Neutral Red Uptake Research Articles

Cytotoxic and genotoxic effects of leaking chemicals from serum infusion sets: an in-vitro study.

Safety concerns about medical devices playing important role in health sciences and bioengineering research are rising day by day. Although there are specific standards regarding disposable medical materials, the information is updating with the toxicological studies. In this study, cytotoxic/genotoxic effects of chemicals leaking from serum infusion sets that have an important place in the clinic were investigated. Media containing leakage chemicals were prepared from equal samples taken from the plastic line sections of 13 different brands of serum infusion sets containing phthalates and the effects on the cultured cells were compared with the untreated control groups. To obtain leaking chemicals, extracting period was selected as 72h, a routine set-change time in the clinic as indicated in various publications. Neutral red uptake and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests were performed in L929 cells to determine cytotoxicity, and cytokinesis blocked micronucleus technique was performed in lymphocytes to determine genotoxicity. Cytotoxic and genotoxic damage levels were compared by evaluating cell-viability rates relative to control, micronucleus frequency, and nuclear division index values. The results showed that all sets caused a decrease in cell viability revealing the effects both on lysosomal and mitochondrial activity and increase in micronucleus frequencies in general. The number of similar studies is extremely limited, and in this study in addition to the short-term effects of using the serum infusion sets, the information about the sample tests to determine the biosecurity of disposable medical materials is given.

RAW 264.7 Macrophage Cell Line: In Vitro Model for the Evaluation of the Immunomodulatory Activity of Zingiberaceae

The immune response plays an essential role in the body's defense against infection. Macrophages are promising targets against which to screen agents that modulate immune responses. In vitro analysis, primarily using cell lines, is preferred to screen bioactivity initially. RAW 264.7 cells comprise a model macrophage cell line close to primary murine macrophages. Indonesian people often use the Zingiberaceae family or “empon-empon" in the favorable treatment of several diseases, including immune disorders. This review highlights several studies of Zingiberaceae using RAW 264.7 cells, focusing on the observed immunomodulatory activity and assay methods. The research on RAW 264.7 macrophage cell line in Zingiberaceae was gathered using Scopus, PubMed, ScienceDirect, and Google Scholar from 2012-2021. Based on the reviews, the Zingiberaceae family has immunomodulatory effects by increasing or decreasing inflammatory mediators such as NO, IL-6, TNF-α, IL-1β, IL-10, and phagocytosis activity in RAW 264.7 cells. Several methods can assess the activation of RAW 264.7 cells, including cell viability assays using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT); measuring nitric oxide production (Griess reaction), cytokine production (enzyme-linked immunosorbent assay [ELISA]), and phagocytosis (neutral red uptake assay); and detecting DNA (real-time quantitative polymerase chain reaction [qPCR]). The information provided in this review presents avenues for future investigations of the immunomodulatory activity of Zingiberaceae.

Ciprofloxacin-Loaded Composite Granules Enriched in Silver and Gallium Ions—Physicochemical Properties and Antimicrobial Activity

Various calcium phosphates (hydroxyapatite, α- and β-tricalcium phosphate, and brushite) containing silver or gallium ions were synthesized via standard methods and subjected to physicochemical analysis by Fourier transform infrared spectroscopy (FT-IR), powder X-ray diffractometry (PXRD), and atomic absorption spectrometry (AAS). In the next step, the obtained calcium phosphate powders, sodium alginate, and chondroitin were used to produce composite granules. Ciprofloxacin, a broad-spectrum antibiotic that can be used in local delivery systems targeting bone tissue, was loaded into the granules. The release of silver and gallium ions as well as ciprofloxacin was then examined by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC), respectively. The cytotoxicity of the granules was studied using a neutral red uptake (NRU) test and mouse embryonic fibroblasts. Moreover, preliminary antibacterial activity against Staphylococcus aureus and Escherichia coli was measured. The study showed that the type of calcium phosphates enriched in silver or gallium significantly affects the release profile of these ions. Biphasic calcium phosphates also have an impact on the morphology of the granules. Most of the granules turned out to be non-toxic to mammalian cells. Microbiological tests showed high antibacterial activity against both strains of bacteria.

Cytotoxicity, endocrine disrupting activity, and chemical analysis of 42 food contact silicone rubber products

After migration in 95 % ethanol (food simulant) at 70 °C for 2 h (accelerated conditions), the cytotoxicity and endocrine-disruption activity of 42 food contact silicone products (FCSPs) obtained from the Chinese market were studied. Of 31 kitchenwares, 96 % showed mild or above cytotoxicity (relative growth rate < 80 %) using the HeLa neutral red uptake test; and 84 % showed estrogenic (64 %), anti-estrogenic (19 %), androgenic (42 %), and anti-androgenic (39 %) activities by the Dual-luciferase reporter gene assay. The mold sample induced late phase HeLa apoptosis as detected by Annexin V-FITC/PI double staining flow cytometry, in addition, the migration of mold sample has a higher risk of endocrine disruption at high temperature usage. Encouragingly, 11 bottle nipples had neither cytotoxic nor hormonal activity. Utilizing multiple mass spectrometry techniques, non-intentionally added substances (NIASs) in 31 kitchenwares were analyzed, and the migration levels of 26 organic compounds and 21 metals were quantified, furthermore, the safe risk of single migrant was evaluated through their special migration limit (SML) or threshold of toxicological concern (TTC). Using “nchoosek” statement and Spearman's correlation analysis in MATLAB, the migration of 38 compounds or combinations including metals, plasticizers, methylsiloxanes, and lubricants, had strong correlation with cytotoxicity or hormonal activity. The coexistence of various chemical substances in migrants leads to complex biological toxicity of FCSPs, so it is very important to detect the toxicity of the final products. The combination of bioassays and chemical analyses are valuable tools to facilitate the identification and analyses of FCSPs and migrants that have potential safety risks.

In vitro immunomodulatory activity study of Garcinia cowa Roxb. fraction.

The objective of this study is to determine the activity of Garcinia cowa Roxb. n-hexane, ethyl acetate, and butanol fractions as an immunomodulator invitro and obtain the fraction that has the potential as an immunomodulator. Raw 264.7 macrophages were used to asses G.cowa Roxb. immunomodulatory activity. The MTT assay was chosen to measure cell viability to evaluate the cytotoxic effect on cells. ELISA method was used to measure the concentration of Interleukin-6 (IL-6) and Tumor Necrosis Factor Alpha (TNF-α) secreted by cells after being treated with G.cowa Roxb. fraction. The neutral red uptake assay determined the effect of Garcinia cowa Roxb. on the phagocytic activity. After Raw 264.7 macrophages were given the Hexan fraction (Hex) at concentrations of 12.5 and 25μg/mL, there was a decrease in the concentration of IL-6, TNF-α, and the phagocytosis index of cells. Administration of the Ethyl Acetate fraction (EtOAc) at concentrations of 12.5 and 25μg/mL on cells caused a decrease in IL-6 and TNF-α levels but did not affect the phagocytosis index. There was an increase in the level of TNF-α and the phagocytosis index after being given the Butanol fraction (BuOH) with concentrations of 12.5 and 25μg/mL but there was a slight decrease in the level of IL-6. Both Hex and EtOAc fractions could suppress immune responses through decreasing IL-6, TNF-α, and slightly decreased phagocytic activity. BuOH fraction could stimulate immunomodulatory activities through enhanced TNF-α levels and phagocytic index, but less potent in enhancing IL-6 production. The BuOH fraction could be developed as an immunostimulant.

Antifungal activity of the ethanolic extract and flavonoid avicularin from Myrcia tomentosa (Aubl.) DC. on virulence factors of Candida species

In this work, the mechanism of the action of the ethanolic extract and avicularin isolated from Myrcia tomentosa was evaluated against Candida albicans and Candida parapsilosis. The effects of ethanolic extract and avicularin regarding adhesion in epithelial cells and on yeast kinetic growth were tested. A neutral red uptake (NRU) assay was used to test cytotoxicity against the 3T3-A31 cells of Balb/c. The morphology and ultrastructural analysis were examined by scanning and transmission electron microscopy. The results showed good activity for the ethanolic extract and the flavonoid avicularin, which inhibited the yeasts in concentrations of 32 and 16 µg/mL, respectively. The plant samples promoted inhibition of the adhesion in epithelial cells and showed a reduction in the number of the yeasts in kinetic growth. Avicularin and ethanolic extract from M. tomentosa showed low in vitro toxicity for the 3T3-A31 cells. The scanning and transmission electron microscopy showed changes in morphology and cell arrangement of Candida species in the presence of avicularin. Considering the results, avicularin obtained from M. tomentosa demonstrates potential for the future treatment of candidiasis.

Does kisspeptin exert a local modulatory effect on bovine ovarian steroidogenesis?

Kisspeptin, a hypothalamic neuropeptide encoded by the KISS1 gene, has a pivotal role in promoting GnRH secretion in mammals. Kisspeptin and its receptor (KISS1R) are also expressed in certain peripheral tissues including gonads, suggesting intra-gonadal roles. Such actions at the level of the bovine ovary have not been explored previously. The current aims were to determine whether KISS1 and its receptor (KISS1R) are expressed in the bovine ovary and whether kisspeptin or a kisspeptin antagonist can modulate ovarian steroid production by cultured ovarian cells. Granulosa (GC) and theca interna (TC) were collected from antral follicles (3-18 mm) categorized into five class sizes. Early, mid and regressing corpora lutea (CL) were also collected for RT-qPCR analysis of KISS1 and KISS1R expression. Bovine TC and GC cultured under both non-luteinizing (serum-free) and luteinizing (serum-supplemented) conditions were treated for 4 days with kisspeptin-10 (10-10-10-6M) or kisspeptin antagonist (p234; 10-10-10-6M), alone and in combination with either FSH (GC), LH (TC) or forskolin (luteinized GC/TC). Steroid secretion (GC: oestradiol, progesterone; TC: androstenedione, progesterone; luteinized GC/TC: progesterone) was measured by ELISA and viable cell number determined by neutral red uptake assay. KISS1 and KISS1R transcripts were detected in TC, GC and CL with significant differences between follicle categories and CL stages. However, neither kisspeptin-10 nor kisspeptin antagonist affected steroid secretion or viable cell number in any of the four ovarian cell culture models. As such, the hypothesis that kisspeptin has a direct intra-ovarian role to modulate follicular or luteal steroidogenesis, or cell proliferation/survival, is not supported.

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).&#x0D; 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.&#x0D; 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.

Development of a GCN-based model to predict &lt;i&gt;in vitro&lt;/i&gt; phototoxicity from the chemical structure and HOMO-LUMO gap

The interaction between sunlight and drugs can lead to phototoxicity in patients who have received such drugs. Phototoxicity assessment is a regulatory requirement globally and one of the main toxicity screening steps in the early stages of drug discovery. An in silico-in vitro approach has been utilized mainly for toxicology assessments at these stages. Although several quantitative structure-activity relationship (QSAR) models for phototoxicity have been developed, in silico technology to evaluate phototoxicity has not been well established. In this study, we attempted to develop an artificial intelligence (AI) model to predict the in vitro Neutral Red Uptake Phototoxicity Test results from a chemical structure and its derived information. To accomplish this, we utilized an open-source software library, kMoL. kMoL employs a graph convolutional neural networks (GCN) approach, which allows it to learn the data for the specified chemical structure. kMoL also utilizes the integrated gradient (IG) method, enabling it to visually display the substructures contributing to any positive results. To construct this AI model, we used only the chemical structure as a basis, then added the descriptors and the HOMO-LUMO gap, which was obtained from quantum chemical calculations. As a result, the assortment of chemical structures and the HOMO-LUMO gap produced an AI model with high discrimination performance, and an F1 score of 0.857. Additionally, our AI model could visualize the substructures involved in phototoxicity using the IG method. Our AI model can be applied as a toxicity screening method and could enhance productivity in drug development.

Homo- and heterometallic complexes of Zn(II), {Zn(II)Au(I)}, and {Zn(II)Ag(I)} with pentadentate Schiff base ligands as promising anticancer agents.

Two families of homo- and heterometallic complexes, [Zn2L1(μ-OH)(H2O)2](ClO4)2, [Zn2L2(μ-OH)(H2O)2](ClO4)2, [Zn2L3(μ-OH)(H2O)2](ClO4)2, 1∞[{L1Zn2(μ-OH)}{μ-[Ag(CN)2]}](ClO4), [{L1Zn2(μ-OH)}2{μ-[Au(CN)2]}{[Au(CN)2]2}](ClO4)·H2O, 1∞[{L2Zn2(μ3-OH)}2(H2O){μ-[Ag(CN)2]}](ClO4)3·THF·0.5MeOH, 1∞[{L2Zn2(μ3-OH)}2(H2O){μ-[Au(CN)2]}](ClO4)3·THF·H2O, and 1∞[{L3Zn2(μ-OH)}{μ-[Ag(CN)2]}][Ag(CN)2]·H2O, respectively, have been synthesized and characterized. The Schiff bases used as ligands were obtained by condensation reactions of 2,6-diformyl-p-cresol with N,N-dimethyl-ethylenediamine (HL1), 2-aminomethyl-pyridine (HL2), and 2-aminoethyl-pyridine (HL3), respectively. The cytotoxic/cytostatic and genotoxic effects in cultured human MCF-7 (luminal type A breast cancer), MDA-MB-231 (triple negative breast cancer), HeLa (cervical carcinoma), and Lep-3 (non-tumor embryonal fibroblastoid cells) were studied. The investigations were performed by thiazolyl blue tetrazolium bromide test (MTT test), neutral red uptake cytotoxicity assay, crystal violet staining, hematoxylin and eosin staining, double staining with acridine orange and propidium iodide, AnnexinV/FITC, and Comet assay in short-term experiments (24-72 h, with monolayer cell cultures) as well as by 3D colony-forming method in long-term experiments (28 days, with 3D cancer cell colonies). The results obtained revealed that: (i) applied at a concentration range of 0.1-100 μg mL-1, the compounds investigated decrease in a time- and concentration-dependent manner the viability and/or proliferation of the treated cells; (ii) complexes of {Zn(II)Au(I)} show relatively higher cytotoxic/genotoxic activity and antitumor potential as compared to {Zn(II)Ag(I)}; (iii) some of the complexes demonstrate more pronounced cytotoxic potential than commercially available antitumor agents cisplatin, oxaliplatin, and epirubicin.

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.

Acetone-precipitated zein protein hydrolysates from blue-maize selectively target hepatocellular carcinoma and fibroblasts in a dose-dependent manner

Herein, we investigated the effects of acetone precipitation on the bioactivity of blue-maize zein hydrolysates (BmapZDH) against hepatocarcinoma (HepG2) and non-tumor cells (fibroblasts). Zein proteins were extracted from blue-maize flour hydrolyzed with Alcalase for 6 h, and the peptide fractions were filtered (<5 KDa). Acetone was used to precipitate flowthroughs overnight, and cell viability was tested using the neutral red uptake method. Acetone precipitation promoted the selection of bioactive BmapZDH. At a dosage of 1500 ng/mL, hepatocarcinoma cells lost 51.9% of their viability after 24 h. When exposed to a 125 ng/mL dose for 48 h, cell viability diminished to 4.3%. Fibroblast viability was alleviated to 77.3% with a 93.75 ng/mL dose. Our findings imply that BmapZDH could be a potential candidate for treating hepatic carcinoma, and its peptide sequences and antibacterial properties should be investigated further.

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.

Multiple endpoint in vitro toxicity assessment of a prototype heated tobacco product indicates substantially reduced effects compared to those of combustible cigarette

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37–39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8–22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90–97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.

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.

Macrophage migration inhibitory factor is vital for inflammatory properties and survival of peripheral blood leukocytes via enhancing mitochondrial function in Ctenopharyngodon idellus

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein implicated in a broad spectrum of inflammatory and proliferative disorders. The gene sequence of grass carp (Ctenopharyngodon idella) was identified and the expression level of it was regulated by cadmium exposure in our previous study. To further clarify the immune-regulatory activity of grass carp MIF, MIF was over-expressed and interfered in grass carp peripheral blood leukocytes via transfection of plasmids pcDNA3.1-MIF-EGFP and pLKO.1-shRNA-EGFP-puro, respectively. Subsequently, survival, phagocytic capacity, mitochondrial function and cytokine production of the transfected leukocytes were assayed. The results shown that grass carp MIF was necessary for leukocyte survival, because it enhanced leukocyte viability and inhibited cell apoptosis, while MIF interference disrupted the cell viability and induced leukocyte apoptosis. The effect might benefit from improved mitochondrial function as evidenced by increased ATP production, which was due to maintained mitochondrial trans-membrane potential. In addition, MIF is essential for neutral red uptake into leukocyte, and it provoked chemokine monocyte chemotactic protein-1 (MCP-1), pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα), interleukin 1β (IL1β), interleukin 6 (IL6), interleukin 8 (IL8), and suppressed anti-inflammatory cytokine interleukin 10 (IL10) production. These results indicated that grass carp MIF played a vital role in regulating inflammatory properties and survival of peripheral blood leukocytes.

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.