Chinese Hamster Lung Fibroblast Cells Research Articles

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Abrogation of methyl methanesulphonate (MMS)-induced cytotoxic and genotoxic effects of tropical fruit juice mixture on fibroblast cells

Tropical fruit juice mixture consisting of pomegranate, guava, and roselle has been proven to possess high polyphenolic composition and antioxidant capacity. The present work aimed to evaluate the cytoprotective and antigenotoxic potentials of juice mixture in methyl methanesulphonate (MMS)-induced V79 Chinese hamster lung fibroblast cell line. MTT assay showed that the IC50 value of the juice mixture was 193.33 ± 46.40 µg/mL. Cells pretreated with 6, 12, and 25 µg/mL juice mixture showed significant increment in viability (p < 0.05) following induction with MMS. However, only cells co-treated with 6 and 12 µg/mL juice mixture showed protective effect (p < 0.05) against MMS-induced cytotoxicity under the co-treatment setting. Comet assay showed that the tail moment and percentage of DNA in tail in cells pretreated with the juice mixture significantly decreased compared with those in positive control groups. However, under the co-treatment setting, only 12 µg/mL juice mixture showed significant reduction (p < 0.05) in tail moment compared with MMS alone. In conclusion, the tropical fruit juice mixture can abrogate and protect cells from the cytotoxic and genotoxic effects of MMS, and has the potential to be developed as beneficial formulation for health preservation.

Evaluation of in vitro irradiation setup: Designed for the horizontal beamline at the Danish Centre for Particle Therapy.

Radiobiological experimental setups are challenged by precise sample positioning along depth dose profile, scattering conditions, and practical difficulties that must be addressed in individual designs. The aim of this study was to produce cell survival curves with several irradiation modalities, by using a setup designed at the Danish Centre for Particle Therapy (DCPT) for in vitro proton irradiations using a horizontal beam line and thereby evaluating the setups use for in vitro irradiations experiments. The setup is a water phantom suitable for in vitro research with multiple irradiation modalities, in particular the pencil scanning proton beam available from a horizontal experimental beamline. The phantom included a water tank of 39.0 × 17.0 × 20.5 cm. Cell survival-curves were produced using the cell line V79 Chinese hamster lung fibroblast cells (V79s) in biological triplicates of clonogenic assays. Cell survival curves were produced with both a 18 MeV electron beam, 6 MV photon beam, and a Spread-Out Bragg Peak (SOBP) proton beam formed by pristine energies of 85-111 MeV where three positions were examined. Survival curves with uncertainty areas were made for all modalities. Dosimetric uncertainty amounted to, respectively, 4%, 3% and 3% for proton, electron, and high energy photon irradiations. Cell survival fraction uncertainty was depicted as the standard deviation between replications of the experiment. Cell survival curves could be produced with acceptable uncertainties using this novel water phantom and cellular laboratory workflow. The setup is useful for future in vitro irradiation experiments.

Thallium Toxicity and its Interference with Potassium Pathways Tested on Various Cell Lines.

Thallium (Tl) is a highly toxic heavy metal whose mechanism of toxicity is still not completely understood. The aim of this study was to test Tl cytotoxicity on several cell lines of different tissue origin in order to clarify specific Tl toxicity to a particular organ. In addition, possible interference of Tl with cell potassium (K) transport was examined. Human keratinocytes (HaCaT), human hepatocellular carcinoma (HepG2), porcine kidney epithelial cells (PK15), human neuroblastoma (SH-SY5Y) and Chinese hamster lung fibroblast cells (V79) were treated with thallium (I) acetate in a wide concentration range (3.9-500µg/mL) for 24h, 48 and 72h. To assess competitive interaction between Tl and K, the cells were treated with four Tl concentrations close to IC50 (15.63, 31.25, 62.50, 125µg/mL) in combination with/or without potassium (I) acetate (500µg/mL). The cells' morphology was monitored, and cytotoxic effect was assessed by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. The most sensitive to Tl exposure were SH-SY5Y cells, while HepG2 were the most resistant. The combined exposure to thallium (I) acetate and potassium (I) acetate for every cell line, except V79 cells, resulted in higher cell viability compared to thallium (I) acetate alone. The results of our study indicate that cell sensitivity to Tl treatment is largely affected by tissue culture origin, its function, and Na+/K+-ATPase activity.

