble copper (copper sulfate) or copper oxide nanoparticles (nano-CuO) during specific early developmental stages of sea urchin embryos were analyzed. Soluble copper caused significant malformations in embryos (skeletal malformations, delayed development or gut malformations) when present at any given stage, while cleavage stage was the most sensitive to nano-CuO exposure causing skeletal malformations and decreased total antioxidant capacity. The stage specificity was linked to higher endocytic activity during the first hours of development that leads to higher accumulation of copper in specific cells critical for development. Results indicate that nano-CuO results in higher accumulation of copper inside of embryos and this intracellular copper is more persistent as compared to soluble copper. The possible implications later in development are discussed.

Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 µg/mL) and GO (<20 µg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 µg/mL graphene and 0.05 µg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos.

Copper oxide nanomaterials (nano-CuOs) are widely used and can be inadvertently introduced into estuarine and marine environments. We analyzed the effects of different nano-CuOs (a synthesized and a less-pure commercial form), as well as ionic copper (CuSO4) on embryo development in the white sea urchin, a well-known marine model. After 96 h of development with both nano-CuO exposures, we did not detect significant oxidative damage to proteins but did detect decreases in total antioxidant capacity. We show that the physicochemical characteristics of the two nano-CuOs play an essential role in their toxicities. Both nano-CuOs were internalized by embryos and their differential dissolution was the most important toxicological parameter. The synthesized nano-CuO showed greater toxicity (EC50 = 450 ppb of copper) and had increased dissolution (2.5% by weight over 96 h) as compared with the less-pure commercial nano-CuO (EC50 = 5395 ppb of copper, 0.73% dissolution by weight over 96 h). Copper caused specific developmental abnormalities in sea urchin embryos including disruption of the aboral-oral axis as a result in changes to the redox environment caused by dissolution of internalized nano-CuO. Abnormal skeleton formation also occurred.

Copper oxide and zinc oxide nanomaterials act as inhibitors of multidrug resistance transport in sea urchin embryos: their role as chemosensitizers.

The ability of engineered nanomaterials (NMs) to act as inhibitors of ATP-binding cassette (ABC) efflux transporters in embryos of white sea urchin (Lytechinus pictus) was studied. Nanocopper oxide (nano-CuO), nanozinc oxide (nano-ZnO), and their corresponding metal ions (CuSO4 and ZnSO4) were used as target chemicals. The results showed that nano-CuO, nano-ZnO, CuSO4, and ZnSO4, even at relatively low concentrations (0.5 ppm), significantly increased calcein-AM (CAM, an indicator of ABC transporter activity) accumulation in sea urchin embryos at different stages of development. Exposure to nano-CuO, a very low solubility NM, at increasing times after fertilization (>30 min) decreased CAM accumulation, but nano-ZnO (much more soluble NM) did not, indicating that metal ions could cross the hardened fertilization envelope, but not undissolved metal oxide NMs. Moreover, nontoxic levels (0.5 ppm) of nano-CuO and nano-ZnO significantly increased developmental toxicity of vinblastine (an established ABC transporter substrate) and functioned as chemosensitizers. The multidrug resistance associated protein (MRP, one of ABC transporters) inhibitor MK571 significantly increased copper concentrations in embryos, indicating ABC transporters are important in maintaining low intracellular copper levels. We show that low concentrations of nano-CuO and nano-ZnO can make embryos more susceptible to other contaminants, representing a potent amplification of nanomaterial-related developmental toxicity.

Laccase-mediated transformations of endocrine disrupting chemicals abolish binding affinities to estrogen receptors and their estrogenic activity in zebrafish.

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17ß-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hERα) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfERαLBD). E2, BPA, NP, and TCS showed higher affinity for the zfERαLBD than for hERα. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfERαLBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.

Halogenated pesticide transformation by a laccase-mediator system.

The transformation of organic halogenated pesticides by laccase-mediator system has been investigated. Twelve pesticides were assayed in the presence of nine different mediators. Acetosyringone and syringaldehyde showed to be the best mediators. The halogenated pesticides bromoxynil, niclosamide, bromofenoxim and dichlorophen were transformed by the laccase-syringaldehyde system showing catalytic activities of 48.8, 142.0, 166.2 and 1257.6nmolmin(-1)U(-1), respectively. The highest pesticide transformation rates were obtained with a mediator-substrate proportion of 5:1, one of the lowest reported so far for the laccase-mediator systems. The analysis of the main product from the dichlorophen transformation showed that an oxidative dehalogenation is involved in the catalytic mechanism. Adduct formation between the mediator syringaldehyde and the pesticides dichlorophen or bromoxynil was also found after enzymatic oxidation. The main goal of this work is to evaluate environmental-friendly mediators for the pesticide transformation, and the potential of laccase-mediator system to efficiently reduce the environmental impact of organic halogenated pesticides is discussed.