Neutral Red Uptake Assay Research Articles

EVALUATION OF ROUNDUP® TOXICITY IN HUMAN LUNG CELLS

Objective: In this study, toxic effects of Roundup, one of the most common glyphosate-based herbicides (GBHs), were assessed on human bronchial epithelial cells (BEAS-2B).
 Material and Method: MTT and neutral red uptake assays were implemented for evaluation of cell viability at 24 and 48 h. Apoptosis detection was made by Muse analyzer while Hoechst staining was employed to detect apoptotic nuclear changes. In addition, dichlorofluorescein diacetate assay was used for the assessment of reactive oxygen species (ROS) formation.
 Result and Discussion: Similar half maximal inhibitory concentrations (IC50) were obtained from cytotoxicity assays. Results showed that significant reduction in the viability of BEAS-2B cells started to occur from 200 µM at 24 h and 50 µM at48 h treatment times. Roundup treatments for both time points were found to dose-dependently cause apoptosis and were also observed to induce cellular ROS formation. These findings suggest that GBHs can stimulate ROS production, as well as apoptosis on healthy human lung cells which is important considering inhalation is one of the primary exposure routes to these group of chemicals.

Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro.

The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.

Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 μM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 μM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 μM) and CLO (0.01 and 1 μM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 μM. HAL and CLO present cytotoxicity at 0.1 μM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics.

Mutagenic and antimutagenic evaluation of Asparagus laricinus Burch., Senecio asperulus DC., and Gunnera perpensa L. to hepatic cells

Introduction. The use of traditional medicinal plant concoctions to cure or treat different diseases daily in African folk medicine. However, the effects of most medicinal plants on human health or genetic material remain unknown. This study thus aimed to evaluate the mutagenic and antimutagenic potentials of Asparagus laricinus Burch. cladodes, Senecio asperulus DC., and Gunnera perpensa L. roots extract in vitro. Methods. Neutral red uptake assay, alkaline comet assay, and the VITOTOX test was used with plant extract dilutions of 4, 20, 50, and 100 µg/ml, respectively, on hepatic (C3A) cells and Salmonella Typhimurium TA104 strains. Ethyl methane-sulfonate and 4-nitroquinoline oxide were used as positive controls for the comet and VITOTOX assays, respectively. Results. In vitro cytotoxicity and genotoxicity were not observed from all tested extracts, except for the two dichloromethane (DCM) extracts of S. asperulus and G. perpensa, which appeared to be cytotoxic with S9 metabolic activation, but not genotoxic or mutagenic. From the VITOTOX test results, none of the extracts appeared to have antimutagenic properties after treating S. Typhimurium strains with a known mutagen. Conclusions. These results confirm that previously reported anticarcinogenic properties of A. laricinus, S. asperulus, and G. perpensa did not result from the protective mechanism against genotoxicity but from other ones. Moreover, the negative mutagenic and cytotoxic activities of the tested plants highlighted the safe use of these medicinal plants in vitro. Therefore, S. asperulus and G. perpensa DCM extracts require further investigation for their possible in vivo cytotoxic effects on humans.

Design, Synthesis, Docking Studies and Biological Evaluation of Novel Benzochromenopyrimidines via Silica Sulfuric Acid Catalyzed Reaction on Apoptosis in Human Cancer Cells

Via one-pot reaction of 2-naphthol, thiobarbituric acid and aldehyde in the presence of silica sulfuric acid (SSA) as a catalyst to synthesis benzochromenopyrimidines derivatives. Then we studied the reaction of benzochromenopyrimidines derivatives with bromoacetic acid to obtain benzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidine-11,13(10H,14H)-dione derivatives. Meanwhile, dichloroethane was reacted with 12-(4-fluorophenyl)-9-thioxo-9,10-dihydro-8H-benzo[5,6]chromeno[2,3-d]pyrimidin-11(12H)-one to form 14-(4-fluorophenyl)-10,11-dihydrobenzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidin-13(14H)-one. All new products structure was elucidated based on analytical and spectroscopic analyses. Anti-cancer activity of newly synthesized structures was investigated. Cancer cells (MDA-MB-231 and HepG2) were used to evaluate the anti-cancer activity of tested compounds using neutral red uptake assay. Treatment of MDA-MB-231 and HepG2 cells with tested compound 6h revealed more inhibitory influence after 48 h. The expression levels of BCL2, BAX, Caspase3, Survivin and P53 genes were investigated using QRT-PCR. This study exhibited that compound 6h revealed significant pro-apoptotic effect via down regulation of BCL2 and Survivin genes and up-regulation of BAX, Caspase3 and P53 genes when treated with MDA-MB-231 and HepG2 cells as compared to control values. The biological studies of these compounds were proved through molecular docking studies with human cyclin-dependent kinase 2 (PDB code: 2A4L) and compound 6h showed low binding energy and shortage bond length with different amino acids. Noteworthy, the tested compound 6h exhibited the most pronounced effect in this respect.

