Search Results (12)

The widespread use of silver nanoparticles in many industries is increasing every year. Along with this use, there is growing concern about the potential unintentional exposure of human and animal organisms to these nanomaterials. It has been shown that AgNPs have the ability to penetrate organisms and can have harmful effects on cells and organs in the body. In order to reduce the effects of AgNPs on living organisms, newer solutions are being investigated, such as particle stabilization or other methods of synthesizing these particles. The physical synthesis of AgNPs using high-voltage arc discharge (HVAD) may be one of these alternatives. To determine the effect of silver nanoparticles obtained by this method, cytogenetic analysis was performed on domestic dog somatic cells using a cytokinesis-blocking micronucleus assay. In the experiments performed, peripheral blood cells of the domestic dog were exposed in vitro for 3 and 24 h to three tested colloidal silver compounds (unstable AgNP-HVAD, sodium citrate-stabilized silver nanoparticles—AgNP+C, and silver nitrate). The toxicity of these compounds was evaluated at concentrations of 5, 10, and 20 µg/L, and the presence of the following cellular abnormalities was analyzed: micronuclei, nuclear buds, nucleoplasmic bridges, or multinucleated cells. The study showed a significant increase in the number of micronuclei compared to the control sample, as well as the presence of nuclear buds and nucleoplasmic bridges in somatic cells of the domestic dog, confirming the genotoxic nature of the particles. However, there was no cytotoxic effect due to the lower number of multinucleated cells and the absence of apoptotic or necrotic cells in the samples analyzed. Further studies are needed to better understand the mechanisms of toxicity of AgNPs produced by the HVAD method and the extent of their effects on mammalian somatic cells. Full article

The widespread applications of ZnO NPs in the different areas of science, technology, medicine, agriculture, and commercial products have led to increased chances of their release into the environment. This created a growing public concern about the toxicological and environmental effects of the nanoparticles. The impact of these NPs on the genetic materials of living organisms is documented in some cultured cells and plants, but there are only a few studies regarding this aspect in animals. In view of this, the present work regarding the assessment of the genotoxicity of zinc oxide nanoparticles using the mosquito Culex quinquefaciatus has been taken up. Statistically significant chromosomal aberrations over the control are recorded after the exposure of the fourth instar larvae to a dose of less than LD₂₀ for 24 h. In order to select this dose, LD₂₀ of ZnO NPs for the mosquito is determined by Probit analysis. Lacto-aceto-orcein stained chromosomal preparations are made from gonads of adult treated and control mosquitoes. Both structural aberrations, such as chromosomal breaks, fragments, translocations, and terminal fusions, resulting in the formation of rings and clumped chromosomes, and numerical ones, including hypo- and hyper-aneuploidy at metaphases, bridges, and laggards at the anaphase stage are observed. The percentage frequency of abnormalities in the shape of sperm heads is also found to be statistically significant over the controls. Besides this, zinc oxide nanoparticles are also found to affect the reproductive potential and embryo development as egg rafts obtained from the genetic crosses of ZnO nanoparticle-treated virgin females and normal males are small in size with a far smaller number of eggs per raft. The percentage frequencies of dominant lethal mutations indicated by the frequency of unhatched eggs are also statistically significant (p < 0.05) over the control. The induction of abnormalities in all of the three short-term assays studied during the present piece of work indicates the genotoxic potential of ZnO NPs, which cannot be labeled absolutely safe, and this study pinpoints the need to develop strategies for the protection of the environment and living organisms thriving in it. Full article

Biosorption successfully remediates saline water contaminated with legacy contaminants, but its effects on the health of marine organisms remain unclear. Therefore, our aim was to address this knowledge gap with data on the accumulation ability, as well as the cytogenetic and biochemical effects in turbot (Scophthalmus maximus). To this end, we exposed turbot for seven days to a mixture of remediated metals (Rem treatments: Cd, Hg, and Pb), with and without the presence of nanoparticles (NP), and compared them with the maximum allowable concentrations (MAC treatment) for effluent discharges. We determined the metal accumulation in the blood and kidney and evaluated haematological changes (red blood cell count, haemoglobin, and mean cell haemoglobin (MCH)) and genotoxicity (erythrocytic nuclear abnormalities assay) in the blood. The results showed that remediation with non-living macroalgae significantly reduced the metallic blood and kidney burdens in the Rem treatments. Furthermore, no genotoxic potential occurred in the Rem and MAC treatments in parallel with the reduction in MCH levels in the Rem treatments, which would reflect hematopoietic disturbances in the MAC. Our results validate biosorption remediation as we achieved a considerable reduction in metal loads while maintaining the health status of fish, highlighting the importance of testing water remediation methods in the biota. Full article

Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 μM), DAS (≥0.006 μM), and REG (≥0.8 μM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs. Full article

The blocking of programmed death-ligand 1 (PD-L1) in tumor cells represents a powerful strategy in cancer immunotherapy. Using viral vectors to deliver the cargo for inactivating the PD-L1 gene could be associated with host cell genotoxicity and concomitant immune attack. To develop an alternative safe gene delivery method, we designed a unique combination for miRNA34a delivery using a transgene carrier in the form of iron oxide magnetic nanoparticles (IONPs) via magnetofection to downregulate PD-L1 expression in cancer cells. We synthesized IONPs of multiple shapes (IONRs (iron oxide nanorods), IONSs (iron oxide nanospheres), and ITOHs (iron oxide truncated octahedrons)), surface-functionalized with polyethyleneimine (PEI) using the ligand exchange method, as gene delivery systems. Under the guidance of an external magnetic field, PEI@IONPs loaded with plasmid DNA (DNA/PEI@IONPs) encoding GFP showed high transfection efficiency at different weight ratios and time points in A549 and MDA-MB-231 cells. Additionally, the DNA/PEI@IONPs with miRNA34a inserts under a static magnetic field resulted in significant knockdown of the PD-L1 gene, as demonstrated via immunoblotting of the PD-L1 protein. Among the three shapes of IONPs, IONRs showed the highest PD-L1 knockdown efficiency. The genetic expression of miRNA34a was also studied using qPCR and it showed high expression of miRNA in cells treated with PEI@IONRs. Flow cytometry and a live/dead assay confirmed apoptosis after transfection with miRNA34a. To conclude, in this paper, a promising transgene carrier with low cost, negligible cytotoxicity, and high transfection efficiency has been successfully established for miRNA gene delivery in the context of cancer immunotherapy. Full article

A genotoxic study was conducted with 101 elementary school children (56 girls and 45 boys) in the 6–7, 8–9, and 10–12 age ranges from El Fraile rural community, which is located beside the El Fraile mine tailings in Taxco of Alarcon City, in northern Guerrero State, Mexico. For this, we used the alkaline comet assay in exfoliated buccal mucosa cells, scoring three genotoxic parameters: tail intensity, tail moment, and tail length. Additionally, we detected oxidative DNA damage through urinary 8-OHdG levels by enzyme-linked immunosorbent assay. We also evaluated a control group consisting of 101 children in the same age ranges from Chilpancingo City, Guerrero, who had never lived near mining zones. Genotoxic results showed that there was a significant increase in three genotoxic parameters and urinary 8-OHdG levels in the exposed children group compared with the control group. Analysis of MANOVA revealed that boys aged 8 and 9 years had higher DNA damage than girls from the same exposure group, and Spearman’s analysis identified a positive correlation between DNA damage and sex and age. This study provides the first valuable genotoxic data in children living in areas with environmental pollution. Full article

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds. Full article

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions. Full article

The interactions between pharmaceuticals and nanomaterials and its potentially resulting toxicological effects in living systems are only insufficiently investigated. In this study, two model compounds, acetaminophen, a pharmaceutical, and cerium dioxide, a manufactured nanomaterial, were investigated in combination and individually. Upon inhalation, cerium dioxide nanomaterials were shown to systemically translocate into other organs, such as the liver. Therefore we picked the human liver cell line HuH-7 cells as an in vitro system to investigate liver toxicity. Possible synergistic or antagonistic metabolic changes after co-exposure scenarios were investigated. Toxicological data of the water soluble tetrazolium (WST-1) assay for cell proliferation and genotoxicity assessment using the Comet assay were combined with an untargeted as well as a targeted lipidomics approach. We found an attenuated cytotoxicity and an altered metabolic profile in co-exposure experiments with cerium dioxide, indicating an interaction of both compounds at these endpoints. Single exposure against cerium dioxide showed a genotoxic effect in the Comet assay. Conversely, acetaminophen exhibited no genotoxic effect. Comet assay data do not indicate an enhancement of genotoxicity after co-exposure. The results obtained in this study highlight the advantage of investigating co-exposure scenarios, especially for bioactive substances. Full article

