Raman imaging of cellular uptake and studies of silver nanoparticles effect in BJ human fibroblasts cell lines.

Silver nanoparticles (AgNPs) have been widely studied for their beneficial antimicrobial effect and have been considered by some to be a safe ingredient, as penetration of metal nanoparticles through the skin in vivo has not been proven. However, AgNPs are becoming a commonly applied nanomaterial for surface modifications of medical products which come into contact with damaged skin. In our experiments, we tested two commercially available AgNPs samples manufactured by electrolysis. AFM was used to characterize tested AgNPs morphology and their mean particle size which was assessed as 30.6nm and 20.4nm. An important mechanism of AgNPs cytotoxicity is generation of reactive oxygen species (ROS), chemically reactive species containing oxygen. Although ROS occur in cell metabolism naturally, their overproduction can induce oxidative stress - imbalance between production and antioxidant defenses. This can be associated with cytotoxicity and DNA damage. Conventional in vitro tests were used to evaluate the cytotoxic potential and DNA damage in BJ human fibroblasts cell lines. We found that both tested AgNPs samples induced ROS generation and caused the DNA damage in fibroblasts. One of the key concerns about the association with cytotoxic or genotoxic responses of nanoparticles is the capability of these materials to penetrate through cellular membrane. Cellular uptake studies were performed using Raman imaging as a label-free microscopic technique. In combination with a univariate image analysis, results demonstrate cellular uptake and distribution of the AgNPs which were taken up by BJ cells within 24h of incubation in a growth medium. The study demonstrates the potential of Raman imaging to unambiguously identify and localize AgNPs in fixed cells.

The effect of silver nanoparticles and silver ions on mammalian and plant cells in vitro.

Silver nanoparticles (AgNPs) are the most frequently applied nanomaterials. In our experiments, we tested AgNPs (size 27 nm) manufactured by the Tollens process. Physico-chemical methods (TEM, DLS, AFM and spectrophotometry) were used for characterization and imaging of AgNPs. The effects of AgNPs and Ag(+) were studied in two experimental models (plant and mammalian cells). Human keratinocytes (SVK14) and mouse fibroblasts (NIH3T3) cell lines were selected to evaluate the cytotoxicity and genotoxicity effect on mammalian cells. Higher sensitivity to AgNPs and Ag(+) was observed in NIH3T3 than in SVK14 cells. AgNPs accumulated in the nucleus of NIH3T3 cells, caused DNA damage and increased the number of apoptotic and necrotic cells. Three genotypes of Solanum spp. (S. lycopersicum cv. Amateur, S. chmielewskii, S. habrochaites) were selected to test the toxicity of AgNPs and Ag(+) on the plant cells. The highest values of peroxidase activity and lipid peroxidation were recorded after the treatment of S. habrochaites genotype with AgNPs. Increased ROS levels were likely the reason for observed damaged membranes in S. habrochaites. We found that the cytotoxic and genotoxic effects of AgNPs depend not only on the characteristics of nanoparticles, but also on the type of cells that are treated with AgNPs.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action.

7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.

Reprint of: Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450.

BACKGROUND: We studied the interaction of oxaliplatin derivatives involving cytotoxic adenine-based cyclin-dependent kinase inhibitors, with human liver microsomal cytochrome P450. METHODS AND RESULTS: The activities of 9 human liver microsomal CYP forms (CYPs 1A2, 7-ethoxyresorufin O-deethylation; 2A6, coumarin 7-hydroxylation; 2B6, 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation; 2C8, luciferin-6´ methyl ether demethylation; 2C9, diclofenac 4´-hydroxylation, 6´-deoxyluciferin hydroxylation; 2C19, (S)-mephenytoin 4´-hydroxylation; 2D6, bufuralol 1´-hydroxylation, 2E1, chlorzoxazone 6-hydroxylation; 3A4, testosterone 6ß-hydroxylation, luciferin-6´ benzyl ether debenzylation) were tested using HPLC, fluorescence and luminescence product detection. At 100 µM platinum(II) oxalato complex concentration, CYP inhibition was in general 25%-50%, except for the CYP3A4 form which showed roughly twice the inhibition (72%-95%). At low complex concentration (10 µM), the difference in inhibition of CYP3A4 and other forms was even more pronounced. Dixon and Lineweaver-Burk plots indicated a partially noncompetitive mechanism of CYP3A4 inhibition. CONCLUSIONS: The tested complexes significantly inhibit human liver microsomal CYP3A4 activity even at clinically relevant concentrations. This could be a serious drawback for the use of these compounds in clinical practice.