Cytotoxic effects of individual and binary combinations of zearalenone and ochratoxin a on liver

Co-contamination with mycotoxin represents a serious concern for human and animal health. In this in vitro study, we investigated the combined effects of ZEA and OTA, mycotoxins which frequently contaminate cereals, in binary mixtures on the viability of human liver cancer cell line (HepG2). Cell viability was assessed after 24 h using a neutral red assay. An antagonistic effect was observed for binary toxins combinations affecting 25% of cell viability (CI=4.18), which turn into a synergistic effect as followed: slight at IL50 (CI=1.51), moderate at IL75 (CI=0.554) and strong at IL90 (CI=0.203). In conclusion, our results show an important additive and even synergistic cytotoxic effect of two commonly occurred mycotoxins: zearalenone and ochratoxin when they are present simultaneously in food or feed. The co-exposure to mycotoxins lead to a higher toxicity than the exposure to single toxin. Our study provides important data for mycotoxins risk assessment. In this context, a re-evaluation of the guidance levels for mycotoxins will be required in the future, in order to reduce the health risk associated with the possible consumption of mycotoxin co-contaminated food or feed.

Phytochemical Profile, Antioxidant and Antitumor Activities of Green Grape Juice

(1) Plants, due to their phytochemicals, have long been known for their pharmacological potential and medicinal value. Verjuice, the acidic juice of unripe green grape, is still poorly characterized in terms of its chemical composition and biological activities. (2) In this study, we characterized the chemical composition, antioxidant and antitumor potential of verjuice extract. Folin–Ciocalteu and aluminum chloride reagents were used to identify the total phenol and total flavonoid composition. Various conventional methods were used to quantify the alkaloids and tannins. DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay and Neutral Red assay were used to assess the antioxidant and antitumor activities, respectively. (3) We showed that the verjuice extract contains alkaloids, tannins, and a high quantity of total flavonoids and total phenols. Besides its antioxidant activity, verjuice significantly repressed human pulmonary adenocarcinoma (A549) cells’ viability in both dose- and time-dependent manners. Moreover, verjuice extract significantly enhanced the anticancer potential of cisplatin. (4) Altogether, these observations suggest a potential use of verjuice as a natural antitumor remedy.

Fridericia platyphylla (Cham.) L.G. Lohmann root extract exerts cytotoxic and antiproliferative effects on gastric tumor cells and downregulates BCL-XL, BIRC5, and MET genes

Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL−1 and 69.38, 41.73, and 52.39 µg mL−1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL−1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL−1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL−1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated.

413. Differences in Inflammatory Mechanisms in Pseudomonas aeruginosa and Staphyloccoccus auereus Infections in Cystic Fibrosis

Background Chronic bacterial lung infections are the primary cause of morbidity and mortality in cystic fibrosis (CF). The most common CF pathogens, Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus), are common commensal or environmental organisms that adapt to the CF lung. We sought to investigate whether adaptation from early lung colonizer to chronic pathogen alters the bacterial effects on host inflammation. Methods P. aeruginosa (n = 25) and S. aureus (n = 25) isolates from CF patients with early and chronic infections were acquired from Seattle Children’s CF. Environmental (n = 8) and clinical, non-CF P. aeruginosa (n = 8) isolates were obtained from the University of Ottawa. P. aeruginosa reference strain PA14 and PA14 transposon mutants for T3SS and flagellin were used to observe the relationship between cell death and cytokine production. We infected THP-1-derived macrophages (PMA differentiated) in vitro for 3 hours with various MOIs. We subsequently measured cell death of THP-1-derived macrophages using neutral red assay and cytokine production using ELISAs. Results Infections with PA14 mutants and non-CF P. aeruginosa isolates demonstrated that rapid cell death of THP-1-derived macrophages caused a reduction in cytokine production relative to strains that did not cause as much cell death. At 10 MOI, early P. aeruginosa isolates from CF patients induced more THP-1-derived macrophage cell death compared with chronic isolates (P < 0.0001). Chronic P. aeruginosa isolates induced greater production of TNF, IL-8, and IL-6 (P < 0.01, P < 0.0001, and P < 0.0001, respectively) compared with early strains. No difference in IL-1β production was observed. When controlling for cell death between the two groups by using heat-killed bacteria, the only difference maintained was in TNF production (P < 0.01). Between early and chronic S. aureus isolates, the one difference observed was greater IL-8 production among early isolates (P < 0.01). Conclusion Chronic P. aeruginosa isolates from CF patients induce less cell death but more TNF, IL-8, and IL-6 production compared with early isolates. This suggests that P. aeruginosa producing chronic infections induce inflammatory signals that may contribute to increased morbidity among CF patients. Disclosures All authors: No reported disclosures.

