Bioelectrochemical platform with human monooxygenases: FMO1 and CYP3A4 tandem reactions with phorate.

It is highly advantageous to devise an in vitro platform that can predict the complexity of an in vivo system. The first step of this process is the identification of a xenobiotic whose monooxygenation is carried out by two sequential enzymatic reactions. Pesticides are a good model for this type of tandem reactions since in specific cases they are initially metabolised by human flavin-containing monooxygenase 1 (hFMO1), followed by cytochrome P450 (CYP). To assess the feasibility of such an in vitro platform, hFMO1 is immobilised on glassy carbon electrodes modified with graphene oxide (GO) and cationic surfactant didecyldimethylammonium bromide (DDAB). UV-vis, contact angle and AFM measurements support the effective decoration of the GO sheets by DDAB which appear as 3 nm thick structures. hFMO1 activity on the bioelectrode versus three pesticides; fenthion, methiocarb and phorate, lead to the expected sulfoxide products with KM values of 29.5 ± 5.1, 38.4 ± 7.5, 29.6 ± 4.1 µM, respectively. Moreover, phorate is subsequently tested in a tandem system with hFMO1 and CYP3A4 resulting in both phorate sulfoxide as well as phoratoxon sulfoxide. The data demonstrate the feasibility of using bioelectrochemical platforms to mimic the complex metabolic reactions of xenobiotics within the human body.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate.

Out of the five isoforms of human flavin-containing monooxygenase (hFMO), FMO1 and FMO3 are the most relevant to Phase I drug metabolism. They are involved in the oxygenation of xenobiotics including drugs and pesticides using NADPH and FAD as cofactors. Majority of the characterization of these enzymes has involved hFMO3, where intermediates of its catalytic cycle have been described. On the other hand, research efforts have so far failed in capturing the same key intermediate that is responsible for the monooxygenation activity of hFMO1. In this work we demonstrate spectrophotometrically the formation of a highly stable C4a-hydroperoxyflavin intermediate of hFMO1 upon reduction by NADPH and in the presence of O2. The measured half-life of this flavin intermediate revealed it to be stable and not fully re-oxidized even after 30 min at 15 °C in the absence of substrate, the highest stability ever observed for a human FMO. In addition, the uncoupling reactions of hFMO1 show that this enzyme is <1% uncoupled in the presence of substrate, forming small amounts of H2O2 with no observable superoxide as confirmed by EPR spin trapping experiments. This behaviour is different from hFMO3, that is shown to form both H2O2 and superoxide anion radical as a result of ∼50% uncoupling. These data are consistent with the higher stability of the hFMO1 intermediate in comparison to hFMO3. Taken together, these data demonstrate the different behaviours of these two closely related enzymes with consequences for drug metabolism as well as possible toxicity due to reactive oxygen species.

Biotransformation of Food-Grade and Nanometric TiO2 in the Oral-Gastro-Intestinal Tract: Driving Forces and Effect on the Toxicity toward Intestinal Epithelial Cells.

Background: Oral exposure to titanium dioxide (TiO2) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO2. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO2. This might be due to the different biological identity of TiO2 in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO2 and two nanometric TiO2 samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering-DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering-ELS-, Electron Paramagnetic Resonance Spectroscopy-EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO2 particles surface and reactivity. Pristine and treated TiO2 nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.

Identification of physicochemical properties that modulate nanoparticle aggregation in blood.

