Biological effects of cerium oxide nanoparticles. Implications at the Bio-Nano interface

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 15-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 15-03-2019This work has been possible due to the “Formación de Profesorado Universitario” fellowship (FPU 2012 program) of Ministerio de Educación, Gobierno de España (Ref: FPU12/01796), the research projects: CTM2013-45775-C2-1-R and CTM2013-45775-C2-2-R grants from MINECO and the Dirección General de Universidades e Investigación de la Comunidad de Madrid Research Network S2013/MAE-2716

Nanoplastic toxicity towards freshwater organisms

The fragmentation of plastic litter into smaller fragments, known as microplastics and nanoplastics, as well as their toxicity and environmental distribution have become issues of high concern. Furthermore, the popularization of bioplastics as a greener substitute of conventional plastics represents a challenge for the scientific community in view of the limited information concerning their potential environmental impact. Here, we systematically review the recent knowledge on the environmental fate and toxicity of nanoplastics in freshwater environments, discuss the results obtained thus far, and identify several knowledge gaps. The sources and environmental behaviors of nanoplastics are presented considering in vitro, in vivo, and in silico studies with a focus on real exposure scenarios. Their effects on organisms are classified based on their impact on primary producers, primary consumers, and secondary consumers. This review covers the main results published in the last four years, including all relevant experimental details and highlighting the most sensitive toxicity endpoints assessed in every study. We also include more recent results on the potential environmental impact of biodegradable plastics, a type of material belonging to the category of bioplastics for which there are still scarce data. This review identifies a need to perform studies using secondary nanoplastics rather than synthetic commercial materials as well as to include other polymers apart from polystyrene. There is also an urgent need to assess the possible risk of nanoplastics at environmentally realistic concentrations using sublethal endpoints and long-term assay

Luminescent microbial bioassays and microalgal biosensors as tools for environmental toxicity evaluation

This chapter deals with toxicity bioassays and biosensors based on luminescent microorganisms that report on global toxicity of a sample in such a way that luminescence is reduced or inhibited in the presence of toxic compounds that impair metabolism. Both natural as well as recombinant microorganisms are considered. A detailed description of their main characteristics and environmental applications is reported. A few examples of bioassays for detecting oxidative stress (both bioluminescent and fluorescent bioreporters) are also mentioned and discussed as reactive oxygen species (ROS) formation and subsequent oxidative stress if the antioxidant defenses of the cells are surpassed is one of the main mechanisms of toxicity for most pollutants. There is also a section dedicated to microalgal–based biosensors given their ecological relevance as primary producers, their easiness of culture and immobilization in different matrices, ability to acclimate to low nutrients conditions and ubiquity in aquatic environments. The most used toxicity endpoints for this type of biosensors are the alteration of photosynthetic activity (optical and amperometric biosensors) and the inhibition of enzymes such as alkaline phosphatase (APA) or sterases (mostly conductometric biosensors). The main information is shown in tables that include the microorganisms, their main characteristics (reporter gene systems, transducer types for biosensors) and their main environmental applications as well as relevant references. Although some of these bioassays have already been standardized by different international organizations, there are still many which are also promising tools for environmental global toxicity evaluation and should be fully validated and standardized for regulatory purposesThis research was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grant CTM2016-74927-C2-2-

Physicochemical and biological interactions between cerium oxide nanoparticles and a 1,8-naphthalimide derivative

