Forskolin and Phorbol 12-myristate 13-acetate modulates the expression pattern of AP-1 factors and cell cycle regulators in estrogen-responsive MCF-7 cells.

Activator protein-1 (AP-1) transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus. The phorbol 12-myristate 13-acetate and Forskolin (Fo) are well-known kinase activators/stimulators of Protein Kinase C (PKC) and Protein Kinase A (PKA) respectively. Importantly, these kinases are found to be present in transitional points of many cell signaling pathways, especially those involved in proliferation. The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized. Hence, the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated. Where, cells treated with PMA showed increased cell proliferation, while Fo had no effect, but inhibited the PMA induced proliferation. The RT-PCR results showed the PMA induced c-Jun, c-Fos and Fra-1 expressions compared to control and Fo. However, Fo in combination with PMA, inhibit the PMA induced above mRNA expressions where Fo alone has no effect. Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells. Further, PMA increases the mRNA expression of Cyclin-E1, Cyclin-D1, and CDK-4, whereas Fo decreases their expressions. Thus, mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.

Protein kinases orchestrate cell cycle regulators in differentiating BeWo choriocarcinoma cells.

Choriocarcinoma, a trophoblastic neoplasia, occurs in women as an incidence of abnormal pregnancy. BeWo choriocarcinoma cells derived from the abnormal placentation are a suitable model system to study the factors associated with differentiation, invasion and other cellular events as an alternative to clinical samples. Many protein kinases orchestrate the complex events of cell cycle and in case of malignancy such regulators are found to be mutated. In the present study, BeWo cells treated with forskolin (Fo) and phorbol 12-myristate 13-acetate (PMA) were used to study the role of PKA (protein kinase A) and PKC (protein kinase C), respectively, on the expression pattern of differentiation-related genes, membrane markers, PKC isoforms and cell cycle regulators. The effect of Fo and PMA on the cell proliferation was assessed. Progressive induction of alkaline phosphatase level and formation of multinucleated differentiated cells were observed in the cells treated with Fo. Exposure of cells to Fo and PMA induced the mRNA transcripts of α-hCG, ß-hCG and endoglin and down-regulates E-cadherin at mRNA and protein levels. Synergistic levels of both up- and down-regulated genes/proteins were observed when cells were treated with the combination of Fo and PMA. The mRNA levels of cyclin D1, cyclin E1, p21, Rb, p53, caspase-3 and caspase-8 decreased gradually during differentiation. Fo significantly inhibited the protein levels of PCNA, Rb, PKC-α and PMA stimulated mRNA expression of PKC-ε and PKC-δ. Further, failure in the activation of essential components of the cell cycle machinery caused G2/M phase arrest in differentiating BeWo cells.

Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells.

The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8 and TNF-α), cyclo­oxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1ß, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases.

Apoptosis and expression of apoptosis-related genes in mouse intestinal tissue after whole-body proton exposure.

Energetic protons are the most abundant particle type in space and can pose serious health risks to astronauts during long-duration missions. The health effects of proton exposure are also a concern for cancer patients undergoing radiation treatment with accelerated protons. To investigate the damage induced by energetic protons in vivo to radiosensitive organs, 6-week-old BALB/c male mice were subjected to 250 MeV proton radiation at whole-body doses of 0.1, 1, and 2 Gy. The gastrointestinal (GI) tract of each exposed animal was dissected 4 h post-irradiation, and the isolated small intestinal tissue was analyzed for histopathological and gene expression changes. Histopathologic observation of the tissue using standard hematoxylin and eosin (H&E) staining methods to screen for morphologic changes showed a marked increase in apoptotic lesions for even the lowest dose of 0.1 Gy, similar to X- or γ rays. The percentage of apoptotic cells increased dose-dependently, but the dose response appeared supralinear, indicating hypersensitivity at low doses. A significant decrease in surviving crypts and mucosal surface area, as well as in cell proliferation, was also observed in irradiated mice. Gene expression analysis of 84 genes involved in the apoptotic process showed that most of the genes affected by protons were common between the low (0.1 Gy) and high (1 and 2 Gy) doses. However, the genes that were distinctively responsive to the low or high doses suggest that high doses of protons may cause apoptosis in the small intestine by direct damage to the DNA, whereas low doses of protons may trigger apoptosis through a different stress response mechanism.