The effect of exposure to radiofrequency LTE signal and coexposure to mitomycin-C in Chinese hamster lung fibroblast V79 cells.

This study aims to investigate the cellular effects of radiofrequency exposure, 1950 MHz, long-term evolution (LTE) signal, administered alone and in combination with mitomycin-C (MMC), a well-known cytotoxic agent. Chinese hamster lung fibroblast (V79) cells were exposed/sham exposed in a waveguide-based system under strictly controlled conditions of both electromagnetic and environmental parameters, at specific absorption rate (SAR) of 0.3 and 1.25 W/kg. Chromosomal damage (micronuclei formation), oxidative stress (reactive oxygen species [ROS] formation), and cell cycle progression were analyzed after exposure and coexposure. No differences between exposed samples and sham-controls were detected following radiofrequency exposure alone, for all the experimental conditions tested and biological endpoints investigated. When radiofrequency exposure was followed by MMC treatment, 3 h pre-exposure did not modify MMC-induced micronuclei. Pre-exposure of 20 h at 0.3 W/kg did not modify the number of micronuclei induced by MMC, while 1.25 W/kg resulted in a significant reduction of MMC-induced damage. Absence of effects was also detected when CW was used, at both SAR levels. MMC-induced ROS formation resulted significantly decreased at both SAR levels investigated, while cell proliferation and cell cycle progression were not affected by coexposures. The results here reported provide no evidence of direct effects of 1950 MHz, LTE signal. Moreover, they further support our previous findings on the capability of radiofrequency pre-exposure to induce protection from a subsequent toxic treatment, and the key role of the modulated signals and the experimental conditions adopted in eliciting the effect.

Inhibition of Neuromyelitis Optica Immunoglobulin G Binding to Aquaporin-4 by the Small Molecule Blocker Melanthioidine.

Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease affecting the optic nerves and spinal cord. The binding of neuromyelitis optica immunoglobulin G (NMO-IgG) and aquaporin-4 (AQP4) on the surface of astrocytes in the serum and cerebrospinal fluid is the main pathogenesis of NMO. Currently, therapeutic strategies for NMO include a reduction of the secondary inflammation response and the number of NMO-IgG, which can only alleviate clinical symptoms rather than fundamentally preventing a series of pathological processes caused by NMO-IgG binding to AQP4. The purpose of this study was to investigate the blocking effect of melanthioidine on the binding of NMO-IgG to AQP4 and its potential cytotoxicity. The current study developed a cell-based high-throughput screening approach to identify a molecular blocker of NMO-IgG binding to AQP4 using the Chinese hamster lung fibroblast (V79) cells expressing M23-AQP4. By screening ~400 small molecules, we identified melanthioidine with blocking effects without affecting AQP4 expression or its water permeability. Melanthioidine effectively blocked the binding of NMO-IgG to AQP4 in immunofluorescence assays and reduced complement-dependent cytotoxicity against both NMO-IgG/complement-treated Fischer rat thyroid-AQP4 cells and primary astrocytes. The docking computations identified the putative sites of blocker binding at the extracellular surface of AQP4. This study serves as proof of a potential NMO therapy by using a small-molecule blocker to target NMO pathogenesis.

Cobalt oxide (Co3O4) nanoparticles induced genotoxicity in Chinese hamster lung fibroblast (V79) cells through modulation of reactive oxygen species.

