Assessment of Cell Death and Genotoxic Potential of Glyphosate and Cypermethrin Formulations, Individually and in Combination, in HEp-2 Cells.

13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

IntroductionWithaferin-A(WFA) is a natural compound extracted from Wihania somnifera plant which showed tumour growth inhibiting effects in both in vitro and in vivo models.of various cancers such as colorectal cancer, ovarian cancer, breast cancer, gastric cancers, glioma and hepatocellular carcinoma. Current commonly used therapeutic agents such as cisplatin (CDDP) are effective on cancer cells however there is a lack of efficiency on cancer stem cells (CSCs) which are considered to be responsible of recurrence and metastasis. Although anti-proliferative effect of WFA on non-small cell lunger cancer (NSCLC) cells was shown in previous studies, its effect on NSCLC CSCs remains unclear. Based on this fact, the aim of this study was to evaluate the in vitro effect of WFA on CD133 +NSCLC CSCs.Material and methodsA549 NSCLC cell line was incubated in DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Penicilline/Streptomycin at 5% CO2 and 37°C. Cells were incubated with WFA 24, 48, 72 hours of 2.5,10,20 and 50 µM WFA and 75,100 µM CDDP and their combinations. Cell viability was determined by the WST-1 assay.CD133+ A549 cells were isolated with magnetic beads. The purity of CD133 positivity was assessed by flow cytometry. A549 cells and CD133+ A549 cells were treated with 50 µM WFA, 100 µM CDDP and incombination for 24 hours. Apoptosis was evaluated by flow cytometry with annexin V and PI staining. Furthermore DNA damage and cell proliferation of CD133+ A549 cells were determined by flow cytomerty with BrdU and H2AX stainings.Results and discussionsWFA caused 18,6% early apoptosis (EA), 36,2% late apoptosis (LA), 56% anti-proliferation while CDDP caused 34,3% EA, 2,5% LA,%31,3% anti-proliferation and WFA+CDDP caused 45,6% EA, 34,5% LA 63,8% anti-proliferation on A549 cells. Also WFA 21,6% EA 38,3% LA, 6,6% DNA damage while CDDP caused 30% EA, 4,4% LA, 7,0% DNA damage and WFA+CDDP caused 21,9% EA, 42,9% LA,6,1% anti-proliferation on A549 CSCs.ConclusionWFA showed anti-proliferative and apoptosis inducing effects of both NSCLCs and their CD133 +CSCs. In addition WFA did not change anti-tumour effect of CDDP and their combination showed more anti-proliferative and apoptosis inducing effect than their administration alone. In addition, WFA caused DNA damage in NSCLC CSCs. Although CDDP has critical anti-tumour effects, it might lead to adverse effects such as ototocity, neprotoxicity, peripheral neuropathy. Therefore, systemic effects of the natural compound WFA are suggested to be evaluated in animal models in future studies.

The effects of propolis and phenolic compounds (caffeic acid - Caf; dihydrocinnamic acid - Cin; p-coumaric acid - Cou) in the same quantity found in our propolis sample were investigated on human laryngeal epidermoid carcinoma (HEp-2) cells. Cell viability, apoptosis/necrosis and cell cycle arrest, P53 and CASPASE-3 gene expression, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect on HEp-2 cells, whereas isolated compounds had no effect on cell viability. Higher concentrations were tested and Caf induced late apoptosis or necrosis in HEp-2 cells, while propolis induced apoptosis, both probably due to ROS generation. P53 expression was downregulated by propolis but not by Caf. CASPASE-3 expression was correlated with induction of both early and late apoptosis, with both propolis and Caf alone upregulating its expression. Propolis induced cell cycle arrest at G2/M phase and Caf at S phase. Propolis but not Caf may act as a P-gp inhibitor by modulating P-gp activity and inhibiting DOX efflux. Propolis exerted cytotoxic effects on HEp-2 cells, and the mechanisms are discussed, showing its potential as an antitumour drug.

Ultra-high dose rate FLASH irradiation (FLASH-IR) has got extensive attention since it may provide better protection on normal tissues while maintain tumor killing effect compared with conventional dose rate irradiation. The FLASH-IR induced protection effect on normal tissues is exhibited as radio-resistance of the irradiated normal cells, and is suggested to be related to oxygen depletion. However, the detailed cell death profile and pathways are still unclear. Presently normal mouse embryonic fibroblast cells were FLASH irradiated (∼109 Gy/s) at the dose of ∼10–40 Gy in hypoxic and normoxic condition, with ultra-fast laser-generated particles. The early apoptosis, late apoptosis and necrosis of cells were detected and analyzed at 6, 12, and 24 h post FLASH-IR. The results showed that FLASH-IR induced significant early apoptosis, late apoptosis and necrosis in normal fibroblast cells, and the apoptosis level increased with time, in either hypoxic or normoxic conditions. In addition, the proportion of early apoptosis, late apoptosis and necrosis were significantly lower in hypoxia than that of normoxia, indicating that radio-resistance of normal fibroblast cells under FLASH-IR can be enhanced by hypoxia. To further investigate the apoptosis related profile and potential pathways, mitochondria dysfunction cells resulting from loss of cytochrome c (cyt c–/–) were also irradiated. The results showed that compared with irradiated normal cells (cyt c+/+), the late apoptosis and necrosis but not early apoptosis proportions of irradiated cyt c–/– cells were significant decreased in both hypoxia and normoxia, indicating mitochondrial dysfunction increased radio-resistance of FLASH irradiated cells. Taken together, to our limited knowledge, this is the first report shedding light on the death profile and pathway of normal and cyt c–/– cells under FLASH-IR in hypoxic and normoxic circumstances, which might help us improve the understanding of the FLASH-IR induced protection effect in normal cells, and thus might potentially help to optimize the future clinical FLASH treatment.

