Cross-species comparisons of nanoparticle interactions with innate immune systems: a methodological review

Many components of the innate immune system are evolutionarily conserved and shared across many living organisms, from plants and invertebrates to humans. Therefore, these shared features can allow the comparative study of potentially dangerous substances, such as engineered nanoparticles (NPs). However, differences of methodology and procedure between diverse species and models make comparison of innate immune responses to NPs between organisms difficult in many cases. To this aim, this review provides an overview of suitable methods and assays that can be used to measure NP immune interactions across species in a multidisciplinary approach. The first part of this review describes the main innate immune defense characteristics of the selected models that can be associated to NPs exposure. In the second part, the different modes of exposure to NPs across models (considering isolated cells or whole organisms) and the main endpoints measured are discussed. In this synergistic perspective, we provide an overview of the current state of important cross-disciplinary immunological models to study NP-immune interactions and identify future research needs. As such, this paper could be used as a methodological reference point for future nano-immunosafety studie

Immunotoxicity of metal nanoparticles in Eisenia andrei earthworms

En los últimos años, la producción de nanopartículas (NPs) ha aumentado dando lugar a que su liberación al medio ambiente haya incrementado. Tanto la liberación ambiental como su desecho se está convirtiendo en una preocupación emergente ya que las NPs alcanzarían los ecosistemas terrestres como acuáticos, presentando problemas de toxicidad para los organismos vivos. En los ecosistemas terrestres, las lombrices de tierra Eisenia andrei son invertebrados típicamente utilizados como organismos modelo en estudios de ecotoxicidad con un sistema inmunológico innato que protege a los animales contra contaminantes, bacterias, hongos y virus que se encuentran en el suelo. El objetivo general de esta tesis doctoral fue evaluar los efectos tóxicos de las NPs de TiO2, las NPs de CuO y las NPs de Ag2S en E. andrei y estudiar la interacción de las NPs con las células inmunocompetentes de E. andrei llamadas celomocitos mediante ensayos in vitro e in vivo. Los puntos finales evaluados durante la tesis doctoral fueron el estrés oxidativo (producción de ROS, de NO y de MDA), la genotoxicidad, los biomarcadores inmunitarios (actividad feneloxidasa, actividad fagocítica) y los niveles de ARN mensajero (ARNm). Los experimentos de toxicidad de las NPs de TiO2 y las NPs de CuO fueron realizadas in vitro con celomocitos de E. andrei a 1, 10 y 100 µg mL-1, a las 2, 6 y 24 h de exposición. Los experimentos realizados con las NPs de Ag2S se llevaron a cabo in vivo donde 180 lombrices de tierra E. andrei fueron expuestas a 5 mg Ag Kg-1 de suelo de las NPs de Ag2S durante 14 días siguiendo la directriz OECD 207. La caracterización de las NPs se realizó mediante la dispersión de luz de difracción de múltiples ángulos (MADLS), microscopio electrónico de transmisión (TEM) y de barrido (SEM). Además, el TEM y SEM se utilizaron en los análisis in vitro para localizar las NPs de TiO2 y las NPs de CuO en los celomocitos. Los resultados de las pruebas in vitro mostraron que la disponibilidad de las NPs de TiO2 y las NPs de CuO difería entre ellas dentro del medio de cultivo celular. Las NPs de TiO2 fueron más estables y tendieron a precipitar a las 24 h, mientras que las NPs de CuO fueron más inestables. A través del TEM, se observó que los celomocitos fueron capaces de engullir las NPs de CuO. Las NPs de TiO2 y Ag2S no produjeron tantos daños a los celomocitos como las NPs de CuO en ninguno de los puntos finales mencionados anteriormente. En conclusión, se demostró que las NPs de TiO2 y NPs de CuO y las NPs de Ag2S pueden afectar a las lombrices de tierra. Se destacó que las células HAs y las células GAs fueron objetivos de las NPs, ya que pudieron engullir a las NPs. En cuanto al mecanismo de defensa de los celomocitos, se observó que los HAs y los GAs fueron capaces de aumentar las enzimas antioxidantes para disminuir el estrés oxidativo producido por la exposición con las NPs.In recent years, the production of nanoparticles (NPs) has increased exponentially, leading to an increase in their release into the environment. The environmental release of NPs and their disposal is becoming an emerging concern since they can reach both terrestrial and aquatic ecosystems, producing toxicity problems for living organisms. In terrestrial ecosystems, Eisenia andrei earthworms are invertebrates typically used as model organisms in ecotoxicity studies that possess an innate immune system that protects animals against contaminants, bacteria, fungi, and viruses that can be found in the soil. The general objective of this doctoral thesis was to evaluate the toxic effects of TiO2 NPs, CuO NPs and Ag2S NPs in E. andrei and to study the interaction of NPs with immunocompetent cells of E. andrei called coelomocytes by means of assays in vitro and in vivo. The endpoints evaluated during the doctoral thesis were oxidative stress (ROS, NO and MDA production), genotoxicity, immune biomarkers (phenoloxidase activity, phagocytic activity) and messenger RNA (mRNA) levels. The toxicity experiments of the TiO2 NPs and the CuO NPs were carried out in vitro with E. andrei coelomocytes at 1, 10 and 100 µg mL-1 and at 2, 6 and 24 h of exposure. The experiments carried out with the Ag2S NPs were carried out in vivo where 180 E. andrei earthworms were exposed to 5 mg Ag Kg-1 of the soil of the Ag2S NPs for 14 days following the OECD 207 guideline. The characterization of NPs was performed using multi-angle diffraction light scattering (MADLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, TEM and SEM were used in in vitro analyses to localize TiO2 NPs and CuO NPs in coelomocytes. The results of the in vitro tests showed that the availability of TiO2 NPs and CuO NPs differed from each other within the cell culture medium. TiO2 NPs were more stable and tended to precipitate after 24 h, while CuO NPs were more unstable. Through TEM, it was observed that coelomocytes were able to engulf CuO NPs. TiO2 NPs and Ag2S NPs did not cause as much damage to coelomocytes as CuO NPs at any of the endpoints mentioned above. In conclusion, it was shown that TiO2 NPs and CuO NPs and Ag2S NPs can affect earthworms. It was highlighted that HAs and GAs were key targets of NPs, as they were able to engulf NPs. Regarding the defence mechanism of coelomocytes, it was observed that HAs and GAs were able to increase antioxidant enzymes to reduce the oxidative stress produced by exposure to NPs