Incessant production, pervasive applications in different fields, and eventually unintended exposure of cobalt oxide nanoparticles (Co3O4 NPs) lead to rise in their toxicity studies toward human health. However, the information regarding the potential toxicity mechanisms of Co3O4 NPs especially genotoxicity is still sparse with missing interconnections. So far, only solitary reports on Co3O4 NPs are at hand, bearing witness to reactive oxygen species (ROS)-mediated DNA damage in lung cells. To address this, we evaluated the Co3O4 NP-induced cytotoxic and genotoxic potential in Chinese hamster lung fibroblast cell line (V79). Our preliminary results demonstrate that Co3O4 NPs at concentrations of 20-100 µg/ml induced moderate mortality after 24-h exposure. However, these low concentrations caused a significant reduction in various organelles' activity in a concentration-dependent manner. Mitochondrial activity and membrane potential were found to be compromised due to NP exposure in a concentration-dependent manner. The study affirms that Co3O4 NPs inhibited lysosomal activity in V79 cells. In addition to this, Co3O4 NPs are also found to stimulate free oxygen radical generation. Genotoxicity studies revealed a potent and dose-dependent effect of non-cytotoxic concentrations of Co3O4 NPs in the induction of DNA lesions. Interestingly, N-acetylcysteine, a free oxygen radical scavenger (5, 10mM, pretreatment) inhibited the progression of free oxygen radicals and induction of Co3O4 NP-mediated DNA lesions. This suggests the ROS-mediated genotoxic potential of Co3O4 NPs.

Genetic Toxicology and Safety Pharmacological Evaluation of Forsythin.

Introduction Forsythin is the main ingredient of Forsythia suspensa and is widely used in treatment of fever, viral cold, gonorrhea, and ulcers clinically. This study aimed to evaluate the potential genetic toxicity of forsythin and its safety for human use. Methods Based on the Good Laboratory Practice regulations and test guidelines, the genetic toxicity of forsythin was assessed by the Ames test, chromosome aberration (CA) test, and bone marrow micronucleus (MN) test in vivo. In the Ames test, five strains of Salmonella typhimurium were exposed to different concentrations of forsythin in the presence or absence of the S9 mixture, and then, the number of His + revertant colonies was counted. In the CA test, Chinese hamster lung (CHL) fibroblast cells were treated with different concentrations of forsythin, mitomycin C, or cyclophosphamide in the presence or absence of the S9 mixture, and the chromosomal aberrations were determined. In the MN test, bone marrow was isolated from the mice with different treatments, and the ratios of polychromatic erythrocytes (PCE) and erythrocytes (PCE/(PCE + NCE)) were measured. Finally, beagle dogs were divided into four groups (negative control, low dose, medium dose, and high dose groups), and then, a telemetry system was used to evaluate the safe use of forsythin. Results Ames test results showed that the number of colonies in all test strains with different treatments showed no significantly dose-dependent increase in the presence or absence of the S9 mixture (p > 0.05). In the CA test, the number of cells with aberrations in the CHL fibroblast cells treated with low, medium, and high doses of forsythin for 24/48 h in the absence of the S9 mixture was, respectively, 5.0/2.5, 4.5/1.5, and 5.0/5.0, and in the presence of the S9 mixture, the number was, respectively, 5.0, 5.0, and 4.5. These results showed that there was no significant difference compared to the negative control group either in the presence (2.0) or in the absence (4.0/2.5 for 24/48 h) of the S9 mixture (p > 0.05). The MN test showed that the values of PCE/(PCE + NCE) in the negative, positive controls, and forsythin treatment groups were all more than 20%, which indicated that forsythin had no cytotoxicity. Additionally, no significant toxicological effects of forsythin on blood pressure, respiration, temperature, electrocardiogram, and other physiological indicators in the conscious beagle dogs of different groups were observed by the telemetry method. Conclusion Our findings showed that forsythin has low probability of genetic toxicity and no significant toxicological effects, which implied that forsythin is suitable for further development and potential application.

Genotoxic effect induced by dried nicotiana tabacum leaves from tobacco barns (kiln-houses) in chinese hamster lung fibroblast cells (V79)

ABSTRACT Nicotiana tabacum is the most cultivated tobacco species in the state of Rio Grande do Sul, Brazil. Workers who handle the plant are exposed to the leaf components during the harvesting process and when separating and classifying the dried leaves. In addition to nicotine, after the drying process, other components may be found including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, as well as pesticides residues. The objective of this study was to examine the genotoxicity attributed to the aqueous extract of dried tobacco leaves obtained from tobacco barns using Chinese hamster lung fibroblast cells (V79) as a model system by employing alkaline comet assay, micronucleus (MN) and Ames test. MTT assay was used to assess cytotoxicity and establish concentrations for this study. Data demonstrated cell viability > 85% for concentrations of 0.625–5 mg/ml while the comet assay indicated a significant increase in DNA damage at all concentrations tested. A significant elevation of MN and nuclear buds (NBUD) was found for 5 mg/ml compared to control and other dry tobacco leaves concentrations (0.625–2.5 mg/ml). Mutagenicity was not found using the Salmonella/Microsome test (TA98, TA100, and TA102 strains) with and without metabolic activation. The concentration of inorganic elements was determined employing the PIXE technique, and 13 inorganic elements were detected. Using CG/MS nicotine amounts present were 1.56 mg/g dry tobacco leaf powder. Due to the observed genotoxicity in V79 cells, more investigations are needed to protect the health of tobacco workers exposed daily to this complex mixture of toxic substances present in dry tobacco leaves.

Nonclinical Toxicological Studies of Brazilian Red Propolis and Its Primary Botanical Source Dalbergia ecastaphyllum.

Propolis is one of the most widely used products in traditional medicine. One of the most prominent types of Brazilian propolis is the red one, whose primary botanical source is Dalbergia ecastaphyllum (L.) Taub. Despite the potential of Brazilian red propolis for developing new products with pharmacological activity, few studies guarantee safety in its use. The objective of this study was the evaluation of the possible toxic effects of Brazilian red propolis and D. ecastaphyllum, as well as the cytotoxicity assessment of the main compounds of red propolis on tumoral cell lines. Hydroalcoholic extracts of the Brazilian red propolis (BRPE) and D. ecastaphyllum stems (DSE) and leaves (DLE) were prepared and chromatographed for isolation of the major compounds. RP-HPLC-DAD was used to quantify the major compounds in the obtained extracts. The XTT assay was used to evaluate the cytotoxic activity of the extracts in the human fibroblast cell line (GM07492A). The results revealed IC50 values of 102.7, 143.4, and 253.1 μg/mL for BRPE, DSE, and DLE, respectively. The extracts were also evaluated for their genotoxic potential in the micronucleus assay in Chinese hamster lung fibroblasts cells (V79), showing the absence of genotoxicity. The BRPE was investigated for its potential in vivo toxicity in the zebrafish model. Concentrations of 0.8-6.3 mg/L were safe for the animals, with a LC50 of 9.37 mg/L. Of the 11 compounds isolated from BRPE, medicarpin showed a selective cytotoxic effect against the HeLa cell line. These are the initial steps to determine the toxicological potential of Brazilian red propolis.

Interaction of curcumin on cisplatin cytotoxicity in HeLa and HepG2 carcinoma cells

Background and Aims: Our study aimed to evaluate how curcumin affect cisplatin cytotoxicity in human cervical carcinoma (HeLa), human hepatocellular carcinoma (HepG2), and Chinese hamster lung fibroblast (V79) cells. Methods: The cytotoxicity was evaluated by MTT assay. Results: The IC50 values of curcumin were 404 μM and 320 μM in HeLa cells; 236 μM and 98.3 μM in HepG2 cells; 877 μM and 119 μM in V79 cells; for 24 h and 48 h, respectively. The IC50 values of cisplatin were 22.4 μM and 12.3 μM in HeLa cells; 25.5 μM and 7.7 μM in HepG2 cells; 15.4 μM and 4.9 μM in V79 cells; for 24 h and 48 h, respectively. Curcumin significantly decreased cisplatin cytotoxicity at 500 μM in HeLa cells and above 250 μM and 125 μM in HepG2 cells, for 24 h and 48 h, respectively. In V79 cells, curcumin significantly decreased the IC50 values of cisplatin above 500 μM and 125 μM for 24 h and 48 h. Conclusion: The results might contribute to the anticancer effect of the curcumin-cisplatin combination in cervical and hepatocellular carcinoma, but in order to support this result and determine its interactions with antineoplastic drugs, further studies are needed.

