Periodic Reporting for period 1 - STARNANO (Spheroids as a Tool to Assess Realistic long term effects of mixtures of nanomaterials and chemicals)
Reporting period: 2018-02-01 to 2020-01-31
Among the many nanomaterials (NMs), graphene-related materials (GRM) have become a subject of intensive scientific and industrial interest. The exceptional mechanical, electrical, thermal and optical properties of these materials make them tremendously attractive for a multitude of different applications. To take advantage of these promising materials without hindering their innovation processes, specific safety issues for human health and the environment have to be addressed. GRM are inevitably released into the environment where aquatic systems tend to be their ultimate sink. Because of their particular physico-chemical properties, GRM are likely to interact with existing environmental contaminants. This may lead to remarkable changes in their fate and therefore bioavailability and impact on biota. Adverse effects of some NMs on aquatic organisms, in particular fish, have already been reported. Although conventional in vivo tests have been routinely used to assess the potential risks of NMs to fish, European regulatory bodies encourage and favor the use of alternative methods for obtaining this kind of data. Isolated rainbow trout hepatocytes maintained in 3D cellular structure mimic the tissue/organ environment, thus represent a more powerful tool to investigate the in vivo effects of NMs. The main objective of the present project was to assess the combined effects of NMs and environmentally relevant pre-existing contaminants. To achieve this goal, two specific objectives were delineated:
Specific Objective 1. To determine the mechanisms of toxic action of individual NMs or chemicals
Specific Objective 2. Determination of the effect of co-exposure of one NM and an organic substance on their cytotoxicity
Specific Objective 1. To determine the mechanisms of toxic action of individual NMs or chemicals
Specific Objective 2. Determination of the effect of co-exposure of one NM and an organic substance on their cytotoxicity
To understand the variables influencing the effect of the NM-organic chemical combinations, first it was necessary to study and characterize the toxicity of single substances. Three GRM, including two sheet-like graphene oxides and a tubular-shaped carbon nanofiber were chosen for the study. Benzo(k)fluoranthene (BkF), a polycyclic aromatic hydrocarbon that is able to activate the aryl hydrocarbon receptor was selected as a representative environmental organic toxicant.
Spheroids were exposed to ranges of concentrations of GRM or organic toxicant separately. Cytotoxicity and enzymes related to detoxification processes (including those related with cytochrome P4501A, CYP1A) were measured to assess the individual effects of GRM and chemicals. BkF exposure activated the detoxification mechanisms in spheroids, however none of the GRM alone induced the CYP1A system at the enzymatic or transcriptional levels. Electron microscopic techniques were applied to evaluate the intracellular fate and any morphological alterations caused by these NMs. Because GRM if any, were localized only in a few cells at the periphery of the spheroids at the tested concentrations and apparently the inner part of the spheroid structure was intact, to simulate possible mechanisms of toxicity taking place in the peripheral (single) layer, our study was extended to hepatocytes cultured in monolayers. Once the individual effects of GRM or BkF on cells had been identified, spheroids and monolayer cultures were exposed to the organic contaminant over a range of concentrations in the presence or absence of GRM. After exposure to BkF, the exposure to a particular graphene oxide provoked a significant and additional increase of CYP1A related detoxification activities, with respect to the induction provoked by BkF alone. These findings suggest that detoxification mechanisms induced by BkF could be intensified by the presence of this graphene oxide and cells are able to defend themselves against the NM.