In Vitro Study of the Toxicity Mechanisms of Nanoscale Zero-Valent Iron (nZVI) and Released Iron Ions Using Earthworm Cells

During the last two decades, nanomaterials based on nanoscale zero-valent iron (nZVI) have ranked among the most utilized remediation technologies for soil and groundwater cleanup. The high reduction capacity of elemental iron (Fe0) allows for the rapid and cost-efficient degradation or transformation of many organic and inorganic pollutants. Although worldwide real and pilot applications show promising results, the effects of nZVI on exposed living organisms are still not well explored. The majority of the recent studies examined toxicity to microbes and to a lesser extent to other organisms that could also be exposed to nZVI via nanoremediation applications. In this work, a novel approach using amoebocytes, the immune effector cells of the earthworm Eisenia andrei, was applied to study the toxicity mechanisms of nZVI. The toxicity of the dissolved iron released during exposure was studied to evaluate the effect of nZVI aging with regard to toxicity and to assess the true environmental risks. The impact of nZVI and associated iron ions was studied in vitro on the subcellular level using different toxicological approaches, such as short-term immunological responses and oxidative stress. The results revealed an increase in reactive oxygen species production following nZVI exposure, as well as a dose-dependent increase in lipid peroxidation. Programmed cell death (apoptosis) and necrosis were detected upon exposure to ferric and ferrous ions, although no lethal effects were observed at environmentally relevant nZVI concentrations. The decreased phagocytic activity further confirmed sublethal adverse effects, even after short-term exposure to ferric and ferrous iron. Detection of sublethal effects, including changes in oxidative stress-related markers such as reactive oxygen species and malondialdehyde production revealed that nZVI had minimal impacts on exposed earthworm cells. In comparison to other works, this study provides more details regarding the effects of the individual iron forms associated with nZVI aging and the cell toxicity effects on the specific earthworms’ immune cells that represent a suitable model for nanomaterial testing