Green toxicological investigation for biofuel candidates

To avoid potential risks of biofuels on the environment and human, ecotoxicity investigation should be integrated into the early design stage for promising biofuel candidates. In the present study, a green toxicology testing strategy combining experimental bioassays with in silico tools was established to investigate the potential ecotoxicity of biofuel candidates. Experimental results obtained from the acute immobilisation test, the fish embryo acute toxicity test and the in vitro micronucleus assay (Chinese hamster lung fibroblast cell line V79) were compared with model prediction results by ECOSAR and OECD QSAR Toolbox. Both our experimental and model prediction results showed that 1-Octanol (1-Oct) and Di-n-butyl ether (DNBE) were the most toxic to Daphnia magna and zebrafish among all the biofuel candidates we investigated, while Methyl ethyl ketone (MEK), Dimethoxymethane (DMM) and Diethoxymethane (DEM) were the least toxic. Moreover, both in vitro micronucleus assay and OECD QSAR Toolbox evaluation suggested that the metabolites present higher genotoxicity than biofuel candidates themselves. Overall, our results proved that this green toxicology testing strategy is a useful tool for assessing ecotoxicity of biofuel candidates.

Investigation of Post‐Translational Modification on NHE1 Transport Function

The sodium hydrogen exchanger isoform 1 (NHE‐1) is a sodium hydrogen antiporter that regulates intracellular pH (pHi), cell volume, dynamic actin remodeling processes, and coordination of cell migration in mammalian tissues. NHE‐1 is post‐translationally modified by phosphorylation and lipid modification (palmitoylation). The intracellular C‐terminal domain of NHE‐1 is reversibly phosphorylated at multiple sites byseveral kinases including: extracellular signal‐regulated kinase (ERK), protein kinase B (AKT), B‐Raf, p90 Ribosomal S6 Kinase (p90rsk), RhoA Kinase (ROCK), and other protein kinases. We recently identified that NHE‐1 is reversibly palmitoylated (S‐acetylation), in which a 16‐carbon palmitic acid is covalently attached to a cysteine residue on the C‐terminal domain via a thioester linkage. Such modifications are reported to regulate protein trafficking, membrane micro localization, and protein‐protein interactions. Steady‐state pHi assays incubated with pH‐sensitive dye were conducted to measure the change in pHi of Chinese Hamster Lung Fibroblast Cells due to NHE‐1. Investigating agonists and stimulating factors that increase NHE‐1 palmitoylation allows us to determine the impact of palmitoylation on NHE‐1 transport. Palmitoyl‐transferases (PATs) catalyze the lipidation of proteins and are inhibited by 2‐bromo‐palmitate (2BP). The presence of 2BP causes a loss of palmitoylation to occur, effectively inhibiting LPA‐induced NHE‐1 activity by 0.1 pH units. Fetal Bovine Serum (FBS), Lysophosphatidic acid (LPA), Phorbol 12‐myristate 13‐acetate (PMA), and Insulin effectively stimulate NHE‐1 by increasing pHi by 0.1 pH units. In the presence of kinase inhibitors, NHE‐1 activity significantly decreases compared to agonist or serum treatment alone. The impact of the combination of kinase inhibitors and palmitoylation inhibitors on NHE‐1 mediated pHi change was determined. The data supports the hypothesis that palmitoylation and phosphorylation coordinate to subtly modify NHE1 activity in a novel rheostat of the cell.

Treatment with 3-Aminobenzamide Negates the Radiofrequency-Induced Adaptive Response in Two Cell Models.