BackgroundTo determine whether ceramide is responsible for the induction of p53-independent early or late apoptosis in response to high- and low-Linear-Energy-Transfer (LET) irradiation.MethodsFour cell lines displaying different radiosensitivities and p53-protein status were irradiated with photons or 33.4 or 184 keV/μm carbon ions. The kinetics of ceramide production was quantified by fluorescent microscopy or High-Performance-Liquid-Chromatogaphy and the sequence of events leading to apoptosis by flow cytometry.ResultsRegardless of the p53-status, both low and high-LET irradiation induced an early ceramide production in radiosensitive cells and late in the radioresistant. This production strongly correlated with the level of early apoptosis in radiosensitive cells and delayed apoptosis in the radioresistant ones, regardless of radiation quality, tumor type, radiosensitivity, or p53-status. Inhibition of caspase activity or ceramide production showed that, for both types of radiation, ceramide is essential for the initiation of early apoptosis in radiosensitive cells and late apoptosis following mitotic catastrophe in radioresistant cells.ConclusionsCeramide is a determining factor in the onset of early and late apoptosis after low and high-LET irradiation and is the mediator of the p53-independent-apoptotic pathway. We propose that ceramide is the molecular bridge between mitotic catastrophe and the commitment phase of delayed apoptosis in response to irradiation.

Both apoptosis and micronuclei formation reflect cytogenetic damage in cells and could contribute to cell homeostasis. The aim of this study was to evaluate apoptosis in peripheral blood lymphocytes (PBLs) of patients with differentiated thyroid cancer (DTC) before and after 131-iodine (131-I)-therapy and its correlation with micronuclei (MN) frequency. The study population included 18 DTC patients and 18 healthy donors. Apoptotic cells were detected using the Annexin V-FITC/7-AAD kit and MN frequency by cytokinesis-block MN assay. The difference between early apoptosis in PBLs of DTC patients before therapy and controls (9.88 ± 4.99% vs. 6.64 ± 2.07%, p = 0.003) was significant, as well as between early apoptosis in PBLs of DTC patients before and after 131-I-therapy (9.88 ± 4.99% vs. 13.53 ± 6.57%, p = 0.008). The MN frequency and early apoptosis in PBLs of DTC patients was positively correlated before (r = 0.540, p= 0.021) and after 131-I-therapy (r = 0.585, p= 0.014). Thyroid cancer patients had a significantly increased early apoptosis in PBLs, which further increased after 131-I-therapy in association with MN frequency.

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

N-acetyl cysteine: could it be an effective adjuvant therapy in ICSI cycles

The apoptotic process is an important step in the process of T-lymphocytes maturation and differentiation. This work is devoted to the analysis of changes in the composition of oligosaccharides of CBA mice thymocytes glycocalyx during the hydrocortisone induced apoptosis. A panel of 23 fluorescein isothiocyanate (FITC)-labeled lectins specific for mannose, mannose and glucose, galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, fucose and N-acetylneuraminic acid residues was used. Flow cytometry was used for evaluation the binding of lectins to thymocytes of intact mice, as well as mice after administration of hydrocortisone. Based on the results of TMRM and 7AAD staining, the cells were divided into living thymocytes, cells in early and late apoptosis. It has been established that living cells carry carbohydrates on the surface of glycocalyx containing terminal residues of galactose and N-acetyl-D-galactosamine. With the transition of thymocytes to late apoptosis, the binding of all lectins increases, except for fucose-specific ones. The glycocalyx structures of living thymocytes are low in density and contain groups of oligosaccharides with N-acetyl-D-galactosamine and D-galactose in the terminal position. The membrane layer of glycocalyx is characterized by a high density and a wide variety of oligosaccharide structures that persist at the stage of late apoptosis of thymocytes. The results indicate nonuniform density and heterogeneity of oligosaccharides in glycocalyx, a significant part of which is lost in early apoptosis.