Evaluation of the cytotoxic and genotoxic/antigenotoxic effects of resveratrol in human limbal explant cultures.

Resveratrol (RSV) is a natural polyphenol phytoalexin compound and has long been considered to possess antioxidant and anti-inflammatory effects. In order to exploit the protective potential of RSV in anterior segment diseases, we investigated the possible cytotoxic, genotoxic/antigenotoxic effects of human limbal explant cultures to RSV and MMC or H2O2 alone and in combination. A total of 18 limbal explant tissues obtained from three corneal donors were placed on the 12 well tissue culture polystyrene plates and cultured for 14days. Cell growth from limbal explants was observed by inverted phase contrast microscopy. The cytotoxic effects of RSV was studied by neutral red uptake assay. For the assessment of the genotoxic and antigenotoxic effects, basic alkaline technique of comet assay was performed. It was found that the concentrations of RSV up to 100μM did not significantly affect the viability of outgrowth cells of limbal explant during 24h exposure. When compared to negative control, all concentrations of RSV alone caused an increase in DNA strand breakage. Interestingly, 10μg/mL MMC alone caused similar tail intensity and tail moment values with RSV alone. On the other hand, RSV treatment in all doses seemed to decrease the DNA damage induced by either H2O2 or MMC. RSV is an attractive natural compound for the purpose of oxidative stress reduction in ocular surface and can be utilized as a supplement to promote ocular surface regeneration in vivo.

IN-VITRO MELANOGENESIS, CYTOTOXICITY AND ANTIOXIDANT ACTIVITIES OF Peltophorum pterocarpum LEAF EXTRACTS

Melanin is a protective pigment against cellular damage and skin cancer. Peltophorum pterocarpum has been used for centuries to treat skin diseases like eczema and psoriasis. However, whether P. pterocarpum leaf extract can affect melanin synthesis with the advantage of antioxidant defense against ultraviolet (UV) radiation remains to be investigated. We aimed to examine the melanogenesis, cytotoxicity, and antioxidant activities of P. pterocarpum leaf extracts. A two-dimensional (2D) cell culture model was employed to demonstrate the effect of P. pterocarpum extracts on melanin synthesis. The cell viability of B16-F1 melanoma cells was measured by Neutral Red Uptake (NRU) assay. Antioxidant activity was accessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assays. The ethanol extract of P. pterocarpum dose-dependently increased the melanin content and displayed cytotoxicity to B16-F1 melanoma cells at the highest concentration. In contrast, the equal amounts of the aqueous extract significantly inhibited melanin synthesis and did not show any cytotoxic effects on B16-F1 melanoma cells. The P. pterocarpum ethanol extract had a significantly higher total phenolic and flavonoid content than the aqueous extract and was more effective at scavenging DPPH free radicals and intracellular ROS induced by UVB, with IC50 of 519.24 ± 122.57 vs 1798.45 ± 143.09 µg/mL and 878.00 ± 23.50 vs 1379.00 ± 21.81 µg/mL, respectively. These findings suggest that the pro-melanogenic and anti-melanogenic activities of P. pterocarpum extract with antioxidant capacity against UVB-induced cellular damage are affected by the total phenolic and flavonoid contents.

