Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

Lack of cytotoxic and genotoxic effects of mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium despite cellular uptake in cancerous and noncancerous lung cells.

Cytotoxic and genotoxic effects of novel mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) have been investigated on human adenocarcinomic alveolar basal epithelial (A549) and human normal bronchial epithelial (BEAS-2B) cells. In the studies several molecular and cellular targets addressing to cell membrane, cytoplasm organelles and nucleus components were served as toxicological endpoints. The as-synthesized nanoparticles were found to be stable in the cell culture media and were examined for different concentration and exposure times. No cytotoxicity of the tested nanoparticles was found although these nanoparticles slightly increased reactive oxygen species in both cell types studied. Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles did not produce any DNA strand breaks and oxidative DNA damages in A549 and BEAS-2B cells. Different concentration of Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles and different incubation time did not affect cell migration. The lung cancer cells' uptake of the nanoparticles was more effective than in normal lung cells. Altogether, the results evidence that mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium do not elucidate any deleterious effects on human normal and cancerous lung cells despite cellular uptake of these nanoparticles. Therefore, it seems reasonable to conclude that these novel biocompatible nanoparticles are promising candidates for further development towards medical applications.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Genotoxic effects of occupational exposure to glass fibres - A human biomonitoring study.

As part of a large human biomonitoring study, we conducted occupational monitoring in a glass fibre factory in Slovakia. Shopfloor workers (n = 80), with a matched group of administrators in the same factory (n = 36), were monitored for exposure to glass fibres and to polycyclic aromatic hydrocarbons (PAHs). The impact of occupational exposure on chromosomal aberrations, DNA damage and DNA repair, immunomodulatory markers, and the role of nutritional and lifestyle factors, as well as the effect of polymorphisms in metabolic and DNA repair genes on genetic stability, were investigated. The (enzyme-modified) comet assay was employed to measure DNA strand breaks (SBs) and apurinic sites, oxidised and alkylated bases. Antioxidant status was estimated by resistance to H2O2-induced DNA damage. Base excision repair capacity was measured with an in vitro assay (based on the comet assay). Exposure of workers to fibres was low, but still was associated with higher levels of SBs, and SBs plus oxidised bases, and higher sensitivity to H2O2. Multivariate analysis showed that exposure increased the risk of high levels of SBs by 20%. DNA damage was influenced by antioxidant enzymes catalase and glutathione S-transferase (measured in blood). DNA repair capacity was inversely correlated with DNA damage and positively with antioxidant status. An inverse correlation was found between DNA base oxidation and the percentage of eosinophils (involved in the inflammatory response) in peripheral blood of both exposed and reference groups. Genotypes of XRCC1 variants rs3213245 and rs25487 significantly decreased the risk of high levels of base oxidation, to 0.50 (p = 0.001) and 0.59 (p = 0.001), respectively. Increases in DNA damage owing to glass fibre exposure were significant but modest, and no increases were seen in chromosome aberrations or micronuclei. However, it is of concern that even low levels of exposure to these fibres can cause significant genetic damage.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment-A Review.

Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000-2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO2, and TiO2.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO2; NM-101), and silica (SiO2; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO2 (NM-203) > TiO2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO2 (NM-101) > SiO2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

The comet assay applied to HepG2 liver spheroids.

In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of human toxicity of industrial chemicals and environmental pollutants. In this study we compared cytotoxic and genotoxic responses induced by chemicals in 2D and 3D spheroidal cultures of the human liver cancer cell line HepG2. HepG2 spheroids were prepared by hanging drop technology. Both 3D spheroids and 2D monolayer cultures were exposed to different chemicals (colchicine, chlorpromazine hydrochloride or methyl methanesulfonate) for geno- and cytotoxicity studies. Cytotoxicity was investigated by alamarBlue assay, flow cytometry and confocal imaging. DNA damage was investigated by the comet assay with and without Fpg enzyme for detection of DNA strand breaks and oxidized or alkylated base lesions. The results from the cyto- and genotoxicity tests showed differences in sensitivity comparing the 2D and 3D HepG2 models. This study shows that human 3D spheroidal hepatocellular cultures can be successfully applied for genotoxicity testing by the comet assay and represent a promising advanced in vitro model for toxicity testing.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.