Modulatory effects of pioglitazone as a ligand for the peroxisome proliferator-activated receptor on semen quality and fertility potential of broiler breeder roosters

Purpose: prevention of bacterial complications correction apoptosis of T lymphocytes with human recombinant granulocyte colony-stimulating factor (G-CSF-par). Materials and Methods: In randomized, controlled, blind clinical study included 69 neonates with respiratory pathology on mechanical ventilation who were born at term 39,2 ± 1,1 weeks, with a mass of 3650 ± 421, Apgar score 2,3±1,1, no clinical signs of infection, containing lymphocytes in early (AnnexinV-FITC + PI-) and late (AnnexinV-FITC + PI +) apoptosis than 9.59% and 0.56% (respectively) distributed in groups: patients I ( n = 39) received G-CSF is a par-10 mg / kg / day intravenously for the first 3 days, in group II (n = 30) par-G-CSF was not given. On admission, for 3-5 days and in the outcome of the disease by ELISA studied the content of G-CSF, FGF, sFas-L; subpopulations of lymphocytes by flow cytometry on a flow cytometer, Beckman Coulter Epics XL (USA). Results: Statistical analysis of the results confirmed the reduction of the relative amount of lymphocytes activated Fas-receptor (CD95) and lymphocytes repositories early and late apoptosis, increasing stem cells in peripheral blood, elevated levels of FGF and antiapoptogennogo sFAS after administration par-GCSF, decrease the incidence of septic complications. Summary: Timely correction of apoptosis of T-lymphocytes par-G-CSF is effective and safe profilaktiruet onset sepsis in neonates with respiratory disorders who are on mechanical ventilation.

Inhibition of DLEU1 Following siRNA Transfection in Burkett's Lymphoma (BL): Implication for Tumor Repressor Role of DLEU1 in C-Myc-Activated BL Lymphomagenesis

Comparative toxicities of bismuth oxybromide and titanium dioxide exposure on human skin keratinocyte cells

The aim was to compare granulosa cell's (GCs) apoptosis rate with (group A) or without (group B) luteinising hormone (LH) supplementation in poor ovarian responders (PORs) during controlled ovarian stimulation (COS). After oocyte retrieval, the follicular fluid was analysed by cytoflowmetry. Primary outcomes were GCs apoptosis rate in terms of viability, early apoptosis, late apoptosis and necrosis. Secondary outcome was clinical pregnancy rate. The viability was 96.7{IQR: 8} and 83.5{IQR: 20} for groups A and B, respectively (p < .001). Late apoptosis rates were significantly lower in group A (median 1.5, {IQR: 3.1}) than group B (median 9.5, {IQR: 20.6}) (p < .001). Median early apoptosis rates were 1.4 {IQR: 2.9} and 5.2 {IQR: 6.5} for group A and B respectively (p = .04). No significant difference was observed in the clinical pregnancy rate. Although LH seems necessary in PORs to decrease late granulosa apoptosis rates, this does not improve clinical pregnancy rates. IMPACT STATEMENT What is already known on this subject? LH supplementation during COS has long been an issue in PORs to overcome the rFSH responsiveness due to the LH polymorphism. LH receptors have also been on GCs and their expression increases in preovulatory follicles. GCs apoptosis rates may show the oocyte quality and reproductive potential of oocyte retrieved and the requirement for LH supplementation. What do the results of this study add? The present study shows that LH supplementation during COS for PORs promotes the GC viability and reduces early/late apoptosis rates. Similarly, the number of MII oocytes was significantly higher in the LH regimen group. However, there was no significant difference in terms of clinical pregnancy rates. What are the implications of these findings for clinical practice and/or further research? The oocyte quality parameters such as higher GC viability and lower GC early/late apoptosis rates verify the LH supplementation in PORs during COS. However, the limited size of this study requires further multi-centre research in a larger cohort of patients. Results obtained with a sensitive and validated method will help clinicians to make better decisions in patient care.

The EU and its Member States have been working on reducing the use and risk of pesticides for decades. This has largely been achieved by regulating the authorisation of pesticides under Directive 91/414/EEC and Regulation (EC) 1107/2009. Consequently, the number of active ingredients authorised for pest and disease control in ornamental crops (e.g. flowers) has dramatically decreased. Now, growers face a shortfall of control options, and there are growing numbers of reports on unlawful use of pesticides in ornamental crops. The Food Inspection Authorities try to change this by imposing fines on these growers. Furthermore, retail companies start to impose restrictions on pesticide residues on ornamental products. In case of exceeding they reject the products supplied. On the one hand, the growers are thus justifiably punished for unlawful use of pesticides. On the other hand, their violations indicate that growers find themselves in a desperate position. The question is how this unsatisfactory situation can be solved. The objective of this paper is to improve understanding of the positions and interests of the involved parties in relation to pesticides and pest control. We therefore study how pesticide use in ornamental crops is framed by the various parties involved. Furthermore, power relations in both the knowledge and value chain are studied. Examples of framing pesticide use are: my pests are difficult to control, surface water quality is below standards, working in greenhouses should be safe, authorisation of control agents is too expensive, ornamental crops should be safe for consumers, chemical pesticides close the door to biological control agents, growers should apply decision support systems. These examples illustrate the frictions among the parties on the playing field of crop protection. The aim is to explore some options for sustainable development of crop protection in floriculture. Our suggestion is that new interactions and initiatives have to be developed between flower growers, value chain partners and/or knowledge partners. Bringing partners together for collective action under a national agreement or in a public-private partnership for plant health research are considered to be the most promising options. The lesson learned is that social innovation needs special attention in governance of sustainable crop production.