This study aimed to assess the association of exposure to particle-bound (PM_2.5) polycyclic aromatic hydrocarbons (PAHs) with potential genotoxicity and cancer risk among children living near the petrochemical industry and comparative populations in Malaysia. PM_2.5 samples were collected using a low-volume sampler for 24 h at three primary schools located within 5 km of the industrial area and three comparative schools more than 20 km away from any industrial activity. A gas chromatography–mass spectrometer was used to determine the analysis of 16 United States Environmental Protection Agency (USEPA) priority PAHs. A total of 205 children were randomly selected to assess the DNA damage in buccal cells, employing the comet assay. Total PAHs measured in exposed and comparative schools varied, respectively, from 61.60 to 64.64 ng m⁻³ and from 5.93 to 35.06 ng m⁻³. The PAH emission in exposed schools was contributed mainly by traffic and industrial emissions, dependent on the source apportionment. The 95th percentiles of the incremental lifetime cancer risk estimated using Monte Carlo simulation revealed that the inhalation risk for the exposed children and comparative populations was 2.22 × 10⁻⁶ and 2.95 × 10⁻⁷, respectively. The degree of DNA injury was substantially more severe among the exposed children relative to the comparative community. This study reveals that higher exposure to PAHs increases the risk of genotoxic effects and cancer among children. Full article

Redox-directed pharmacophores have shown potential for the apoptotic elimination of cancer cells through chemotherapeutic induction of oxidative stress. Phenazine methosulfate (PMS), a N-alkylphenazinium cation-based redox cycler, is used widely as an electron transfer reactant coupling NAD(P)H generation to the reduction of tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a superoxide generating chemotherapeutic for the pro-oxidant induction of cancer cell apoptosis. In a panel of malignant human melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1–10 μM, 24 h) displayed pronounced apoptogenicity as detected by annexin V-ITC/propidium iodide flow cytometry, and PMS-induced cell death was suppressed by antioxidant (NAC) or pan-caspase inhibitor (zVAD-fmk) cotreatment. Gene expression array analysis in A375 melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced superoxide production was substantiated by MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating NADPH-driven (SOD-suppressible) superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375 melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of malignant melanoma cells. Full article

The economy of the state of Tabasco is based on oil extraction. However, this imposes major effects to the environment and communities. Examples are the Polycyclic Aromatic Hydrocarbons (PAHs) that may be found in the soil, water and sediment of the region. Their volatility makes them available to living beings and results in genotoxic activity. The purpose of this study was to quantify the levels of PAHs in the air at several points in the state, and to analyze their relationship with possible damage to DNA on local inhabitants. Single Cell Gel Electrophoresis Assay (Comet Assay) was applied to peripheral blood lymphocytes of five groups of children between six and 15 years of age. PAH samples were analyzed following US/EPA TO-13-A method. Results indicated the presence in the air of most of the 16 PAHs considered as high priority by EPA, some of which have been reported with carcinogenic activity. Differences (p<0.05) were found between PAHs concentration in the gaseous component and in the particulate component of air samples, with the greatest values for the gaseous component. Greatest PAH concentrations were detected in areas with high oil extraction activities. Children groups from high oil activity areas presented genotoxic damage labeled from moderate to high according to DNA migration from nuclei (Tail Length: 14.2 - 42.14 μm and Tail/Head: 0.97 - 2.83 μm) compared with control group (12.25 and 0.63 μm, respectively). The group with greatest cell damage was located in the area with the greatest oil activity. We conclude that the presence of PAHs in the air may represent a health risk to populations that are chronically exposed to them at high oil activity regions. Full article