Exploration of (hetero)aryl Derived Thienylchalcones for Antiviral and Anticancer Activities

Background: Search for new antiviral and anticancer agents are essential because of the emergence of drug resistance in recent years. In continuation of our efforts in identifying the new small molecule antiviral and anticancer agents, we identified chalcones as potent antiviral and anticancer agents. Objective: With the aim of identifying the broad acting antiviral and anticancer agents, we discovered substituted aryl/heteroaryl derived thienyl chalcones as antiviral and anticancer agents. Method: A focused set of thienyl chalcone derivaties II-VI was screened for selected viruses Hepatitis B virus (HBV), Herpes simplex virus 1 (HSV-1), Human cytomegalovirus (HCMV), Dengue virus 2 (DENV2), Influenza A (H1N1) virus, MERS coronavirus, Poliovirus 1 (PV 1), Rift Valley fever (RVF), Tacaribe virus (TCRV), Venezuelan equine encephalitis virus (VEE) and Zika virus (ZIKV) using the National Institute of Allergy and Infectious Diseases (NIAID)’s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. Additionally, a cyclopropylquinoline derivative IV has been screened for 60 human cancer cell lines using the Development Therapeutics Program (DTP) of NCI. Results: All thienyl chalcone derivatives II-VI displayed moderate to excellent antiviral activity towards several viruses tested. Compounds V and VI were turned out be active compounds towards human cytomegalovirus for both normal strain (AD169) as well as resistant isolate (GDGr K17). Particularly, cyano derivative V showed very high potency (EC50: <0.05 µM) towards AD169 strain of HCMV compared to standard drug Ganciclovir (EC50: 0.12 µM). Additionally, it showed moderate activity in the secondary assay (AD169; EC50: 2.30 µM). The cyclopropylquinoline derivative IV displayed high potency towards Rift Valley fever virus (RVFV) and Tacaribe virus (TCRV) towards Rift Valley fever virus (RVFV). The cyclopropylquinoline derivative IV is nearly 28 times more potent in our initial in vitro visual assay (EC50: 0.39 µg/ml) and nearly 17 times more potent in neutral red assay (EC50: 0.71 μg/ml) compared to the standard drug Ribavirin (EC50: 11 µg/ml; visual assay and EC50: 12 µg/ml; neutral red assay). It is nearly 12 times more potent in our initial in vitro visual assay (EC50: >1 µg/ml) and nearly 8 times more potent in neutral red assay (EC50: >1.3 µg/ml) compared to the standard drug Ribavirin (EC50: 12 µg/ml; visual assay and EC50: 9.9 µg/ml; neutral red assay) towards Tacaribe virus (TCRV). Additionally, cyclopropylquinoline derivative IV has shown strong growth inhibitory activity towards three major cancers (colon, breast, and leukemia) cell lines and moderate growth inhibition shown towards other cancer cell lines screened. Conclusion: Compounds V and VI were demonstrated viral inhibition towards Human cytomegalovirus, whereas cyclopropylquinoline derivative IV towards Rift Valley fever virus and Tacaribe virus. Additionally, cyclopropylquinoline derivative IV has displayed very good cytotoxicity against colon, breast and leukemia cell lines in vitro.

Dihydrocaffeic Acid Prevents UVB-Induced Oxidative Stress Leading to the Inhibition of Apoptosis and MMP-1 Expression via p38 Signaling Pathway