Inorganic materials are receiving significant interest in medicine given their usefulness for therapeutic applications such as targeted drug delivery, active pharmaceutical carriers and medical imaging. However, poor knowledge of the side effects related to their use is an obstacle to clinical translation. For the development of molecular drugs, the concept of safe-by-design has become an efficient pharmaceutical strategy with the aim of reducing costs, which can also accelerate the translation into the market. In the case of materials, the application these approaches is hampered by poor knowledge of how the physical and chemical properties of the material trigger the biological response. Hemocompatibility is a crucial aspect to take into consideration for those materials that are intended for medical applications. The formation of nanoparticle agglomerates can cause severe side effects that may induce occlusion of blood vessels and thrombotic events. Additionally, nanoparticles can interfere with the coagulation cascade causing both pro- and anti-coagulant properties. There is contrasting evidence on how the physicochemical properties of the material modulate these effects. In this work, we developed two sets of tailored carbon and silica nanoparticles with three different diameters in the 100-500 nm range with the purpose of investigating the role of surface curvature and chemistry on platelet aggregation, activation and adhesion. Substantial differences were found in the composition of the protein corona depending on the chemical nature of the nanoparticles, while the surface curvature was found to play a minor role. On the other hand, large carbon nanoparticles (but not small carbon nanoparticles or silica nanoparticles) have a clear tendency to form aggregates both in plasma and blood. This effect was observed both in the presence or absence of platelets and was independent of platelet activation. Overall, the results presented herein suggest the existence of independent modes of action that are differently affected by the physicochemical properties of the materials, potentially leading to vessel occlusion and/or formation of thrombi in vivo.

Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS).

The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO2, SiO2, and CeO2 were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1ß production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO2 and CeO2 were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1ß releases were found for the SiO2, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

Insight into ultrasound-mediated reactive oxygen species generation by various metal-porphyrin complexes.

Ultrasound is used to trigger the cytotoxicity of chemical compounds, known as sonosensitisers, in an approach called sonodynamic therapy (SDT), which is under investigation herein. The generation of reactive oxygen species (ROS) has been proposed as the main biological occurrence that leads to the cytotoxic effects, which are achieved via the synergistic action of two components: the energy-absorbing sonosensitiser and ultrasound (US), which are both harmless per se. Despite some promising results, a lack of investigation into the mechanisms behind US sonosensitiser-mediated ROS generation has prevented SDT from reaching its full potential. The aim of this work is to investigate the US-responsiveness of a variety of metal-porphyrin complexes, free-base porphyrin and Fe(III), Zn(II) and Pd(II) porphyrin, by analyzing their ROS generation under US exposure and related bio-effects. All experiments were also carried out under light exposure and the results were used as references. Our results show that porphyrin ultrasound-responsiveness depends on the metal ion present, with Zn(II) and Pd(II) porphyrin being the most efficient in generating singlet oxygen and hydroxyl radicals. ROS production efficiency is lower after ultrasound exposure than after light exposure, because of the various physico-chemical mechanisms involved in sensitiser activation. US and porphyrin-mediated ROS generation is oxygen-dependent and the activation of porphyrin by US appears to be more compatible with sonoluminescence-based photo-activation rather than a radical path process that occurs via the homolytic bond rupture of water. Notably, the cytotoxicity results reported herein, which are mirrored by ex-cellulo data, confirm that the type of ROS generation achieved by the US activation of intracellular porphyrins is pivotal to the effectiveness of cancer cell killing.

Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 signalling pathway.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-ß/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-ß-mediated signalling pathway. METHODS: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-ß production. Experiments with neutralizing anti-TGF-ß antibody, specific inhibitors of GSK-3ß and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test. RESULTS: Fully characterized MWCNT (mean length < 5 µm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-ß secretion, Akt activation and GSK-3ß inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear ß-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure. CONCLUSIONS: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-ß and its intracellular effectors Akt/GSK-3ß that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

Surface reactivity and in vitro toxicity on human bronchial epithelial cells (BEAS-2B) of nanomaterials intermediates of the production of titania-based composites.

Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide in large quantities for use in a wide range of applications. Evaluating the hazards associated with TiO2 NPs is crucial as it enables risk assessment related to human and environmental exposure. In this study the in vitro human toxicity of a set of TiO2 NPs modified with acetic, oleic and boric acids were studied in order to assess the hazard in view of a future scale-up of the synthesis. The surface reactivity of the powders under simulated solar illumination and in the dark has been evaluated by means of EPR spectroscopy. Human bronchial epithelial cells (BEAS-2B) have been chosen as a model for lung epithelium. Cytotoxicity has been assessed by measuring the cells membrane integrity by lactate dehydrogenase (LDH) assay, and the inflammatory response evaluated as nitric oxide (NO) and TNF-α production, and oxidative stress measured as intracellular reduced glutathione (GSH) levels, and induced lipoperoxidation. Aeroxide P25 was used for comparison. The results demonstrated a low photoreactivity and toxic effects lower than Aeroxide P25 of the nano-TiO2 powders, probably as a consequence of the presence of acidic moieties at the surface.