Anti-proliferative action, molecular investigation and computational studies of novel fused heterocyclic cellulosic compounds on human cancer cells

Reactivity of MCEC (3) with diazonium chloride, DMF-DMA and NH2NH2 to afford fused heterocyclic cellulosic derivative which confirmed through spectral analysis. Also, the cellulosic compounds were evaluated their growth inhibitory activity contra cancer cells A549 and Caco2 using neutral red uptake assay. Compounds, MCEN(6), MCPT(5a), MCPT(5b) showed better growth inhibitory activity on A549 and Caco2 growth compared to control values. While compound MCPY(7) exerted the best cytotoxic activity against A549 cells after 48 h. The expression levels of HIF-1α, β-Catenin, MYC, Cyclin D1 and MMP7 genes in A549 cells were examined using QRT-PCR. The compounds MCEN(6) and MCPY(7) down regulated levels of β-Catenin, MYC, Cyclin D1 and MMP7 genes and up-regulated levels of HIF-1α when treated with A549 cells compared to control values. Also, these biological studies confirmed through docking stimulation with different proteins and showed least binding energy with amino acids and attached with NH2 and OH of cellulose with hydrogen bond interaction. Moreover, optimization of compounds using DFT/6-311(G) showed the stability of them and identifies their physical descriptors which showed excellent correlation with experimental results. Noteworthy, compounds, MCEN(6) and MCPY(7) were most promising anticancer agents against lung cancer through the regulation of apoptosis, cell cycle, proliferation, progression, chemo-resistance, and angiogenesis.

Fabrication of a Patterned Scaffold Using Soft Lithography Technique to be Used in Cell Growth Applications.

In recent years, within tissue engineering, cell growth on patterned surfaces have gained significant attention. Growing cells in patterns is important to manufacture polymeric tissues that can be used within the medical field. For this reason, the main focus of this study was to prepare patterned scaffolds using Titanium (Ti) and polyvinyl chloride (PVC) covered on microscope lamellas and examine their liability for cell growth. A polydimethylsiloxane stamp was initially prepared which was then used to transfer a predefined pattern onto PVC- and Ti-covered surfaces. Cell growth experiments were performed on the prepared materials by seeding L929 mouse fibroblasts. The growth of cells seeded on the surface of the scaffolds were spectroscopically followed using Neutral Red uptake assay. The results showed cell proliferation on both patterned surfaces, however, it was higher on Ti-covered samples. In addition, three different alkanethiols were tested for cell adhesion on patterned surfaces. A higher number of cell proliferation was observed with undecanethiol, which has a shorter alkane group among them. The morphological properties of the samples before and after cell-seeding were analyzed via scanning electron microscope and optical microscopy. Significant amount of cell proliferation was observed on all of the prepared samples.

Combined Effect of Neutron Radiation and Curcumin on Breast Cancer Cells Cytotoxicity in 3D Culture Medium

Introduction: Chemotherapy, biotherapy, and radiotherapy play a limited but important role in treating breast cancer. For more efficient treatment, combination therapy could be an appropriate option. In this study, radiotherapy using neutron radiation emitted from a 241Am-Be neutron source, as well as biotherapy using curcumin (80 μM) was combined to investigate the efficiency of treatment towards MCF-7 breast cancer in a three dimensional (3D) culture medium. Methods: MTT, neutral red uptake assay, nitric oxide, glutathione assay, catalase, cytochrome c, comet assay, and caspase-3 were used to determine the effect of neutron radiation and also neutron and curcumin combination on the viability of cancer cells. Results: The results of cytotoxicity test showed that neutron irradiation with or without curcumin at 5, 10, 15, and 20 h reduced the survival of tumor cells. Moreover, the rate of apoptosis due to the neutron effect at different irradiation times enhanced with the increasing time. Conclusion: Due to the significant anticancer effect of curcumin in 3D culture, using this molecule before or after neutron therapy is recommended.