Chronic UVB exposure promotes oxidative stress, directly causes molecular damage, and induces aging-related signal transduction, leading to skin photoaging. Dihydrocaffeic acid (DHCA) is a phenolic compound with potential antioxidant capacity and is thus a promising compound for the prevention of UVB-induced skin photodamage. The aim of this study was to evaluate the antioxidant and protective effect of DHCA against oxidative stress, apoptosis, and matrix metalloproteinase (MMP) expression via the mitogen-activated protein kinase (MAPK) signaling pathway on L929 fibroblasts irradiated with UVB. DHCA exhibited high antioxidant capacity on 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+), and xanthine/luminol/xanthine oxidase (XOD) assays and reduced UVB-induced cell death in the neutral red assay. DHCA also modulated oxidative stress by decreasing intracellular reactive oxygen species (ROS) and extracellular hydrogen peroxide (H2O2) production, enhancing catalase (CAT) and superoxide dismutase (SOD) activities and reduced glutathione (GSH) levels. Hence, cellular damage was attenuated by DHCA, including lipid peroxidation, apoptosis/necrosis and its markers (loss of mitochondria membrane potential, DNA condensation, and cleaved caspase 9 expression), and MMP-1 expression. Furthermore, DHCA reduced the phosphorylation of MAPK p38. These findings suggest that DHCA can be used in the development of skin care products to prevent UVB-induced skin damage.

A new epithelial cell line, SSK from kidney of striped snakehead Channa striatus (Bloch 1793)

A cell line was established from striped snakehead Channa striatus kidney. The cell line, named as SSK, has been passaged for over 62 times. Growth studies at different temperatures and foetal bovine serum (FBS) concentrations revealed that SSK cells show optimum growth at 28°C in L-15 medium containing 20% FBS. The chromosome number of SSK cells was 2n=40. Partial sequencing of mitochondrial cytochrome oxidase 1 gene of the cells confirmed that the cell line originated from C. striatus. The SSK cells were primarily epithelial, as determined using immunophenotyping. The cells from SSK cell line were transfected with phrGFP II-N mammalian expression vector. The SSK cell line was stored in liquid nitrogen (-196οC) at different passages and was successfully revived after four months of storage. The cell line could be successfullyemployed for cytotoxicity studies, as revealed by neutral red assay. The cell line can be a valuable surrogate to the whole fish studies.

Biocompatibility Evaluation of Electrospun Scaffolds of Poly (L-Lactide) with Pure and Grafted Hydroxyapatite

<p>The objective of this work was to evaluate the biocompatibility of scaffolds of poly(<em>L</em>-lactide) with pure and grafted hydroxyapatite, at various concentrations of reinforcement. The biocompatibility tests were carried out <em>in vivo </em>in Wistar rats by implanting the material into the subcutaneous and muscle tissues from 1 to 14 weeks and evaluating the surrounding tissue stained with hematoxylin-eosin. For <em>in vitro </em>assays, MTT and neutral red assay were used to evaluate any cytotoxicity in Mioblast Muscle C2C12 Cells (ATCC® CRL-1772™) and Bovine Coronary Artery Endothelial Cells (BCAEC); <em>Escherichia coli </em>and <em>Staphylococcus aureus </em>were used to evaluate bacterial adhesion. All variants of scaffolds provoked a mild inflammatory response, without showing necrosis. No evidence of cytotoxicity was presented in cell viability tests and good bacterial cell adhesion was visualized for all of the materials studied.</p>

Tumoricidal Effect of Hematite (α-Fe2O3) and SiO2 Nanoparticles in Human Rhabdomyosarcoma Cell Line

Nanotechnology provides the opportunity for the development of new materials in the nanometer size range with many potential applications in biological sciences and clinical medicine. RD (muscle cancer cell line) was seeded out in 25 cm2plastic tissue culture flasks (NuncWiesbaden Germany) individually, in Minimum Essential Medium (MEM) with Hanks salts, containing 10% fetal bovine serum (FBS) and 2 mM L-Glutamine along with some nonessential amino acids and were incubated for 24 h for proper attachment to the substratum and kept at a 96 wells plate, incubated at 37°C and 5% CO2. SEM was employed to the nanoparticles and size of α-Fe2O3and SiO2nanoparticles were about 66 nm and 250 nm. Moreover 10-80μg/mL of Hematite (α-Fe2O3) and SiO2nanoparticles dispersed solution were labeled for each row of 96 wells plate. The present study evaluates the different parameters, e.g. time of incubation, cytotoxicity and cellular viability of the Human Rhabdomyosarcoma cell line (RD) as an experimental model. The viability of cells was determined by means of neutral red assay (NRA) after the cell-exposition to different concentrations of Hematite (α-Fe2O3) and SiO2nanoparticles into mentioned tumoricidal cells