In previous investigations, we demonstrated that pre-exposure of different cell cultures to radiofrequency fields can reduce the damage induced by genotoxic agents, an effect resembling the so-called adaptive response. In this study, we pre-exposed human peripheral blood lymphocytes and Chinese hamster lung fibroblast cell line to 1950 MHz, UMTS (Universal Mobile Telecommunication System) signal, for 20 h, and then treated cultures with Mitomycin-C. After confirming the induction of an adaptive response in terms of the reduction of micronuclei formation, we observed that such a response was negated by treatments with 3-aminobenzamide. Since 3-aminobenzamide is an inhibitor of poly (ADP-ribose) polymerase enzyme, which is involved in DNA repair, these results support the possible involvement of DNA repair mechanisms in radiofrequency-induced adaptive response.

An In Vitro Study on the Cytotoxicity and Genotoxicity of Silver Sulfide Quantum Dots Coated with Meso-2,3-dimercaptosuccinic Acid.

Silver sulfide (Ag2S) quantum dots (QDs) are highly promising nanomaterials in bioimaging systems due to their high activities for both imaging and drug/gene delivery. There is insufficient research on the toxicity of Ag2S QDs coated with meso-2,3-dimercaptosuccinic acid (DMSA). In this study, we aimed to determine the cytotoxicity of Ag2S QDs coated with DMSA in Chinese hamster lung fibroblast (V79) cells over a wide range of concentrations (5-2000 μg/mL). Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays. The genotoxic and apoptotic effects of DMSA/Ag2S QDs were also assessed by comet assay and real-time polymerase chain reaction technique, respectively. Cell viability was 54.0±4.8% and 65.7±4.1% at the highest dose (2000 μg/mL) of Ag2S QDs using the MTT and NRU assays, respectively. Although cell viability decreased above 400 μg/mL (MTT assay) and 800 μg/mL (NRU assay), DNA damage was not induced by DMSA/Ag2S QDs at the studied concentrations. The mRNA expression levels of p53, caspase-3, caspase-9, Bax, Bcl-2, and survivin genes were altered in the cells exposed to 500 and 1000 μg/mL DMSA/Ag2S QDs. The cytotoxic effects of DMSA/Ag2S QDs may occur at high doses through the apoptotic pathways. However, DMSA/Ag2S QDs appear to be biocompatible at low doses, making them well suited for cell labeling applications.

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 (1-20μg/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 6hours 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.

Effects of silver sulfide quantum dots coated with 2-mercaptopropionic acid on genotoxic and apoptotic pathways in vitro

Quantum dots (QDs) are highly promising nanomaterials in bioimaging system because of their bright fluorescence, broad UV excitation, narrow emission band, and high photostability. Recently, there is a great activity on Ag2S quantum dots for both imaging and drug/gene delivery due to the potential of having a better cytocompatability and near infrared luminescence. 2-Mercaptopropionic acid (2 MP A)-coated silver sulfide (Ag2S) QDs were reported as the most luminescent, stable, anionic Ag2S QDs in the literature. In this study, we aim to determine the cytotoxicity of 2 MP A/Ag2S in Chinese hamster lung fibroblast (V79) cells. The genotoxic and apoptotic effects of 2 MP A/Ag2S QDs were assessed by the alkaline single cell electrophoresis assay and real time polymerase chain reaction techniques, respectively. The cell viability decreased above 200 μg/ml and 800 μg/ml for MTT tetrazolium and neural red uptake assays, respectively. DNA damage was not observed by 2 MP A/Ag2S QDs at the studied concentration levels (5–2000 μg/ml). The levels of mRNA expression of p53, caspase 3, caspase 9, bax, bcl-2, survivin were not changed by 2 MP A/Ag2S QDs below IC50 (around 1000 μg/ml). Hence, 2 MP A/Ag2S QDs did not show any cytotoxic or genotoxic effects in V79 cells at lower doses. We conclude that the biocompatibility of 2 MP A/Ag2SODs makes them suitable for cell labeling applications.