Spheroids were exposed to ranges of concentrations of GRM or organic toxicant separately. Cytotoxicity and enzymes related to detoxification processes (including those related with cytochrome P4501A, CYP1A) were measured to assess the individual effects of GRM and chemicals. BkF exposure activated the detoxification mechanisms in spheroids, however none of the GRM alone induced the CYP1A system at the enzymatic or transcriptional levels. Electron microscopic techniques were applied to evaluate the intracellular fate and any morphological alterations caused by these NMs. Because GRM if any, were localized only in a few cells at the periphery of the spheroids at the tested concentrations and apparently the inner part of the spheroid structure was intact, to simulate possible mechanisms of toxicity taking place in the peripheral (single) layer, our study was extended to hepatocytes cultured in monolayers. Once the individual effects of GRM or BkF on cells had been identified, spheroids and monolayer cultures were exposed to the organic contaminant over a range of concentrations in the presence or absence of GRM. After exposure to BkF, the exposure to a particular graphene oxide provoked a significant and additional increase of CYP1A related detoxification activities, with respect to the induction provoked by BkF alone. These findings suggest that detoxification mechanisms induced by BkF could be intensified by the presence of this graphene oxide and cells are able to defend themselves against the NM.
In Europe, the production, use and disposal of NMs and hence of GRMs is regulated by a number of directives and regulations that apply to particular kinds of products. This is, for instance, the case of regulations for biocides, pesticides, cosmetics, veterinary products and pharmaceuticals. In addition, the REACH regulation concerning the registration, evaluation, authorization and restriction of chemicals (regulation EC 1907/2006) covers most of chemicals manufactured and imported to Europe with the aim to protect human health and the environment, whilst also enhancing industrial competitiveness. This regulation has been modified in the last years with the aim to also cover NMs. Therefore, it has profound implications at the economic, societal and scientific levels.
Once released into the environment, GRM due to their unique physicochemical properties, are likely to interact with other simultaneously present chemicals. Nevertheless, current risk assessments for regulatory purposes mainly rely on the evaluation of the effects of individual chemicals and they do not take into account the mixture toxicities. The present project addresses this issue and the information generated will have an impact on the knowledge about effects of GRM and about the combined NM-chemical effects on the organisms, which is crucial in the development of risk assessment approaches that contemplate the specificities of this kind of substances.
In addition, the generated information will facilitate the regulation and assessment processes of GRMs that are currently being produced or imported in Europe. This is strongly related with a positive economic impact of the project. At the same time, this project can have an important social impact by contributing to generate collective awareness about benefits and hazards of nanomaterials, helping therefore to an appropriate use of these substances.
Nowadays, there is an increasing social demand for the application of the 3Rs principle, replacement, reduction, refinement, in science and toxicology. As a reflection of this, the REACH regulation also encourages the use of alternative methods for obtaining chemical toxicity data. Therefore, this project also has a direct scientific and social impact related with the potentiation of the use of in vitro systems. 3D in vitro systems could generate reliable information about toxicity and mechanisms of toxic action of substances. Taking into account that 3D in vitro systems could be a good reflection of in vivo situations, the generated information could be evaluated in order to be directly applied in the risk assessment of NMs by regulatory bodies.
Once released into the environment, GRM due to their unique physicochemical properties, are likely to interact with other simultaneously present chemicals. Nevertheless, current risk assessments for regulatory purposes mainly rely on the evaluation of the effects of individual chemicals and they do not take into account the mixture toxicities. The present project addresses this issue and the information generated will have an impact on the knowledge about effects of GRM and about the combined NM-chemical effects on the organisms, which is crucial in the development of risk assessment approaches that contemplate the specificities of this kind of substances.
In addition, the generated information will facilitate the regulation and assessment processes of GRMs that are currently being produced or imported in Europe. This is strongly related with a positive economic impact of the project. At the same time, this project can have an important social impact by contributing to generate collective awareness about benefits and hazards of nanomaterials, helping therefore to an appropriate use of these substances.
Nowadays, there is an increasing social demand for the application of the 3Rs principle, replacement, reduction, refinement, in science and toxicology. As a reflection of this, the REACH regulation also encourages the use of alternative methods for obtaining chemical toxicity data. Therefore, this project also has a direct scientific and social impact related with the potentiation of the use of in vitro systems. 3D in vitro systems could generate reliable information about toxicity and mechanisms of toxic action of substances. Taking into account that 3D in vitro systems could be a good reflection of in vivo situations, the generated information could be evaluated in order to be directly applied in the risk assessment of NMs by regulatory bodies.