Giving meaning to alternative methods to animal testing

The 3 rd edition of the advanced theoretical-training course \u201c Giving meaning to alternative methods to animal testing \u201d was held in Genoa on July 6-7, 2017. The theoretical modules included talks by specialists from companies engaged in the field of advanced in vitro technologies, who offered participants the possibility to try out their technologies in the training modules

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -ε isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -ε activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process

Development of in vitro testing strategies for toxicity evaluation of mixtures derived from discarded laboratory chemicals

Our complementary methods can represent an high content sensitive tool to assess the safety of chemical mixtures, that can act on skin, as well on eye and neural functions. Aim of this study was to assess the toxic potential of two mixtures (Cat. EWC160506, one inorganic and one organic), by different in vitro approaches

Evaluation of the Cytotoxic Effects of Humid Lightweight Coal Ash derived from the Disposal of Waste on Normal Human Keratinocyte and Endothelial Cell Lines in 2-D and 3-D Culture

The presence of waste in the environment has frequently been indicated as a significant risk to human health. Therefore, landfill sites and the disposal of urban solid and non-hazardous waste by incineration are subject to much environmental monitoring, in addition to the regulations already in place. However, little action has been taken, and consequently no specific legislation exists, in relation to the assessment of the real biological risk of various substances, including chemical mixtures and ashes, derived from the incineration processes. This study assessed the cytotoxic potential of humid lightweight coal ash (LA) derived from incineration processes and waste management, on two cell lines: NCTC 2544 normal human keratinocytes and HECV endothelial cells. To reach this goal and to assess more-realistic methods for animal replacement, we employed different in vitro experimental approaches: acute and longer exposure to LA, by direct and indirect contact (0\u20132mg/ml and 16mg, respectively), both in 2-D and 3-D cultures. In 2-D HECV cultures, we observed a decrease in the viability index, but only during direct contact with LA doses higher than 0.1mg/ml. Moreover, some striking differences in cytotoxicity were observed between the 2-D and 3-D models. Taken together, these observations indicate that, for studying pollutant toxicity during longer exposure times, 3-D cultures in direct contact with the pollutant seem to offer a more suitable approach \u2014 they mimic the in vivo behaviour of cells more realistically and under strictly controlled conditions. Thus, in readiness for possible forthcoming European regulations, we believe that the proposed study, even in its preliminary phase, can provide new advice on the assessment of the toxic and biological potential of particular chemical compounds derived from waste management processes

ANTI-TUMOR EFFECTS OF LIPOPEROXIDES IN VITRO

The oxidant/antioxidant status of patients could influence tumor growth and proliferation and subsequently overall survival. Lipid hydroperoxides (LOOHs) accumulate, not in young and proliferating cells, but in aging cells, suggesting that this intracellular increasing should restore the capability of tumour cells to age and die. Potential antitumoral effects of LOOHs, were verified in vitro on a human bladder (5637), and normal endothelial (HECV) cell lines by analysis of proliferation rate, oxidative markers and several molecules involved into neoplastic progression. LOOH exposure may exhibit different effects on cancer and normal cell proliferation, and the mechanism on the growth inhibitory activity of LOOHs seems to be associated to the reduction of APRIL and other proangiogenic factors

2D- compared 3D- human cell models: cytotoxic effects of humid lightweight coal ash derived from the disposal of waste

2D- compared 3D- human cell models: cytotoxic effects of humid lightweight coal ash derived from the disposal of wast

Multidisciplinary approach to assess the biological potential of bioactive green extracts from exhaust organic matrices, obtained by microwave irradiation

we have assessed in vitro the biological potential of essential waters (EWs) from grapes, apple and raspberry residues recovered from exhaust plant matrices from the alimentary industry. The aqueous extracts were obtained by a microwaves assisted extraction (MAE), allowing to recover the inner water phases of the plants with different water-soluble principles in order to gain new bioactive compounds with potential nutraceutical and cosmeceutical properties. To assess safety and biological potentials of new green extracts, normal human keratinocytes were exposed for 24 and 48 hours to 0.05-1% (v/v) grapes, apple and raspberry EWs in order to evaluate basic their toxic effects, in terms of viability and proliferation indexes (MTT and NRU tests, and DNA content) as well antioxidant potential (in terms of iron-reducing capacity, by a commercial kit), and pro/anti-inflammatory activity (genic expression of proinflammatory cytokines, such IL-1\u3b1 and IL-8). The obtained results highlight the innovation and originality of our approach in order to recover, worn-out plant matrices, new non toxic aqueous extracts enriched in watersoluble bioactives. Their antioxidant potential together with their null cytotoxicity assessed by means of in vitro studies confirm their interest as new eco-sustainable cosmeceutical and nutraceutical ingredients Taking into account several points of the Green Chemistry Principles in compliance with the 3R policy, this preliminary multidisciplinary project represents a useful tool for the industries involved in a Global Responsible Care Program

Neurogenic-committed human pre-adipocytes express CYP1A isoforms.

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of β-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants