The estimation of pore size distribution of electroporated MCF-7 cell membrane.

The size of the pores created by external electrical pulses is important for molecule delivery into the cell. The size of pores and their distribution on the cell membrane determine the efficiency of molecule transport into the cell. There are very few studies visualizing the presence of electropores. In this study, we aimed to investigate the size distribution of electropores that were created by high intensity and short duration electrical pulses on MCF-7 cell membrane. Scanning Electron Microscopy (SEM) was used to visualize and characterize the membrane pores created by the external electric field. Structural changes on the surface of the electroporated cell membrane was observed by Atomic Force Microscopy (AFM). The size distribution of pore sizes was obtained by measuring the radius of 500 electropores. SEM imaging showed non-uniform patterning. The average radius of the electropores was 12 nm, 51.60% of pores were distributed within the range of 5 to 10 nm, and 81% of pores had radius below 15 nm. These results showed that microsecond (µs) high intensity electrical pulses cause the creation of heterogeneous nanopores on the cell membrane.

Electroporation is a phenomenon in which permeability of the cell membrane to molecules and ions is increased due to externally applied high electric field pulses. The externally applied electric field pulses create pores on the cell membrane, allowing ions and molecules that normally can not pass through the membrane. The transport of molecules into the cell is related to the size and distribution of the pores created on the membrane. Studies visualizing the presence of electropores are very limited. In this study, we aimed to visualize pores and determine the size distribution of pores created due to the application of external electric field pulses on the cell membrane of human breast cancer cells. The membrane pores created by external electric field were visualized and characterized by different imaging techniques. The size distribution of pores was obtained by measuring the radius of 500 pores created on the cell membrane due to the applied electric fields. The surface of the electropermeabilized cells were very rough due to deformation during electroporation. We observed heterogeneous pore populations that were formed due to application of external electrical pulses on the surface of cell membrane. The average radius of the pores was found to be 12 nm.

Treatment of cervical cancer by electrochemotherapy with bleomycin, cisplatin, and calcium: an in vitro experimental study.

Low-dose chemotherapy in advanced stages of cancer does not give a positive response in treatment. The use of high-dose antineoplastic drugs creates significant side effects. The limiting situation in treatment creates a need for new generation drugs with less side effects and new treatment methods that will enable low-dose drug use. Electroporation (EP), a phenomenon, is a technique in which the membrane permeability is increased by the establishment of hydrophilic pores in the cell membrane with short and high-voltage electrical pulses. In the present investigation study, we aimed to inspect the effects of EP plus bleomycin, cisplatin, and calcium administration (CaEP) on cell viability, apoptotic activity, gene expression p53, Bax/Bcl-2 rate mitochondrial membrane potential (ΔΨm), and cell cycle in HeLa cervical cancer cell line. The permeabilization of the membrane was evaluated in flow cytometry with the PI method, and cell viability was measured in an ELISA reader with the WST-8 method. For bleomycin and cisplatin doses applied to HeLa cells, the concentration values (IC50) that inhibited 50% of the cells were found to be 214.11 ± 4.7 µM and 35.16 ± 3.3 µM, respectively. The IC50 values of the groups administered together with EP were calculated as 0.44 ± 0.3 µM for bleomycin and 20.55 ± 4.3 µM for cisplatin. There was no change in cell viability in calcium alone application, but a statistically notable reduction in cell vitality was observed in CaEP application. An increase in ΔΨm was found in bleomycin and CaEP exposure with EP. It was determined that EP exposure caused G0/G1 arrest in the cell cycle at all electric field intensities. It was determined that EP application in HeLa cells increased bleomycin cytotoxicity 487 times and cisplatin cytotoxicity 1.71 times, and CaEP could be an alternative treatment method.

Investigations on effects of titanium dioxide (TiO2) nanoparticle in combination with UV radiation on breast and skin cancer cells.

