Physiological and histopathological alterations in male Swiss mice after exposure to titanium dioxide (anatase) and zinc oxide nanoparticles and their binary mixture.

Existing studies have shown the systemic damage of titanium dioxide (TiO2) or zinc oxide (ZnO) nanoparticles (NPs), but there is little or no existing knowledge on the potential adverse toxic effects of the mixture of the two. In order to investigate the in vivo toxic effect of the mixture of TiO2 NPs and ZnO NPs, the acute toxicities of TiO2 NPs, ZnO NPs by themselves, and their mixture (1:1) were determined. The systemic toxicities of the individual NPs and mixture were evaluated in mice using hematological indices, hepatic, renal, and lipid profile parameters, and histopathology as endpoints. NPs were intraperitoneally administered at doses of 9.38, 18.75, 37.50, 75.00, and 150.00 mg/kg bw each. Individual NPs and their mixture were administered daily for 5 and 10 d, respectively. The LD50 of ZnO NPs was 299.9 mg/kg while TiO2 NPs by themselves or TiO2 NPs + ZnO NPs were indeterminate due to the absence of mortality of the male mice treated. TiO2 NPs, ZnO NPs by themselves and TiO2 NPs + ZnO NPs induced significant alterations in the hematological and biochemical parameters, with higher toxicity at 10 d. Histopathological lesions were observed in the liver, kidneys, spleen, heart, and brain of mice treated with the individual NPs and their mixture. TiO2 NPs + ZnO NPs were able to induce a higher systemic toxicity than TiO2 NPs or ZnO NPs individually. Our data suggest that more comprehensive risk assessments should be carried out on the mixture of NPs before utilization in consumer products.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Sub-chronic effects of AgNPs and AuNPs on Gammarus fossarum (Crustacea Amphipoda): From molecular to behavioural responses.

The aim of the present study was the assessment of the sub-chronic effects of silver (AgNPs) and gold nanoparticles (AuNPs) of 40 nm primary size either stabilised with citrate (CIT) or coated with polyethylene glycol (PEG) on the freshwater invertebrate Gammarus fossarum. Silver nitrate (AgNO3) was used as a positive control in order to study the contribution of silver ions potentially released from AgNPs on the observed effects. A multibiomarker approach was used to assess the long-term effects of AgNPs and AuNPs 40 nm on molecular, cellular, physiological and behavioural responses of G. fossarum. Specimen of G. fossarum were exposed for 15 days to 0.5 and 5 µgL-1 of CIT and PEG AgNPs and AuNPs 40 nm in the presence of food. A significant uptake of both Ag and Au was observed in exposed animals but was under the toxic threshold leading to mortality of G. fossarum. Silver nanoparticles (CIT-AgNPs and PEG-AgNPs 40 nm) led to an up-regulation of Na+K+ATPase gene expression. An up-regulation of Catalse and Chitinase gene expressions due to exposure to PEG-AgNPs 40 nm was also observed. Gold nanoparticles (CIT and PEG-AuNPs 40 nm) led to an increase of CuZnSOD gene expression. Furthermore, both AgNPs and AuNPs led to a more developed digestive lysosomal system indicating a general stress response in G. fossarum. Both AgNPs and AuNPs 40 nm significantly affected locomotor activity of G. fossarum while no effects were observed on haemolymphatic ions and ventilation.

The Food Matrix and the Gastrointestinal Fluids Alter the Features of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are used in the agri-food sector, which can lead to their ingestion. Their interaction with food and their passage through the gastrointestinal tract can alter their properties and influence their fate upon ingestion. Therefore, this study aims at developing an in vitro method to follow the fate of AgNPs in the gastrointestinal tract. After incorporation of AgNPs into a standardized food matrix, a precolonic digestion is simulated and AgNPs are characterized by different techniques. The presence of food influences the AgNPs properties by forming a corona around nanoparticles. Even if the salivary step does not impact significantly the AgNPs, the pH decrease and the digestive enzymes induce the agglomeration of AgNPs during the gastric phase, while the addition of intestinal fluids disintegrates these clusters. AgNPs can thus reach the intestinal cells under nanometric form, although the presence of food and gastrointestinal fluids modifies their properties compared to pristine AgNPs. They can form a corona around the nanoparticles and act as colloidal stabilizer, which can impact the interaction of AgNPs with intestinal epithelium. This study demonstrates the importance of taking the fate of AgNPs in the gastrointestinal tract into account to perform an accurate risk assessment of nanomaterials.