This Accepted Manuscript is available for reuse under a CC BY-NC-ND licence after the 24 month embargo period provided that all the terms of the licence are adhered toCerium (Ce) oxide nanoparticles (CNPs) have attracted attention due to their high bioactivity and unique redox-chemistry. The oxygen vacancies at the surface of the nanoparticle explain the autocatalytic properties of CNPs in which the Ce3+ atoms occupy the center of the oxygen vacancies surrounded by Ce4+ atoms. Until now, CNPs have been associated with organic molecules at the synthesis stage to extend their applications or improve their stability. However, there is a lack of information regarding the post-synthesis interaction of CNPs and organic molecules that could enhance or induce new properties. Due to their unique optical properties and their many uses in different areas such as supramolecular chemistry or biomedicine, we have chosen a derivative from the family of naphthalimides (the 4-amino-1,8-naphthalimide-N-substituted; ANN) to study the interaction with different CNPs (CNP1-4) and their joint bioactivity compared to that of the same compounds alone. ANN-CNP complexes were formed as revealed by spectroscopic studies, but, the interaction was markedly different depending on the physicochemical properties of CNPs and their surface content of Ce3+ sites. The ANN adsorption on all CNPs involved the amino group in the naphthalene moiety as shown by NMR spectroscopy, while the pyrrolidine ring was mainly involved in the specific interaction between ANN and CNP1. The biological effect of each CNP and ANN individually and forming complexes was assessed using a bioluminescent model bacterium. The results showed that ANN and CNP with the higher content of surface Ce3+ (CNP1) when combined acted additively towards the used model organism. In the opposite, ANN-CNP2, ANN-CNP3 and ANN-CNP4 complexes were antagonistic when the nanoparticles dominated the mixture. The results of this study contribute to expand the knowledge of the interaction between nanoparticles and organic molecules which may be useful for understanding the behavior of nanoparticles in complex matricesThis research was supported by CTM2013-45775-C2-1-R and CTM2013-45775-C2-2-R grants from MINECO. Cerium oxide nanoparticles were kindly provided by S. Seal and S. Das (University of Central Florida, U.S.A). Gerardo Pulido-Reyes thanks the Spanish Ministry of Education for the award of an FPU grant (FPU12/01796

Toxicity of superparamagnetic iron oxide nanoparticles to the microalga Chlamydomonas reinhardtii

Superparamagnetic iron oxide nanoparticles (SPION) have been widely studied for different biomedical and environmental applications. In this study we evaluated the toxicity and potential alterations of relevant physiological parameters caused to the microalga Chlamydomonas reinhardtii (C. reinhardtii) upon exposure to SPION. The results showed dose-dependent toxicity. A mechanistic study combining flow cytometry and physiological endpoints showed a toxic response consisting of a decrease in metabolic activity, increased oxidative stress and alterations in the mitochondrial membrane potential. Additionally, and due to the light absorption of SPION suspensions, we observed a significant shading effect, causing a marked decrease in photosynthetic activity. In this work, we demonstrated for the first time, the internalization of SPION by endocytosis in C. reinhardtii. These results demonstrated that SPION pose a potential risk for the environment if not managed properlyThis research is supported by CTM2013-45775-C2-1/2-R, CTM2016-74927-C2-1,2-R and MAT2015-71806-R grants from MINEC

Secondary nanoplastics released from a biodegradable microplastic severely impact freshwater environments

This article is part of the themed collection: Environmental Science: Nano Recent HOT Articles https://pubs.rsc.org/en/journals/articlecollectionlanding?sercode=en&themeid=280a8 9ca-3eed-4abe-ae65-c856206f6c3c Article selected by the Editors in Chief of Environmental Science journals: DOI: 10.1039/D0EM90014A (Editorial)Over the last five decades, plastics production has increased as a consequence of their use in strategic sectors causing damage on aquatic ecosystems. In this context, biodegradable plastics have emerged as an ecological alternative because they are easily degradable in the environment. Despite the recent advances in the field of plastic ecotoxicology, the ecological impact of secondary nanoplastics (nanoplastics resulting from natural degradation of micro and macro plastics) in the environment remains poorly understood. Here, we have investigated the effects of secondary nanoplastics of polyhydroxybutyrate (PHB), a biodegradable plastic, on three representative organisms of aquatic ecosystems. Secondary PHB-nanoplastics were produced from PHB-microplastics by abiotic degradation under environmentally representative conditions. Secondary PHB-nanoplastics induced a significant decrease in cellular growth and altered relevant physiological parameters in all organisms. We investigated whether the observed toxicity was exerted by PHB-nanoplastics themselves or by other abiotic degradation products released from PHB-microplastics. An experiment was run in which PHB-nanoplastics were removed by ultrafiltration; the resulting supernatant was not toxic to the organisms, ruling out the presence of toxic chemicals in the PHB-microplastics. In addition, we have performed a complete physicochemical characterization confirming the presence of secondary PHB-nanoplastics in the 75-200 nm range. All results put together indicated that secondary PHB-nanoplastics released as a consequence of abiotic degradation of PHB-microplastics were harmful for the tested organisms, suggesting that biodegradable plastic does not mean safe for the environment in the case of PH