In this study, we have investigated the chemotherapeutic potential of titanium dioxide (TiO2) nanoparticles on skin and breast cancer cells. The cells have treated with a 75 µg/ml concentration of titanium dioxide because it is a recommended dose with proven effectiveness in vitro studies and then the cells were exposed to UV-A radiation. The combined effects of titanium dioxide and UV-A radiation on cell viability, cell cycle, plasma membrane, mitochondrial membrane potentials and apoptotic activity of the cells were investigated. The viability of SK-MEL 30 cells was measured by MTT assay and apoptotic activity of cells was determined by Annexin-V FITC/PI staining. As a result of the research, an increase was observed in the viability of cells treated with 75 µg/ml titanium dioxide concentration, while a significant decrease in cell viability was observed for both cell types when UV-A radiation and TiO2 were applied together. The results also showed that the percentage of apoptotic cells increased as a result of UV + TiO2 exposure. Accordingly, it can be said that TiO2 nanoparticles may research as potential chemotherapeutic agents for skin and breast cancers, especially in the presence of UV radiation.

The effects of melatonin on possible damage that will occur on adipocytokines and liver tissue by coadministration of fructose and bisphenol a (BPA).

BPA, one of the environmental endocrine disruptors, and fructose, reason of liver steatosis which is frequently encountered in the daily diet, contribute to the formation of metabolic syndrome (MetS). This study examines the possible effects of concurrent fructose and BPA administration on MetS and determines the effects of melatonin on this process. In the seven identified groups, a total of forty-two adult male Sprague Dawley rats were treated by following fructose, BPA, and melatonin amounts, separately and together: group 1 (control), group 2 (10% aqueous fructose), group 3 (25 mg/kg BPA), group 4 (10% fructose + 25 mg/kg BPA), group 5 (10% fructose + 20 mg/kg melatonin), group 6 (25 mg/kg BPA + 20 mg/kg melatonin), and group 7 (10% fructose + 25 mg/kg BPA + 20 mg/kg melatonin). At the end of 60 days, histochemical, immunohistochemical, and biochemical procedures were performed on liver tissue. As a result, it was seen that BPA and fructose + BPA induced morphological alteration and inflammation and increased intracellular lipid quantity and amount of collagen and reticular fibers. The percentage of apoptotic liver cells stained by annexin V-FITC/PI was lower in group 7 compared to the group 4 (p < 0,001) and also in group 6 compared to the group 3 (p = 0.014). Both BPA and fructose application caused an increase in lipid peroxidation level due to the increase of oxidative stress. Application of melatonin induced antioxidant enzyme activity and reduced lipid peroxidation level. Our results indicate that fructose and BPA administration triggered the formation of MetS, whereas melatonin healed these variations, although not entirely.

Effects of Electroporation on Tamoxifen Delivery in Estrogen Receptor Positive (ER+) Human Breast Carcinoma Cells.

Estrogen receptor positive breast cancer is the most common type of breast cancer and in most cases, hormone therapy is considered complementary to surgery. Tamoxifen is one of the most common drugs used in hormone therapy for treating estrogen receptor positive breast cancer cells. However, it has severe side-effects depending on the duration of treating breast cancer and amount of tamoxifen used. In this study, we examined the effects of electroporation on the tamoxifen uptake in estrogen receptor positive MCF-7 breast cancer cells. The survival rate of MCF-7 cells had a negative relationship with energy dissipation in cells. Similarly, the electrical charge delivered to cells during electroporation was inversely proportional to survival rate. The combined application of electroporation and tamoxifen is much more effective than the usage of tamoxifen alone in the treatment of estrogen receptor positive breast cancer. The application of electroporation increased the uptake of tamoxifen into MCF-7 cells and reduced the minimal tamoxifen dosage which, is needed for the treatment of estrogen receptor positive breast cancer.

Does MW Radiation Affect Gene Expression, Apoptotic Level, and Cell Cycle Progression of Human SH-SY5Y Neuroblastoma Cells?