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

Pro-inflammatory effects of crystalline- and nano-sized non-crystalline silica particles in a 3D alveolar model.

BACKGROUND: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression. RESULTS: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1ß (IL-1ß) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments. CONCLUSIONS: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/ß released from the apical epithelial cells and macrophages.

Redox metabolism modulation as a mechanism in SSRI toxicity and pharmacological effects.

Depressive disorders are amongst the greatest mental health challenges, with an increasing number of patients being diagnosed each year. Though it has not yet been fully elucidated, redox metabolism imbalances and oxidative stress seem to play a major role in the pathogenesis of depressive disorders. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressants, considered to have a better tolerability. However, several adverse effects have been reported and the mechanisms involved in their pharmacological activity are not entirely understood. SSRIs have been shown to influence the redox metabolism, which could be involved in their toxicity and pharmacological effects. A comparative analysis of published in vivo and in vitro data regarding the activity of SSRIs on the redox metabolism pathways has been performed in this paper, with an emphasis on mechanistical aspects. Furthermore, a comparison between oxidative stress biomarker levels reported by different studies was attempted. The reviewed data point towards both pro- and antioxidant effects of SSRIs, dependent on tissue/cell type and dose/concentration, suggest a redox modulating potential of these compounds. In hepatic and testicular tissue, the majority of reviewed studies reported pro-oxidant effects, with possible implications towards the hepatotoxicity and sexual dysfunction that were reported following SSRI treatment; while in brain, the most common findings were antioxidant effects that could partially explain their antidepressant activity. However, given the heterogeneity of the reviewed data, further research is needed to fully understand the impact of SSRIs on redox metabolism and its implications.

Alteration of sperm parameters and reproductive hormones in Swiss mice via oxidative stress after co-exposure to titanium dioxide and zinc oxide nanoparticles.

In this study, Swiss male mice were intraperitoneally administered with titanium dioxide (TiO2 ) and zinc oxide (ZnO) nanoparticles (NPs) and their mixture (1:1) at doses between 9.38 and 75 mg/kg for 5 weeks to evaluate reproductive toxicity. Both NPs and their mixture significantly (p < .001) altered sperm motility, reduced sperm numbers and increased abnormalities, while their mixture induced more sperm abnormalities than either TiO2 NPs or ZnO NPs. Both NPs and their mixture significantly (p < .05) reduced the LH level, while ZnO NPs alone and their mixture (p < .001) increased the testosterone levels at tested doses. The testes of exposed mice showed pathological changes and altered histomorphometrics. TiO2 NPs and ZnO NPs individually induced a significant (p < .01) reduction in SOD and CAT activities, while the mixture significantly (p < .001) decreased CAT activity and increased SOD activity. TiO2 NPs alone at 9.38 mg/kg induced a significant (p < .001) reduction in the GSH level, while both NPs and their mixture increased the MDA level significantly (p < .05). The data showed that the mixture had a synergistic interaction to induce testicular damage. Overall, oxidative stress may be involved in the NP-mediated testicular damage observed.

In vitro exposure of a 3D-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires.