Anti-quorum sensing and cytotoxic activity of elemi essential oil

Essential oils have several biological activities such as antimicrobial, antioxidant, proliferative, and anti-inflammatory. This study aimed identification of bioactive compounds found in Elemi essential oil (EO) and to determine the anti-quorum sensing and cytotoxic activities of EO. In this study, bioactive compounds of EO were analyzed using GC-MS, and the antibacterial activity of elemi was screened against Staphylococcus aureus ATCC 25923, Methicillin-Resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa PAO1. Anti-biofilm activity and pyocyanin production on P. aeruginosa PAO1 were also investigated. The effect of EO on cell viability was also analyzed by thiazolyl blue tetrazolium bromide (MTT) and neutral red uptake (NR) assay in fibroblast cells. According to GC results, the major component of EO was determined as limonene (55%). A sub-MIC of elemi essential oil inhibited biofilm formation and pyocyanin production by 43% and 56%, respectively. On the other hand, EO also had an acute effect on the mitochondrial and lysosomal activities of fibroblast cell lines. Mitochondrial and lysosomal activities were significantly decreased when EO concentrations were applied for 24 and 48 hours (p<0.05). In conclusion, EO has inhibitory activity on biofilm formation and pyocyanin production, and also the lower doses of oil have no toxic effects on fibroblast cells. However, higher doses of EO have more cytotoxic effects on mitochondrial activity rather than the lysosomal activity of fibroblast cell lines. It is thought that EO exhibits these activities due to the amount of limonene in its content.

First evaluation of the anxiolytic-like effects of a bromazepam‑palladium complex in mice

A significant fraction of patients are affected by persistent fear and anxiety. Currently, there are several anxiolytic drug options, however their clinical outcomes do not fully manage the symptoms. Here, we evaluated the effects of a bromazepam‑palladium derivative [2-{(7-bromo-2-oxo-1,3-dihydro-2H-1,4-benzodiazepin-5-il)pyridinyl-κ2-N,N}chloropalladium(II)], [(BMZ)PdCl2], on fear/anxiety and memory-related behavior in mice. For this, female Swiss mice were treated intraperitoneally (i.p.) with saline (NaCl 0.9%) or [(BMZ)PdCl2] (0.5, 5.0, or 50 μg/kg). After 30 min, different tests were performed to evaluate anxiety, locomotion, and memory. We also evaluated the acute toxicity of [(BMZ)PdCl2] using a cell viability assay (neutral red uptake assay), and whether the drugs mechanism of action involves the γ-aminobutyric acid type A (GABAA) receptor complex by pre-treating animals with flumazenil (1.0 mg/kg, i.p., a competitive antagonist of GABAA-binding site). Our results demonstrate that [(BMZ)PdCl2] induces an anxiolytic-like phenotype in the elevated plus-maze test and that this effect can be blocked by flumazenil. Furthermore, there were no behavioral alterations induced by [(BMZ)PdCl2], as evaluated in the light-dark box, open field, and step-down passive avoidance tests. In the acute toxicity assay, [(BMZ)PdCl2] presented IC50 and LD50 values of 218 ± 60 μg/mL and 780 ± 80 mg/kg, respectively, and GSH category 4. Taken together, our results show that the anxiolytic-like effect of acute treatment with [(BMZ)PdCl2] occurs through the modulation of the benzodiazepine site in the GABAA receptor complex. Moreover, we show indications that [(BMZ)PdCl2] does not promote sedation and amnesia and presents the same toxicity as the bromazepam prototype.

Preventive potential of Bacillus sonorensis exopolysaccharide upon hepatocellular carcinoma and quantitation of tumor suppressor protein p53.

Exopolysaccharide, a carbohydrate polymer, is known to possess several biological activities. This approach was designed to clarify the cytotoxic mechanism of Bacillus sonorensis exopolysaccharide (EPS-1) on Huh7, HepG2 and BNL cells besides exploring its influence on the expression of the tumor suppressor protein p53. p53 is the biomarker of the prognosis and occurrence of severe stages of the tumor and activation of both cell-cycle arrest and apoptosis in cancer cells which are the most targeted cellular processes for the therapy of tumor patients. The cytotoxic impact of EPS-1 was quantified via neutral red uptake assay and the results were confirmed by a morphology study. The expression level of p53 was analyzed using quantitative real-time PCR. The outcomes of the present study explicated that EPS-1 with IC 50 = 164 and 398 µg ml -1 exhibited an inhibitory influence on Huh7 and HepG2 cells growth after 48 h incubation time respectively. EPS-1 showed no influence on normal BNL cells. Furthermore, the molecular genetic analysis revealed that EPS-1 provoked significant upregulation in the expression level of the p53 gene in the treated Huh7 cell line more than that in HepG2, whereas no significant gene expression was noticed in BNL cells ( P = 0.006, 0.65 and 0.83), respectively. The antitumor activity displayed by this compound may be of interest for further studies of its structure-activity relationship. Before application in phase 1 of the clinical study, in-vivo studies would be needed to confirm the results obtained in the hope of finding more active and selective anticancer agents for drug development in the future.

