In Vitro Effects of Zirconia Nanoparticles: Uptake, Genotoxicity, and Mutagenicity in V-79 cells.

Zirconia nanoparticles are used in various industrial and biomedical applications such as dental implants, thermal barrier sprays, and fuel cells. The interaction of nanoparticles with the environment and humans is inevitable. Despite the enormous application potential of these nanoparticles, there are still some gaps in the literature regarding potential toxicological mechanisms and the genotoxicity of zirconia nanoparticles. The lung is one of the main exposure routes to nanomaterials; therefore, the present study was designed to determine the genotoxic and mutagenic effect of zirconia NPs in V-79 lung cells. Zirconia nanoparticles showed significant internalization in cells at 100 µg/mL and 150 µg/mL concentrations. Zirconia nanoparticles showed low cytotoxicity and were found to generate ROS in V-79 cells. In alkaline comet assay, zirconia nanoparticles (10 µg/mL, 50 µg/mL, and 100 µg/mL) exposed cells exhibited significant DNA strand breaks, while the neutral comet assay, which was used for double-strand break assessment, only revealed significant damage at 100 µg/mL. Chromosomal aberration induced by zirconia nanoparticles mainly resulted in the generation of gaps, few fragments, and breaks which signifies the low clastogenic activity of these nanoparticles in the V-79 cell line. In MN assay, zirconia nanoparticles resulted in no significant micronuclei induction at any given concentration. In the HPRT mutation assay, the particle shows a dose-dependent increase in the mutant frequency. It is evident from the result that zirconia nanoparticles cause dose-dependent cytotoxicity and genotoxicity, but still, more studies are needed to evaluate the clastogenic potential and the possible mechanism involved.

Physicochemical characterization and oxidative potential of size fractionated Particulate Matter: Uptake, genotoxicity and mutagenicity in V-79 cells.

For many years, the impact of Particulate Matter (PM) in the ambient air has been one of the major concerns for the environment and human health. The consideration of the heterogeneity and complexity of different size fractions is notably important for the assessment of PM toxicological effects. The aim of the study was to present a comprehensive size-composition-morphology characterization and to assess the oxidative potential, genotoxicity, and mutagenicity of the atmospheric PM fractions, collected by using MOUDI near a busy roadside in Lucknow, India. Physicochemical characterization of ambient coarse particles (1.8-10 µm), fine particles (0.32-1.8 µm), quasi-ultrafine (0.1-0.32 µm) and ultrafine particles (≤0.1 µm) along with SRM 1649b was done using TEM, SEM, DLS, NTA, ICP-MS, and IC in parallel with the estimation of exogenous Reactive Oxygen Species (ROS) by acellular assays. In this study, two different acellular assays, dithiothreitol (DTT) and the CM-H2DCFDA assay, indicated stronger mass-normalized bioactivity for different size ranges. Enrichment factor analysis indicated that the different size fractions were highly enriched with elements of anthropogenic origin as compared to elements of crustal origin. The endotoxin concentration in different size fractions was also estimated. Cellular studies demonstrated significant uptake, cytotoxicity, ultrastructural changes, cellular ROS generation, and changes in the different phases of the cell cycle (Sub G1, G1, S, G2/M) exposed to different size fractions. The Comet assay and the Micronucleus assay were used to estimate genotoxicity. Mutagenic potential was revealed by the HGPRT gene forward mutation assay in V-97 cells. Conclusively, our results clearly indicate that the genotoxic and mutagenic potential of the coarse PM was greater than the other fractions, and interestingly, the ultrafine PM has higher bioactivity as compared to quasi-ultrafine PM.

Importance of protozoa Tetrahymena in toxicological studies: A review.

Tetrahymena is a single-cell eukaryotic organism present in all aquatic environments and can easily be maintained in laboratory conditions in a cost-effective manner. This review gives a brief description of the physiology of Tetrahymena, culture handling, and maintenance of Tetrahymena species. The review article focuses on various toxicological bioassays at different biological organizational (biochemical, individual, population, and community) levels. Furthermore, some techniques such as single cell gel electrophoresis (SCGE) and microcalorimetry assay are also available to investigate the effect of xenobiotics on the integrity of DNA and metabolic state of Tetrahymena species respectively. The article also discusses how the general physiology, behavioural activities and different organelles of Tetrahymena could be useful in toxicological studies. The strength and limitations of Tetrahymena over other model organisms are also discussed. This article also provides suggestions to overcome some problems related to toxicity assessment. Various aspects associated with variability in results, toxicity endpoints, characteristics of organisms and responses against xenobiotic substances (old and new emerging toxicants) are considered.