Fibrinogen enhances the inflammatory response of alveolar macrophages to TiO2, SiO2 and carbon nanomaterials.

Many studies have shown that the composition of the protein corona dramatically affects the response of cells to nanomaterials (NMs). However, the role of each single protein is still largely unknown. Fibrinogen (FG), one of the most abundant plasma proteins, is believed to mediate foreign-body reactions. Since this protein is absent in cell media used in in vitro toxicological tests the possible FG-mediated effects have not yet been assessed. Here, the effect of FG on the toxicity of three different kinds of inorganic NMs (carbon, SiO2 and TiO2) on alveolar macrophages has been investigated. A set of integrated techniques (UV-vis spectroscopy, dynamic light scattering and sodium dodecyl sulphate-polyacrylamide gel electrophoresis) have been used to study the strength and the kinetics of interaction of FG with the NMs. The inflammatory response of alveolar macrophages (MH-S) exposed to the three NMs associated with FG has also been investigated. We found that FG significantly enhances the cytotoxicity (lactate dehydrogenase leakage) and the inflammatory response (increase in nitric oxide (NO) concentration and NO synthase activation) induced by SiO2, carbon and TiO2 NMs on alveolar macrophages. This effect appears related to the amount of FG interacting with the NMs. In the case of carbon NMs, the activation of fibrinolysis, likely related to the exposure of cryptic sites of FG, was also observed after 24 h. These findings underline the critical role played by FG in the toxic response to NMs.

Possible Chemical Source of Discrepancy between in Vitro and in Vivo Tests in Nanotoxicology Caused by Strong Adsorption of Buffer Components.

In the course of studies of the interaction of proteins with TiO2 nanoparticles, we have investigated the role of the medium employed in cellular tests, by measuring the variation of ζ-potential vs pH in the range 2-9 and bovine serum albumin adsorption on TiO2 P25 in the presence of either HEPES or PBS as buffers, both mimicking the physiological pH, but with different chemical nature. The two buffers yield remarkably dissimilar surface charges and protein uptake, i.e., they impart different surface characteristics to the particles which could affect the contact with cells or tissues. This may account for dissimilar toxicological outcomes among in vitro tests and particularly between in vitro vs in vivo tests, considering the high amount of phosphate ions present in body fluids.

Hydroxyl density affects the interaction of fibrinogen with silica nanoparticles at physiological concentration.

An increasing interest in the interaction between blood serum proteins and nanoparticles has emerged over the last years. In fact, this process plays a key role in the biological response to nanoparticles. The behavior of proteins at the biofluid/material interface is driven by the physico-chemical properties of the surface. However, much research is still needed to gain insight into the process at a molecular level. In this study, the effect of silanol density on the interaction of fibrinogen at physiological concentrations with silica nanoparticle/flat surfaces has been studied. Silica nanoparticles and silica wafers were modified and characterized to obtain a set of samples with different silanols density. The interaction with fibrinogen has been studied by evaluating the extent of coverage (bicinchoninic acid assay) and the irreversibility of adsorption (shift of the ζ potential). To clarify the molecular mechanism of fibrinogen/surface interactions, confocal micro-Raman spectroscopy (nanoparticles) and atomic force microscopy (wafers) were used. Finally the effect of fibrinogen on the agglomeration of nanoparticles has been evaluated by Flow Particle Image Analysis. The data reported here show that a minimal variation in the state of the silica surface modifies the adsorption behavior of fibrinogen, which appears mediated by a competition between protein/protein and protein/surface interactions. By comparing the data obtained on nanoparticles and silicon-supported silica layers, we found that hydrophilicity increases the tendency of fibrinogen molecules to interact with the surface rather than with other molecules, thus inhibiting fibrinogen self-assembly. This study contributes to the knowledge of the processes occurring at the surface/biological fluids interface, needed for the design of new biocompatible materials.