Disposable Puff Bar Electronic Cigarettes: Chemical Composition and Toxicity of E-liquids and a Synthetic Coolant.

The popularity of disposable fourth-generation electronic cigarettes (ECs) among young adults and adolescents has been increasing since the ban on flavored cartridge EC products such as JUUL. Although the constituents and toxicity of some cartridge-based fourth-generation ECs, such as JUUL, have been studied, limited data exist for other disposable ECs such as Puff. The purpose of this study was to determine flavor chemicals, synthetic coolants, and nicotine concentrations in 16 disposable Puff devices, evaluate the cytotoxicity of the different flavors from the Puff brand using in vitro assays, and investigate the health risks of synthetic coolants in EC products. Gas chromatography/mass spectrometry was used to identify and quantify chemicals in Puff EC fluids. One hundred and twenty-six flavor chemicals were identified in Puff fluids, and 16 were >1 mg/mL. WS-23 (2-isopropyl-N,2,3-trimethylbutyramide) was present in all products, and concentrations ranged from 0.8 to 45.1 mg/mL. WS-3 (N-ethyl-p-menthane-3-carboxamide) concentrations ranged from 1.5 to 16.4 mg/mL in 6/16 products. Nicotine concentrations ranged from 40.6 to 52.4 (average 44.8 mg/mL). All unvaped fluids were cytotoxic at dilutions between 0.1 and 10% in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. The cytotoxicity of Puff fluids was highly correlated with total chemical concentrations, nicotine, WS-23, both synthetic coolants, and synthetic coolants plus ethyl maltol. Lower concentrations of WS-23 than those in the fluids adversely affected cell growth and morphology. Concentrations of synthetic coolants exceeded levels used in consumer products. The margin of exposure data showed that WS-3 and WS-23 concentrations were high enough in Puff products to present a health hazard. Our study demonstrates that disposable Puff ECs have high levels of cytotoxic chemicals. The data support the regulation of flavor chemicals and synthetic coolants in ECs to limit potentially harmful health effects.

Toxicity of pesticides widely applied on soybean cultivation: Synergistic effects of fipronil, glyphosate and imidacloprid in HepG2 cells

The transgenic soy monoculture demands supplementation with pesticides. The aim of this study was to evaluate the individual and mixture effects of fipronil, glyphosate and imidacloprid in human HepG2 cells. Cytotoxicity was evaluated after 48-h incubations through MTT reduction and neutral red uptake assays. Free radicals production, mitochondrial membrane potential, DNA damage, and release of liver enzymes were also evaluated. Data obtained for individual agents were used to compute the additivity expectations for two mixtures of definite composition (one equipotent mixture, based in the EC50 values achieved in the MTT assay; the other one based in the acceptable daily intake of each pesticide), using the models of concentration addition and independent action. The EC50 values for fipronil, glyphosate and imidacloprid were 37.59, 41.13, and 663.66 mg/L, respectively. The mixtures of pesticides elicited significant synergistic effects (p < 0.05), which were greater than the expected by both addictive predictions. Decreased in mitochondrial membrane potential and increased in the transaminases enzymatic activities were observed. As they occur simultaneously, interactions between pesticides, even at non-effective single levels, can reverberate in significant deleterious effects, justifying the need for a more realistic approach in safety evaluations to better predict the effects to human health.

Antimicrobial Effects of Nanostructured Rare-Earth-Based Orthovanadates.