BACKGROUND: The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 µm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 µg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated. RESULTS: Significant cytotoxic effect was observed for all three types of AgNMs at the highest tested doses. The increased mRNA levels of the pro-apoptotic gene CASP7 suggests that apoptosis may occur after exposure to AgNWs. All three types of AgNMs increased the mRNA level of the anti-oxidant enzyme HMOX-1 and of the metal-binding anti-oxidant metallothioneins (MTs), with AgNWs being the most potent inducer. Even though all types of AgNMs induced the nuclear translocation of NF-kB, only AgNWs increased the mRNA level of pro-inflammatory mediators. The pro-inflammatory response elicited by AgNWs was further confirmed by the increased secretion of the 10 evaluated interleukins. CONCLUSION: In the current study, we demonstrated that the direct exposure of a complex tetra-culture alveolar model to different types of AgNMs at ALI induces shape- and size-specific biological responses. From the three AgNMs tested, AgNWs were the most potent in inducing biological alterations. Starting from 50 ng/cm2, a dose representative for an acute exposure in a high exposure occupational setting, AgNWs induced prominent changes indicative for a pro-inflammatory response. Even though the acute responses towards a dose representative for a full-lifetime exposure were also evaluated, chronic exposure scenarios at low dose are still unquestionably needed to reveal the human health impact of AgNMs during realistic conditions.

Xenopus laevis as a Bioindicator of Endocrine Disruptors in the Region of Central Chile.

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s).

BACKGROUND: Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. RESULTS: Exposure-relevant concentrations of DEP (0.12 µg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 µg/cm2) induced low to moderate increases in IL-1α, IL-1ß, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. CONCLUSION: Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

Respiratory sensitization: toxicological point of view on the available assays.

Respiratory sensitization as a consequence of exposure to chemical products has increased over the last decades, leading to an increase of morbidity. The increased use of synthetic compounds resulted in an exponential growth of substances to which we are potentially exposed on a daily basis. Some of them are known to induce respiratory sensitization, meaning that they can trigger the development of allergies. In the past, animal studies provided useful results for the understanding of mechanisms involved in the development of respiratory allergies. However, the mechanistic understanding of the involved cellular effects is still limited. Currently, no in vitro or in vivo models are validated to identify chemical respiratory sensitizers. Nonetheless, chemical respiratory sensitizers elicit a positive response in validated assays for skin sensitization. In this review, we will discuss how these assays could be used for respiratory sensitization and if necessary, what can be learnt from these assays to develop a model to assess the respiratory sensitizing potential of chemicals. In the last decades, much work has been done to study the respiratory toxicity of inhaled compounds especially in developing in vitro assays grown at the air-liquid interface. We will discuss how possibly the tests currently used to investigate general particle toxicity could be transformed to investigate respiratory sensitization. In the present review, we describe the most known mechanism involved in the sensitization process and the experimental in vivo and alternative in vitro models, which are currently available and how to adapt and improve existing models to study respiratory sensitization.

Estrogenic and anti-estrogenic activity of butylparaben, butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate and their binary mixtures on two estrogen responsive cell lines (T47D-Kbluc, MCF-7).

The estrogenic and anti-estrogenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) were evaluated for individual compounds as well as for binary mixtures, using an estrogen-dependent reporter gene assay in T47D-Kbluc breast cancer cells and an estrogen-dependent proliferation assay in MCF-7 breast cancer cells. In terms of estrogenicity the potency of the selected compounds increased from BHA < PG < BuPB in the luciferase assay (with BHT showing no significant estrogenic activity), while in the proliferation assay the following order was observed: BHT < BHA < BuPB (with PG showing no significant estrogenic activity). Non-monotonic dose-response curves were obtained for BuPB (in both assays) and PG (in the luciferase assay), respectively. In the presence of estradiol, a significant anti-estrogenic activity was observed in both cell lines for PG, BuPB and BHA, while BHT showed weak anti-estrogenic activity only in T47D-Kbluc cells. The evaluation of binary mixtures confirmed the endocrine disruptive potential of the compounds, their individual potency being correlated with that of the mixtures. All mixtures were able to reduce the estradiol-induced luminescence or cell proliferation, an effect that was accurately predicted by the dose addition mathematical model, suggesting the same (or at least partially overlapping) modes of action for the tested compounds. The results of the present study emphasize the importance of a cumulative risk assessment of endocrine disruptors.

Endothelial responses of the alveolar barrier in vitro in a dose-controlled exposure to diesel exhaust particulate matter.

BACKGROUND: During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells. RESULTS: Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture. CONCLUSION: In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.