In vitro genotoxicity assessment of dinitroaniline herbicides pendimethalin and trifluralin

Pendimethalin and trifluralin are widely used dinitroaniline herbicides. Both compounds can be found as residue levels in agricultural products. This study was conducted in order to provide necessary information for the risk assessment of pendimethalin and trifluralin. In this study, reactive oxygen species (ROS) levels were measured to examine the potential of both compounds to induce oxidative damage in Chinese hamster lung fibroblast (V79) cells. Also, the genotoxic effects of pendimethalin and trifluralin at the concentration range of 1-500 μM was determined. Single cell gel electrophoresis (comet) and micronucleus assays were used on human peripheral lymphocytes and V79 cells for the genotoxicity assessment. The cell viability of two dinitroaniline herbicides were determined by the use of neutral red uptake assay on V79 cells. IC50 values were determined as 66 μM and 128 μM for pendimethalin and trifluralin, respectively. They significantly increased ROS levels on V79 cells for 1-24 h. Both herbicides significantly induced the DNA damage and showed genotoxicity on lymphocytes and V79 cells. Micronucleus frequency increased significantly after pendimethalin and trifluralin treatment of the lymphocytes and V79 cells. Therefore, we concluded that both of the herbicides induced the genotoxicity through the activation of oxidative stress pathway and chromosomal damage.

Toxicological evaluation of β-Galactosidase enzyme produced by Papiliotrema terrestris

β-Galactosidase (EC 3.2.1.23) is a glycoside hydrolase that catalyzes the release of sugar monomers from β-galactoside oligosaccharides via hydrolysis of glycosidic bonds, and has potential uses in the food industry. The safety of this enzyme and its production organism, Papiliotrema terrestris (P. terrestris), are described herein. P. terrestris was non-pathogenic upon intravenous (IV) administration of 2.4 × 108 cfu and oral administration of 1.3 × 109 cfu. Genotoxic results for the enzyme concentrate were negative in a bacterial reverse mutation test (Ames test) and chromosome aberration test in cultured Chinese hamster lung fibroblast (CHL/IU) cells. In a 13-week oral gavage study in Sprague-Dawley rats, no adverse effects were observed in any of the tested group and a No Observed Adverse Effects Level (NOAEL) of 2000 mg/kg bw/day [total organic solids (TOS) 1800 mg/kg bw/day)] was established, which was the highest dosage tested. Allergenicity sequence analysis revealed no evidence suggesting that β-galactosidase is an allergen. The data presented in this study support the conclusion that β-galactosidase produced from P. terrestris is safe for use in food production.

Regulatory role of miR-18a to CCN2 by TGF-β1 signaling pathway in pulmonary injury induced by nano-SiO2.

This research is designed to investigate the regulatory effect of miR-18a to the target gene connective tissue growth factor (CTGF, or CCN2), by participating in TGF-β1 signaling pathway and explore the pathogenic mechanism of miR-18a in pulmonary injury induced by nano-SiO2 based on our early study. miR-18a and expression of TGF-β1 in Chinese hamster lung (CHL) fibroblasts cells stimulated by supernatants of NR8383 cells exposed to 40μg/ml nano-SiO2 for 24h demonstrated 1.58±0.22-fold and 1096.00±2.60pg/ml increase compared with blank control group analyzed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression increase of miR-18a and reduction of CCN2 mRNA expression levels and protein gray value ratio detected by Western blotting in CHL cells transfect miR-18a mimics for 48h. The reverse of CHL cell transfection miR-18a inhibit is also true. The result of miR-18a and CCN2 binding sites tested by luciferase reporter gene assay shows that the report relative fluorescence value of miR-18a mimics wild type on CCN2 is 0.50±0.02 with the control of mimics NC and mutant relative fluorescence report value 0.86±0.04 (P<0.05). Expression levels of miR-18a, CCN2 mRNA, and protein gray value ratio decreased in CHL cells treated by TGF-β1, respectively, and vice versa treated by TGF-β1corepressor. The results suggest that CCN2 is the target gene regulated by miR-18a and miR-18a participates in TGF-β1 signaling pathway by regulating the expression of CCN2 negatively through CCN2 3'UTR site, and thus may be involved in the development process of pulmonary injury.