Neuroblastoma (NB) is a cancer that occurs in sympathetic nervous system arising from neuroblasts and nerve tissue of the adrenal gland, neck, chest, or spinal cord. It is an embryonal malignancy and affects infants and children. In this study, we investigated the effects of microwave (MW) radiation on apoptotic activity, cell viability, and cell cycle progression in human SH-SY5Y NB cells which can give information about MW radiation effects on neural cells covering the period from the embryonic stages to infants. SH-SY5Y NB cells were exposed to 2.1 GHz W-CDMA modulated MW radiation for 24 h at a specific absorption rate of 0.491 W/kg. Control samples were in the same conditions with MW-exposed samples but they were not exposed to MW radiation. The apoptotic activity of cells was measured by Annexin-V-FITC and propidium iodide staining. Moreover, mRNA levels of proliferative and cell cycle proteins were determined by real-time RT-PCR. The change in cell cycle progression was observed by using CycleTest-Plus DNA reagent. No significant change was observed in apoptotic activity of MW-exposed cells compared to control cells. The mRNA levels of c-myc and cyclin D1 were significantly reduced in MW group (p < 0.05). The percentage of MW-exposed cells in G1 phase was significantly higher than the percentage of control cells in G1 phase. MW radiation caused cell cycle arrest in G1 phase. These results showed that 2.1 GHz W-CDMA modulated MW radiation did not cause apoptotic cell death but changed cell cycle progression.

Effects of Cisplatin Electrochemotherapy on Human Neuroblastoma Cells.

Electrochemotherapy is the usage of electroporation to introduce chemotherapeutic drugs through membrane pores into target cells for cancer treatment. The effectiveness of chemotherapeutic drugs would be increased dramatically when they are used in electrochemotherapy than standard chemotherapy. In the present study, we investigated the effects of cisplatin treatment with electroporation on human SH-SY5Y neuroblastoma cells. SH-SY5Y cells were treated with different concentrations (0.15-24 µg/mL) of cisplatin and then exposed to 1500 volts per centimeter (V/cm), 100 microseconds (µs) pulse duration, and 1 Hertz (Hz) electric pulses. Cisplatin alone showed a dose-dependent effect on cell viability. On the other hand, cisplatin + electroporation treatment was more effective than cisplatin treatment alone. Lower doses of cisplatin treatment with electroporation was as effective as higher doses of cisplatin treatment without electroporation. These results indicated that cisplatin cytotoxicity was potentiated after exposure of cells to high intensity electric pulses and low doses of cisplatin can be used with electroporation in the treatment of neuroblastoma.

Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure.

The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15min cellular phone radiation+PTZ treatment+30min cellular phone radiation, Group III: 30min cellular phone radiation+PTZ treatment+30min cellular phone radiation. The RF radiation was produced by a 900MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain.

Tea extracts protect normal lymphocytes but not leukemia cells from UV radiation-induced ROS production: An EPR spin trap study.

PURPOSE: An ex vivo method for detection of free radicals and their neutralization by aqueous tea in human normal lymphocytes and MEC-1 leukemia cells under ultraviolet (UV) irradiation was investigated. MATERIALS AND METHODS: This method is based on the electron paramagnetic resonance (EPR) spectroscopy spin-trapping technique. 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was used as the spin trap. Normal human lymphocytes and leukemia cells were exposed to UVB radiation (290-315 nm) at 47.7 and 159 mJ/cm(2) and to UVA radiation (315-400 nm) at 53.7 J/cm(2). RESULTS: No significant radical production at 47.7 mJ/cm(2) UVB dose in both cell lines was observed. In normal cells, free radical production was observed at 159 mJ/cm(2) UVB and 53.7 J/cm(2) UVA doses. However, both UV sources did not significantly produce free radicals in leukemia cells. A radical scavenging property of tea extracts (black, green, sage, rosehip) was observed in normal lymphocytes after both UVB and UVA exposure. In leukemia cells, the intensities of EPR signals produced in BMPO with tea extracts were found to be increased substantially after UVA exposure. CONCLUSION: These results showed that UV radiation induced free radical formation in normal human lymphocytes and indicated that tea extracts may be useful as photoprotective agents for them. On the other hand, tea extracts facilitated free radical production in leukemia cells.