Untangling the biological effects of cerium oxide nanoparticles: The role of surface valence states

Cerium oxide nanoparticles (nanoceria; CNPs) have been found to have both pro-oxidant and antioxidant effects on different cell systems or organisms. In order to untangle the mechanisms which underlie the biological activity of nanoceria, we have studied the effect of five different CNPs on a model relevant aquatic microorganism. Neither shape, concentration, synthesis method, surface charge (ζ-potential), nor nominal size had any influence in the observed biological activity. The main driver of toxicity was found to be the percentage of surface content of Ce3+ sites: CNP1 (58%) and CNP5 (40%) were found to be toxic whereas CNP2 (28%), CNP3 (36%) and CNP4 (26%) were found to be non-toxic. The colloidal stability and redox chemistry of the most and least toxic CNPs, CNP1 and CNP2, respectively, were modified by incubation with iron and phosphate buffers. Blocking surface Ce3+ sites of the most toxic CNP, CNP1, with phosphate treatment reverted toxicity and stimulated growth. Colloidal destabilization with Fe treatment only increased toxicity of CNP1. The results of this study are relevant in the understanding of the main drivers of biological activity of nanoceria and to define global descriptors of engineered nanoparticles (ENPs) bioactivity which may be useful in safer-by-design strategies of nanomaterialsThis research was supported by CTM2013-45775-C2-1-R and CTM2013-45775-C2-2-R grants from MINECO, the Dirección General de Universidades e Investigación de la Comunidad de Madrid, Research Network S2013/MAE-2716 and National Science Foundation for Nanotechnology Research (EECS – 0901503, CBET-1261956). Gerardo Pulido-Reyes thanks the Spanish Ministry of Education for the award of an FPU grant

Reverse Trojan-horse effect decreased wastewater toxicity in the presence of inorganic nanoparticles

We studied the toxicological interaction of 46 micropollutants from a biologically treated wastewater effluent in mixtures with silica, amine-modified silica, titanium dioxide and magnetite nanoparticles. The pollutants tracked in this work were polar pharmaceuticals belonging to different therapeutic groups, some of their metabolites and artificial sweeteners, the concentrations of which were mostly in the tens to hundreds of ng L-1 range. The results showed particularly high adsorption for furosemide, gemfibrozil and the aminopyrine metabolite 4FAA. There was preferential adsorption of the less polar compounds on the less polar nanoparticles. The total amounts of compounds adsorbed and quantified were 13.4, 4.8, 10.8 and 7.1 μg g-1 for SiO2, SiO2-NH2, TiO2 and Fe3O4, respectively. The toxicity of wastewater-nanoparticle mixtures was assessed using the bioluminescent cyanobacterium Anabaena sp. PCC 7120 CPB4337. The interactions were quantified by means of the combination index (CI)-isobologram method. The binary mixtures of wastewater with SiO2, SiO2-NH2 and TiO2 displayed antagonism for the lower affected fractions, which corresponded to the lower concentrations. For higher effects and for the Fe3O4 nanoparticles over the whole tested range, the mixtures were additive leading to synergism for the higher affected fractions. No internalization was observed. The results showed that the reduced toxicant bioavailability due to the interaction with nanoparticles is relevant for micropollutants at environmental concentrations. The amount of anthropogenic pollutants retained by metal oxide nanoparticles has significant toxicological effectsFinancial support was provided by FP7-ERA-Net Susfood, 2014/00153/001, the Spanish Ministry of Economy, CTM2013-45775-C2-1-R and CTM2013-45775-C2-2-R and the Dirección General de Universidades e Investigación de la Comunidad de Madrid, Network S2013/MAE-271