Effect of difenoconazole fungicide on physiological responses and ultrastructural modifications in model organism Tetrahymena pyriformis.

The continuous and extensive use of pesticides, particularly in the field of agriculture, leads to contamination of all ecosystems (water, soil, and atmosphere). Among pesticides, fungicides constitute a larger group whose impact on the environment are still poorly studied. Difenoconazole belongs to triazole group of fungicides having high photochemical stability and have low biodegradability, which makes them persistent in water bodies. The present study focuses on the physiological and cytotoxic impact of difenoconazole fungicide on ciliated protozoa, Tetrahymena pyriformis with reference to growth, morphology, behaviour and its generation time. Morphological studies showed changes in the shape and size of T. pyriformis. Our result showed an inhibitory effect on population growth of T. pyriformis and the IC50 concentration was found to be 6.8 µg mL-1.The numbers of generations decreased and generation time was found to be extended in a concentration and time dependent manner. Difenoconazole caused significant depletion in phagocytic activity and also ultra-structural changes were observed by Transmission electron microscopy (TEM) analysis. The results indicate that the Tetrahymena toxicity assay could be used as a complementary system to rapidly elucidate the cytotoxic potential of fungicide.

Zinc oxide nanoparticles induced gene mutation at the HGPRT locus and cell cycle arrest associated with apoptosis in V-79 cells.

In recent years, the large-scale production of ZnO nanoparticles (NPs) for various applications is increasing exponentially and may pose serious health issues when inhaled either during occupational exposure or in consumer settings. The mechanisms underlying the toxicity of NPs have recently been studied intensively. Despite the existing studies, the mutagenicity of ZnO NPs in the eukaryotic system is still unclear. Therefore, the aim of the present study was to investigate the mutagenic potential of ZnO NPs using Chinese hamster lung fibroblast cells (V-79) as an in-vitro model. The study has demonstrated a significant uptake of ZnO NPs by flow cytometry with the confirmation of transmission electron microscopy. A reduction in cell viability was observed with a concomitant increase in reactive oxygen species (**P < 0.01, ***P < 0.001) after ZnO NP (1-20 µg/mL) exposure. Excessive reactive oxygen species can induce oxidative stress, which leads to genotoxic insult, and further gene mutation. Apart from measuring the genotoxicity by Comet assay, a change of 2.84-fold in the HGPRT gene mutant frequency was observed by the mammalian gene forward mutation assay. All the genotoxicity endpoints such as chromosomal break, DNA damage and mutagenicity were observed at 6 hours of ZnO NP exposure. Our results also showed that ZnO NPs manifested the cell cycle arrest, ultrastructural modifications and further cell death. A significant (**P < 0.01, ***P < 0.001) increase in the apoptotic cells was detected using annexin V-fluorescein isothiocyanate/propidium iodide double staining by flow cytometry. Our findings presented here clearly stimulate the need for careful regulations of ZnO NPs.

Impact of anatase titanium dioxide nanoparticles on mutagenic and genotoxic response in Chinese hamster lung fibroblast cells (V-79): The role of cellular uptake.

The unique physico-chemical properties of nano crystalline anatase titanium dioxide nanoparticles (TiO2 NPs) render them with different biological and chemical activities. Hence, it is widely used in industrial and consumer applications. Previous studies have shown the genotoxicity of TiO2 NPs. However, there is a paucity of data regarding mutagenicity of these NPs. In the present study, the cellular uptake, sub-cellular localization, cytotoxicity and short term DNA interaction of TiO2 NPs (1-100 µgmL-1) of diameter ranging from 12 to 25 nm on mammalian lung fibroblast cells (V-79) has been studied. The flow cytometric analysis and electron micrographs of V-79 monolayer showed the internalization of TiO2 NPs in the cytoplasm with the confirmation of elemental composition through SEM/EDX analysis. TEM analysis also showed TiO2 NPs induced ultra-structural changes such as swollen mitochondria and nuclear membrane disruption in V-79 cells. TiO2 NPs generated free radicals, which induced indirect mutagenic and genotoxic responses. Apart from measuring the genotoxicity by Comet assay, the mutagenic potential of TiO2 NPs in V-79 cells was evaluated by mammalian HGPRT gene forward mutation assay, showing a 2.98- fold increase in 6TGR HGPRT mutant frequency (*p < 0.05, **p < 0.01, ***p < 0.001) by culture plate method, which is an early indicator of potential carcinogenicity. Hence, TiO2 NPs should be closely monitored and there should be a judicious use and disposal of NPs.