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt's lymphoma (Raji) cells.

We investigated the effects of 1.8 MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt's lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8 GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350 W/kg in a CO2 incubator. The duration of the exposure was 24 h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW + Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.

Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.

Mutagenic and morphologic impacts of 1.8GHz radiofrequency radiation on human peripheral blood lymphocytes (hPBLs) and possible protective role of pre-treatment with Ginkgo biloba (EGb 761).

The mutagenic and morphologic effects of 1.8GHz Global System for Mobile Communications (GSM) modulated RF (radiofrequency) radiation alone and in combination with Ginkgo biloba (EGb 761) pre-treatment in human peripheral blood lymphocytes (hPBLs) were investigated in this study using Sister Chromatid Exchange (SCE) and electron microscopy. Cell viability was assessed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay. The lymphocyte cultures were exposed to GSM modulated RF radiation at 1.8GHz for 6, 8, 24 and 48h with and without EGb 761. We observed morphological changes in pulse-modulated RF radiated lymphocytes. Longer exposure periods led to destruction of organelle and nucleus structures. Chromatin change and the loss of mitochondrial crista occurred in cells exposed to RF for 8h and 24h and were more pronounced in cells exposed for 48h. Cytoplasmic lysis and destruction of membrane integrity of cells and nuclei were also seen in 48h RF exposed cells. There was a significant increase (p<0.05) in SCE frequency in RF exposed lymphocytes compared to sham controls. EGb 761 pre-treatment significantly decreased SCE from RF radiation. RF radiation also inhibited cell viability in a time dependent manner. The inhibitory effects of RF radiation on the growth of lymphoctes were marked in longer exposure periods. EGb 761 pre-treatment significantly increased cell viability in RF+EGb 761 treated groups at 8 and 24h when compared to RF exposed groups alone. The results of our study showed that RF radiation affects cell morphology, increases SCE and inhibits cell proliferation. However, EGb 761 has a protective role against RF induced mutagenity. We concluded that RF radiation induces chromosomal damage in hPBLs but this damage may be reduced by EGb 761 pre-treatment.

Pulse modulated 900 MHz radiation induces hypothyroidism and apoptosis in thyroid cells: a light, electron microscopy and immunohistochemical study.

PURPOSE: In the present study we investigated the possible histopathological effects of pulse modulated Radiofrequency (RF) fields on the thyroid gland using light microscopy, electron microscopy and immunohistochemical methods. MATERIALS AND METHODS: Two months old male Wistar rats were exposed to a 900 MHz pulse-modulated RF radiation at a specific absorption rate (SAR) of 1.35 Watt/kg for 20 min/day for three weeks. The RF signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). To assess thyroid endocrine disruption and estimate the degree of the pathology of the gland, we analysed structural alterations in follicular and colloidal diameters and areas, colloid content of the follicles, and height of the follicular epithelium. Apoptosis was confirmed by Transmission Electron Microscopy and assessing the activites of an initiator (caspase-9) and an effector (caspase-3) caspases that are important markers of cells undergoing apoptosis. RESULTS: Morphological analyses revealed hypothyrophy of the gland in the 900 MHz RF exposure group. The results indicated that thyroid hormone secretion was inhibited by the RF radiation. In addition, we also observed formation of apoptotic bodies and increased caspase-3 and caspase-9 activities in thyroid cells of the rats that were exposed to modulated RF fields. CONCLUSION: The overall findings indicated that whole body exposure to pulse-modulated RF radiation that is similar to that emitted by global system for mobile communications (GSM) mobile phones can cause pathological changes in the thyroid gland by altering the gland structure and enhancing caspase-dependent pathways of apoptosis.