Search

The assessment of the environmental footprint of an organization or product is based on methods published by the European Union Joint Research Centre, which take 16 impact areas into account. Among the listed categories are human and freshwater ecosystem toxicities. Standard protocols utilize just chemical parameters as input data, hindering the determination of the full impact of complex mixes, such as pollutants released into the environment. Biological assays enable us to overcome this gap: in the present work, assays were employed to determine both baseline and specific toxicity to aquatic species (green algae, luminescent bacteria, and crustacean cladocera) as well as specific toxicity (mutagenicity and carcinogenicity). Ecological footprint was estimated with regard to the impact categories “freshwater toxicity” and “human cancer toxicity” following the standard methodology. In parallel, the impact on the above categories was estimated using the results of biological assays as input. Standard and bioassay-based results are not always congruent, and conventional methods generally underestimate the effects. Likewise, the choice of reference substance (metals or organics) influences the quantification of impact. Appropriate batteries of biological assays could therefore be utilized to complement LCA (Life Cycle Assessment) techniques in order to make them more sensitive when considering toxicity in mid-term impact categories.

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand the early metabolic deregulation and events in cadmium-induced carcinogenesis.

In three different biological models, the recombinant protein expressed in E. coli, the neuronal cells SH-SY5Y and the nematode Caenorhabditis elegans, cadmium inhibits SOD1 activity without affecting its expression level.

Epidemiological data have related cadmium exposure to neurotoxicity. The combination of multi-level and complementary approaches provides a comprehensive view of mechanistic processes in neurotoxicity.

Cadmium is a widespread pollutant, which easily accumulates inside the human body with an estimated half-life of 25–30 years. Many data strongly suggest that it may play a role in the pathogenesis of neurodegenerative diseases. In this paper we investigated cadmium effect on human SH-SY5Y neuroblastoma cells metabolism. Results showed that, although SH-SY5Y cells already showed hyperactivated glycolysis, cadmium further increased basal glycolytic rate. Both glycolytic capacity and reserve were also increased following cadmium administration, endowing the cells with a higher compensatory glycolysis when oxidative phosphorylation was inhibited. Cadmium administration also led to an increase in glycolytic ATP production rate, paralleled by a decrease in ATP production by oxidative phosphorylation, due to an impairment of mitochondrial respiration. Moreover, following cadmium administration, mitochondria increased their dependency on glutamine, while decreasing lipids oxidation. On the whole, our data show that cadmium exacerbates the Warburg effect and promotes the use of glutamine as a substrate for lipid biosynthesis. Although increased glutamine consumption leads to an increase in glutathione level, this cannot efficiently counteract cadmium-induced oxidative stress, leading to membrane lipid peroxidation. Oxidative stress represents a serious threat for neuronal cells and our data confirm glutathione as a key defense mechanism.

The ecotoxicological tools reveal to be profitably employable within the assessment of the so-called environmental footprint, which is commonly based on the results of a chemical monitoring. Due to the heterogeneity of biological endpoints and the possibility to explore several exposure frames, as well as to consider higher levels of organization (from cells to organisms and mesocosms), the definition of a protocol is desirable.

La valutazione del potenziale carcinogeno delle sostanze chimiche si basa, a livello regolatorio, sul saggio di cancerogenesi sui roditori (OECD, TG 451). La loro diffusa applicabilità è però messa in discussione a causa delle lunghe tempistiche e dei costi associati alla loro esecuzione, e dal potenzialmente limitato valore predittivo verso l'uomo. I Cell Transformation Assays (CTA) sono tra i metodi in vitro più avanzati e standardizzati a livello regolatorio. I CTA riproducono passaggi chiave della trasformazione in vivo; si basano sulla formazione, in seguito al trattamento con un sospetto carcinogeno, di colonie trasformate (o foci), classificate al microscopio da un esperto sulla base di caratteristiche morfologiche standard. I CTA sono impiegati sia per la valutazione del rischio sia nell'ambito della ricerca; tuttavia, per consentirne un maggior utilizzo, alcuni miglioramenti sono auspicabili. Nel presente lavoro sono stati sviluppati due approcci allo scopo di potenziare i saggi CTA. Il primo ha lo scopo di ottimizzare la fase di riconoscimento dei foci trasformati sulla base di caratteristiche morfologiche, spesso affetta da soggettività. Questo obiettivo è stato raggiunto sviluppando algoritmi e metodi statistici per definire in modo quantitativo le caratteristiche morfologiche dei foci. Il secondo approccio ha come obiettivo il miglioramento della comprensione dei meccanismi coinvolti nella trasformazione in vitro, attraverso la caratterizzazione biochimica e molecolare di cellule isolate da foci e durante fasi iniziali della trasformazione. E' stato acquisito un database di immagini di foci ottenuti tramite saggi CTA condotti da EURL ECVAM (JRC), nell'ambito dello studio di prevalidazione, e da ARPA-ER. Sono stati sviluppati: 1) un algoritmo di segmentazione capace di isolare la regione del focus dal monostrato circostante; 2) descrittori statistici delle immagini dei foci, allo scopo di riassumere le caratteristiche di dimensione, crescita multis, The evaluation of the carcinogenic potential to humans relies at regulatory level on the two-year rodent bioassays (OECD TG451), which are extremely costly in terms of time and animals used, and whose predictive value towards humans has been questioned. The Cell Transformation Assays (CTAs) are the most advanced in vitro methods to identify the chemical carcinogenicity potential, in terms of standardization and validation, and reproduce key stages of in vivo transformation. The endpoint is the formation of transformed colonies (or foci) upon treatment with a carcinogen, which are visually scored by stereomicroscopy, using defined morphological features. These assays offer several advantages in comparison to the in vivo bioassays in rodents, and are used by industry and academia as screening methods for carcinogenicity testing and as a tool for mechanistic studies. Even though OECD Guidance Documents on CTAs have been recently published, further improvements are considered important to enhance the use of the assay. We developed two approaches aiming to: i) increase throughput and reliability of the scoring process, by developing algorithms and statistical methods designed to quantitatively characterize foci morphological features and ii) increase the understanding of in vitro transformation mechanisms, through the molecular characterization of transformed cells from foci, and from initial phases of transformation. A database of digital images of foci from CTAs performed by EURL ECVAM (JRC), in the prevalidation study, and by ARPA-ER, were acquired and foci regions were isolated from the background through an originally developed algorithm. Statistical image descriptors defining the morphological features recognized during visual scoring were developed to cover size, multilayer growth, spindle-shape, invasiveness, and degree of heterogeneity of foci. Statistical models were developed to automatically classify foci, supporting the phase of visual scoring. In addition