BACKGROUND: Interactions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. RESULTS: TiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 µg/ml) is due to blockage while a low dose (0.16 µg/ml) promoted autophagy. Cellular viability was not affected in either case. CONCLUSIONS: The uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.

Case study: Possible differences in phthalates exposure among the Czech, Hungarian, and Slovak populations identified based on the DEMOCOPHES pilot study results.

OBJECTIVE: Phthalates and their metabolites are classified as endocrine modulators. They affect the hormonal balance in both children and adults. The aim of this publication was to compare the urinary levels of phthalate metabolites in selected populations of the Czech Republic (CZ), Slovakia (SK), and Hungary (HU) in relation to the sources of phthalate exposure identified by means of questionnaire (personal care products, floor and wall coverings, plastic toys, and some kinds of foods). METHODS: Data were obtained through the twin projects COPHES (COnsortium to Perform Human biomonitoring on a European Scale) and DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) from 2009 to 2012. The target groups were children aged 6-11 years old and their mothers up to 45 years of age. The metabolites of phthalates (monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), mono-cyclohexyl phthalate (MCHP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), and mono-(2-ethyl-5-oxohexyl) phthalate (5OXO-MEHP)) were analysed in first morning urine samples. After enzymatic glucuronide cleavage, the urine sample analyses were performed using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) in one laboratory that qualified in the External Quality Assessment exercises organised by COPHES. RESULTS: Significant differences in phthalate exposure between countries were revealed for children only but not for mothers. The concentrations of 5-OH-MEHP (P<0.001), 5OXO-MEHP (P<0.001), and their sum (P<0.001) were the highest in SK compared to CZ and HU. The health based guidance values for the sum of DEHP metabolites 5-OH MEHP and 5OXO-MEHP established by the German Commission for biomonitoring of 300 µg/L and 500 µg/L for women adults and children, respectively, were only exceeded in one mother and three boys. A significant difference was also found for MEP (P=0.0149), with the highest concentrations detected in HU. In all countries, the increasing frequency of using personal care products significantly elevated the concentrations of MEP. CONCLUSION: Some differences were observed between countries in the concentrations of individual urinary phthalate metabolites in children. However, the questionnaire results give no direct explanation for the differences between the countries except the variation in using personal care products.

Urinary BPA measurements in children and mothers from six European member states: Overall results and determinants of exposure.

For the first time in Europe, both European-wide and country-specific levels of urinary Bisphenol A (BPA) were obtained through a harmonized protocol for participant recruitment, sampling and quality controlled biomarker analysis in the frame of the twin projects COPHES and DEMOCOPHES. 674 child-mother pairs were recruited through schools or population registers from six European member states (Belgium, Denmark, Luxembourg, Slovenia, Spain and Sweden). Children (5-12 y) and mothers donated a urine sample. Information on socio-demographic characteristics, life style, dietary habits, and educational level of the parents was provided by mothers. After exclusion of urine samples with creatinine values below 300 mg/L or above 3000 mg/L, 653 children and 639 mothers remained for which BPA was measured. The geometric mean (with 95% confidence intervals) and 90th percentile were calculated for BPA separately in children and in mothers and were named "European reference values". After adjustment for confounders (age and creatinine), average exposure values in each country were compared with the mean of the "European reference values" by means of a weighted analysis of variance. Overall geometric means of all countries (95% CI) adjusted for urinary creatinine, age and gender were 2.04 (1.87-2.24) µg/L and 1.88 (1.71-2.07) µg/L for children (n=653) and mothers (n=639), respectively. Multiple regression analysis was used to identify significant environmental, geographical, personal or life style related determinants. Consumption of canned food and social class (represented by the highest educational level of the family) were the most important predictors for the urinary levels of BPA in mothers and children. The individual BPA levels in children were significantly correlated with the levels in their mothers (r=0.265, p<0.001), which may suggest a possible common environmental/dietary factor that influences the biomarker level in each pair. Exposure of the general European population was well below the current health-based guidance values and no participant had BPA values higher than the health-based guidance values.

Exposure determinants of cadmium in European mothers and their children.

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.