The search for novel antimicrobial agents is of huge importance. Nanomaterials can come to the rescue in this case. The aim of this study was to assess the cytotoxicity and antimicrobial effects of rare-earth-based orthovanadate nanoparticles. The cytotoxicity against host cells and antimicrobial activity of LaVO4:Eu3+ and GdVO4:Eu3+ nanoparticles were analyzed. Effects of nanomaterials on fibroblasts were assessed by MTT, neutral red uptake and scratch assays. The antimicrobial effects were evaluated by the micro-dilution method estimating the minimum inhibitory concentration (MIC) of nanoparticles against various strains of microorganisms, DNA cleavage and biofilm inhibition. GdVO4:Eu3+ nanoparticles were found to be less toxic against eukaryotic cells compared with LaVO4:Eu3+. Both nanoparticles exhibited antimicrobial activity and the highest MIC values were 64mg/L for E. hirae, E. faecalis and S. aureus shown by GdVO4:Eu3+ nanoparticles. Nanoparticles demonstrated good DNA cleavage activity and induction of double-strand breaks in supercoiled plasmid DNA even at the lowest concentrations used. Both nanoparticles showed the biofilm inhibition activity against S. aureus at 500mg/L and reduced the microbial cell viability. Taken the results of host toxicity and antimicrobial activity studies, it can be assumed that GdVO4:Eu3+ nanoparticles are more promising antibacterial agents compared with LaVO4:Eu3+ nanoparticles.

Gaz Alafi: A Traditional Dessert in the Middle East With Anticancer, Immunomodulatory, and Antimicrobial Activities.

BackgroundFrom the earliest times, manna has been widely used as a tasty local sweet or folk medicine. The type of manna being investigated in the present study is called Gaz-alafi, a mixture of insect and Quercus brantii leaves secretions from oak forests in the north of Iraq and west of Iran.MethodsAqueous and ethanol extracts were prepared as decoction. Various phytochemical tests were conducted to analyze manna composition, including total phenolic contents using the Folin-Ciocalteu method and LC-MS. Gallic acid and catechin were detected in both extracts, in addition to tiliroside presence in ethanol extract, which added more value to the phenolic content of ethanol extract. Cytotoxic activities of Gaz alafi were evaluated against breast cancer cell lines and compared to normal cell lines and doxorubicin using the MTT assay. Antimicrobial properties were assessed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Candida albicans using the dilution method of the micro-titer plate. Serum levels of IFN-γ, interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were measured using ELISA. The effect of extracts on splenocyte proliferation was evaluated using the lymphocytes proliferation assay. Macrophage function was evaluated using the nitro blue tetrazolium assay, whereas pinocytosis was evaluated using the neutral red uptake assay. Ten days after tumor inoculation, changes in tumor size, survival rates, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were measured.ResultsThe growth of cancer cells was inhibited by Gaz alafi ethanol extract. An alteration in IFN- γ, IL-2, and IL-4 levels toward antiproliferation immune response were reported for both extracts. The aqueous extract efficiently stimulated lymphocyte proliferation, phagocytosis, and pinocytosis, followed by the ethanol extracts with moderate activity. After treating the mice with ethanol extracts, a significant reduction in tumor size and several undetected tumors were recorded.ConclusionsGaz alafi extracts (aqueous and ethanol) are promising sources for anticancer and immunostimulatory agents. Further studies are needed to fully identify the chemical composition of Gaz alafi extracts.

Dose-Dependent Variation in Anticancer Activity of Hexane and Chloroform Extracts of Field Horsetail Plant on Human Hepatocarcinoma Cells.

Horsetail fern plant is botanically known as Equisetum arvense L., and it is a good source of phenolic flavonoids, phenolic acids, and compounds. Anticancer properties of hexane and chloroform extracts of the horsetail fern plant and their mechanisms involved in the anticancer activity on human hepatocarcinoma (HuH-7) cells were examined. Cytotoxicity was evaluated by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and NRU (neutral red uptake) assays. Other parameters such as oxidative stress and apoptosis in pretreated hexane and chloroform extracts of the horsetail fern plant were examined in HuH-7 cells. The observation showed that hexane and chloroform extract of the horsetail fern plant exhibited cytotoxicity against HuH-7 cells. The value of IC50-24 h of hexane and chloroform extract of the horsetail fern plant was determined as 199.0 μg/ml and 161.90 0 μg/ml for HuH-7 cells, respectively, and on the basis of IC50 value, three acute concentrations, viz., 75% of IC50, 50% of IC50, and 25% of IC50, were determined for further study. The lower dose of extracts hexane and chloroform extract of the horsetail fern plant did not show significant toxicity. Higher concentrations of extract induced significant antioxidant effects as well as apoptosis effects. However, exposure to hexane and chloroform extract of the horsetail fern plant upregulated the expression of Bax and p53 in HuH-7 cells. These data suggest that hexane and chloroform extract of the horsetail fern plant plays a significant role in the induction of toxicity via the regulation of oxidative stress in HuH-7 cells. This work may be useful for cancer chemotherapy.