Internalization and toxicological mechanisms of uncoated and PVP-coated cerium oxide nanoparticles in the freshwater alga Chlamydomonas reinhardtii

Due to the wide range of applications of cerium oxide nanoparticles (CeO2NPs), a risk assessment of their biological effects using environmentally relevant species becomes highly important. There are contradictory reports on the effects of CeO2NPs, which may be related to the use of different types of nanoparticles (NPs) and coatings. CeO2NPs may act as an oxidant causing toxicity or as an antioxidant able to scavenge free radicals. As a consequence of such complexity, the toxicological behaviour of these NPs is still poorly understood. Moreover, little is known about the internalization process of CeO2NPs in algae. There is evidence of CeO2NP-internalization by the green alga Chlamydomonas reinhardtii, but the mechanism and route of uptake are still unknown. In this study, we used uncoated and different polyvinylpyrrolidone (PVP)-coated CeO2NPs with the aim of identifying their toxicological mechanisms to C. reinhardtii and exploring their possible internalization. Our results showed that PVP coated-CeO2NPs significantly increased the formation of reactive oxygen species in exposed cells, indicating that oxidative stress is an important toxicity mechanism for these particles. Direct contact and damage of the cellular membrane was identified as the mechanism causing the toxicity of uncoated NPs. From experiments with endocytosis inhibitors, clathrin-dependent endocytosis was revealed as the main internalization route for all NPs. However, as uncoated CeO2NPs led to severe cellular membrane damage, direct passage of NPs through membrane holes could not be discarded. To our knowledge, this is the first report with evidence of direct linking between NP internalization and a specific endocytic pathway. The results presented here will help to unravel the toxicological mechanism and behaviour of CeO2NPs and provide input information for the environmental health and safety assessment of CeO2NPs.This research was supported by CTM2013-45775-C2-1,2-R and CTM2016-74927-C2-1,2-R grants from MINECO. NanoMILE (Grant Agreement no 310451 to EVJ & SMB) and the Endeavour Scholarship Scheme (Group B) (to SMB) are acknowledged.Peer reviewe

Understanding nanoplastic toxicity and their interaction with engineered cationic nanopolymers in microalgae by physiological and proteomic approaches

The amount of plastics produced per year is in constant growth alongside their use in different sectors like the textile industry, agriculture or, more recently, in nanotechnology. Under certain environmental conditions, plastics break down into smaller pieces. Those plastics in the nanosize range are the most difficult to identify, quantify and remove and therefore probably prevail in aquatic ecosystems. Likewise, nanomaterial production has been increasing exponentially and therefore their potential release to the environment poses a threat. There is a lack of knowledge regarding the combined effects of co-occurring nanopolymers on biota. In this work, we have studied the individual toxicity of polystyrene nanoplastics (PS-NPs) as well as their combined effect with generation 7 PAMAM dendrimers (G7) on the filamentous cyanobacterium Anabaena sp. PCC7120, a relevant aquatic primary producer. Exposure to PS-NPs induced the overproduction of reactive oxygen species, lipid peroxidation, membrane disruptions, intracellular acidification and a decrease in photosynthetic activity. Internalization of the nanoplastics was also observed. Combined exposure to PS-NPs and G7 lowered PS-NP toxicity and precluded their internalization. This antagonistic interaction was due to the formation of heteroaggregates. Molecular biomarkers (differentially expressed proteins, DEPs) of the toxic effect of nanoplastics, G7 and their binary mixture were identified for the first time. These molecular biomarkers may be envisaged as a molecular signature of the toxic effect of the nanopolymers and could be predictors of cellular damage caused by exposure to nanopolymer