The inhalation of zinc oxide nanoparticles (nZnO) may induce systemic diseases, damages to the alveolar epithelium and inflammatory response to endothelial cells. In this work the use of an in vitro air-blood barrier (ABB) model provided a tool to elucidate the biological mechanisms underlying the potential effects of inhaled nanoparticles (NPs). The ABB model used is composed of a Transwell co-culture of a lung epithelial cell line (NCI-H441) and an immortalized pulmonary microvascular endothelial cell line (HPMEC-ST1.6R). In addition, a tri-culture model was developed by adding monocytes (THP-1) on the basal compartment of the inserts. These models have been set up to analyse the importance of the interplay among the different cell types on various responses after nZnO exposure: inflammation, endothelial damage and modulation of the immune system. The barrier integrity was assessed by measuring the transepithelial electrical resistance (TEER); the pro-inflammatory and immune cells responses were analysed by ELISA. The results have evidenced that nZnO do not affect the barrier integrity, since no TEER reduction was measured after 24 h of exposure, but an activation of endothelial cells, which released pro-inflammatory mediators (IL-6, IL-8), and endothelial dysfunction markers (sICAM-1 and sVCAM-1) were induced. These results confirm that apical exposure to NPs promote endothelium activation. The in vitro-ABB model here used is thus a useful tool able to evidence the interaction between lung epithelium and endothelium in inducing biological response, and the role of endothelium dysfunction following NPs inhalation.

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling.

Background: In the last decade, nanoparticles (NPs) have brought a huge revolution in the many fields, especially in toxicology studies. Their small size (diameter lower than 100 nm) consent them to have unique chemical and physical properties, which, besides their positive impact in industrial use, may induce adverse health effects too. The production and the emission of zinc oxide nanoparticles (nZnO) is highly increased during the last years, for their industrial, cosmetic, anti-microbial and biomedical applications. Several in vitro and in vivo studies have shown that nZnO are toxic to different mammalian cells inducing inflammation, oxidative stress and systemic effects. However, many questions about the mechanisms of nZnO induced toxicity are still unanswered. Inhalation is the major way of entry and NPs, for their small dimension, can evade the clearance of the respiratory tree and reach the alveoli. NPs can exert their effects through two routes: 1) they may translocate across the air-blood barrier (ABB) and directly enter the endothelial cells; 2) they may induce the epithelial cells to release inflammatory mediators, which subsequently promote the release of endothelial dysfunction markers from the endothelium. According to the REACH regulation (No 1907/2006/EC) and the 3Rs (Refinement, Reduction, Replacement) principle, in vitro alternative strategies are needed to test new chemical compounds, such as NPs. Thus, the choice of an appropriate in vitro model becomes crucial for the evaluation of NPs-induced effects. Co-cultures on Transwell inserts represent a useful tool for the study of NPs effects on different cells. With this model, systemic effects are evaluable, as well as cells exposure to the air-liquid interface (ALI), a more suitable exposure scenario. The aim of this work was to evaluate the effects of nZnO, especially potential effects on inflammation and endothelial dysfunction, using different new in vitro models that mimic the respiratory barr

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX., Funding Agencies|Ministero dellUniversita e della Ricerca; Universita degli Studi di Torino

On May 19, 2015, a meeting was held on the use of alternative methods for the evaluation of hepatotoxicity. It was chaired by Prof. Francesca Caloni and Prof. Giovanna Mazzoleni, and organized by CELLTOX, the Italian Association of in vitro Toxicology, in partnership with the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animal and MISTRAL (Integrated Models for Prevention and Protection in Environmental and Occupational Health) Research Center, University of Brescia, Italy. The meeting was hosted by the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna, Brescia. The conference was introduced by Dr Maura Ferrari, who reported on the activity of the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animals, indicating the need for alternative liver models.

Carcinogenesis is a multi-step process involving genetic alterations and non-genotoxic mechanisms. The in vitro cell transformation assay (CTA) is a promising tool for both genotoxic and non-genotoxic carcinogenesis. CTA relies on the ability of cells (e.g. BALB/c 3T3 mouse embryo fibroblasts) to develop a transformed phenotype after the treatment with suspected carcinogens. The classification of the transformed phenotype is based on coded morphological features, which are scored under a light microscope by trained experts. This procedure is time-consuming and somewhat prone to subjectivity.Herewith we provide a promising approach based on image analysis to support the scoring of malignant foci in BALB/c 3T3 CTA.The image analysis system is a quantitative approach, based on measuring features of malignant foci: dimension, multilayered growth, and invasivity into the surrounding monolayer of non-transformed cells. A logistic regression model was developed to estimate the probability for each focus to be transformed as a function of three statistical image descriptors. The estimated sensitivity of the derived classifier (untransformed against Type III) was 0.9, with an Area Under the Curve (AUC) value equal to 0.90 under the Receiver Operating Characteristics (ROC) curve.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to- in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.