Neodymium oxide nanostructures and their cytotoxic evaluation in human cancer cells

Neodymium oxide exhibits a unique property, which facilitates and largely utilized as an industrial applications. A number of cytotoxic study is available but very limited information is available to understand their biological activity with neodymium oxide at a very low conc- entration of the material. The present work was designed to understand the cytotoxicity against liver (HepG-2) and lung (A-549) cancer cells. Initially, Neodymium oxides (Nd2O3) were prepared and characterized with various instruments. The crystallinity and morphology of Nd2O3 powder were examined with instruments such as X-Ray Diffraction (XRD), scanning electron microscope (SEM), Transmission electron microscopy (TEM), Energy Dispersive X-Ray Analysis (EDX) respectively, revealed the size of curved nanostructure are ~140 ± 2 in diameter whereas length goes upto ~700 nm with elemental composition. The cytotoxicity study was conducted with MTT, NRU assay with genotoxicity study via ROS, cell cycle and qPCR analysis. The cells cytotoxic assessment were analysed via MTT(3-(4,5-Dimethylthiazol-2-yl)− 2,5-Diphenyl tetra zolium Bromide) and Neutral Red Uptake (NRU) assay with neodymium oxide (Nd2O3), which indicates the reduction in cell viability. Additionally, cell-cycle analysis showed an increase in the apoptotic peak after a 24-h. Quantitative real-time PCR (RT-PCR) data revealed that apoptotic genes such as p53, bax, and caspase-3 were up regulated, whereas bcl-2, an anti-apoptotic gene, was down regulated; therefore, apoptosis was mediated through ROS and genotoxicity pathways. The experiments of cytotoxicity was tested and concludes that the Nd2O3 express a moderate and dose dependent effect on cancer cells. The ROS, cell cycle analysis and qPCR showed that Nd2O3 exhibit the capability to cells death via ROS generation and genotoxicity study pathways.

Chemical analysis of selected harmful and potentially harmful constituents and in vitro toxicological evaluation of leading flavoured e-cigarette aerosols in the Chinese market.

Use of electronic cigarettes (e-cigarettes) has increased significantly over the past decade due to consumer perception that these products represent a less risky alternative to combustible cigarettes. E-liquids generally contain a simple mix of vegetable glycerin, propylene glycerol, nicotine, organic acids, and flavourings. Regulators require that harmful and potentially harmful constituents (HPHCs) that might cause harm to the consumer must be monitored in the aerosol generated by e-cigarettes and in cigarette smoke (CS). To quantify HPHCs in aerosols from commercial flavoured e-cigarettes in Chinese market, this study has systematically compared levels of HPHCs, including eight carbonyls, five volatile organic compounds, four tobacco-specific nitrosamines, 16 polycyclic aromatic hydrocarbons, and seven heavy metals, in the aerosols of four market-leading flavoured e-cigarettes and mainstream CS, alongside in vitro cytotoxicity and mutagenicity assays. The vast majority of HPHCs were either undetected or significantly lower in the e-cigarette aerosols than in commercial CS or reference CS (3R4F). Where HPHCs were detected, there were small variations among the different flavoured e-cigarettes. In the neutral red uptake and Ames assays, aqueous extracts of the e-cigarette aerosols did not induce obvious cytotoxicity or mutagenicity, whereas CS aqueous extract showed dose-related cytotoxicity and mutagenicity. Collectively, these results indicate that use of e-cigarettes might potentially lead to a significant reduction in exposure to harmful substances, with fewer cytotoxic and mutagenic effects, as compared with conventional smoking. Further studies based on human puffing conditions and longer evaluation periods will be needed to substantiate this potential.