Combined Effects of Simulated Microgravity and Radiation Exposure on Osteoclast Cell Fusion.

The loss of bone mass and alteration in bone physiology during space flight are one of the major health risks for astronauts. Although the lack of weight bearing in microgravity is considered a risk factor for bone loss and possible osteoporosis, organisms living in space are also exposed to cosmic radiation and other environmental stress factors. As such, it is still unclear as to whether and by how much radiation exposure contributes to bone loss during space travel, and whether the effects of microgravity and radiation exposure are additive or synergistic. Bone is continuously renewed through the resorption of old bone by osteoclast cells and the formation of new bone by osteoblast cells. In this study, we investigated the combined effects of microgravity and radiation by evaluating the maturation of a hematopoietic cell line to mature osteoclasts. RAW 264.7 monocyte/macrophage cells were cultured in rotating wall vessels that simulate microgravity on the ground. Cells under static 1g or simulated microgravity were exposed to γ rays of varying doses, and then cultured in receptor activator of nuclear factor-κB ligand (RANKL) for the formation of osteoclast giant multinucleated cells (GMCs) and for gene expression analysis. Results of the study showed that radiation alone at doses as low as 0.1 Gy may stimulate osteoclast cell fusion as assessed by GMCs and the expression of signature genes such as tartrate resistant acid phosphatase (Trap) and dendritic cell-specific transmembrane protein (Dcstamp). However, osteoclast cell fusion decreased for doses greater than 0.5 Gy. In comparison to radiation exposure, simulated microgravity induced higher levels of cell fusion, and the effects of these two environmental factors appeared additive. Interestingly, the microgravity effect on osteoclast stimulatory transmembrane protein (Ocstamp) and Dcstamp expressions was significantly higher than the radiation effect, suggesting that radiation may not increase the synthesis of adhesion molecules as much as microgravity.

Transcriptomics, NF-κB Pathway, and Their Potential Spaceflight-Related Health Consequences.

In space, living organisms are exposed to multiple stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as bone loss and immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. Over the years, a number of studies have been conducted under real or simulated space conditions. RNA and protein levels in cellular and animal models have been targeted in order to identify pathways affected by spaceflight. Of the many pathways responsive to the space environment, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) network appears to commonly be affected across many different cell types under the true or simulated spaceflight conditions. NF-κB is of particular interest, as it is associated with many of the spaceflight-related health consequences. This review intends to summarize the transcriptomics studies that identified NF-κB as a responsive pathway to ground-based simulated microgravity or the true spaceflight condition. These studies were carried out using either human cell or animal models. In addition, the review summarizes the studies that focused specifically on NF-κB pathway in specific cell types or organ tissues as related to the known spaceflight-related health risks including immune dysfunction, bone loss, muscle atrophy, central nerve system (CNS) dysfunction, and risks associated with space radiation. Whether the NF-κB pathway is activated or inhibited in space is dependent on the cell type, but the potential health impact appeared to be always negative. It is argued that more studies on NF-κB should be conducted to fully understand this particular pathway for the benefit of crew health in space.

Differential expression of AP-1 transcription factors in human prostate LNCaP and PC-3 cells: role of Fra-1 in transition to CRPC status.

Androgen receptor (AR) signaling axis plays a vital role in the development of prostate and critical in the progression of prostate cancer. Androgen withdrawal initially regresses tumors but eventually develops into aggressive castration-resistant prostate cancer (CRPC). Activator Protein-1 (AP-1) transcription factors are most likely to be associated with malignant transformation in prostate cancer. Hence, to determine the implication of AR and AP-1 in promoting the transition of prostate cancer to the androgen-independent state, we used AR-positive LNCaP and AR-negative PC-3 cells as an in vitro model system. The effect of dihydrotestosterone or anti-androgen bicalutamide on the cell proliferation and viability was assessed by MTT assay. Expression studies on AR, marker genes-PSA, TMPRSS2, and different AP-1 factors were analyzed by semi-quantitative RT-PCR and expressions of AR and Fra-1 proteins were analyzed by Western blotting. Dihydrotestosterone induced the cell proliferation in LNCaP with no effect on PC-3 cells. Bicalutamide decreased the viability of both LNCaP and PC-3 cells. Dihydrotestosterone induced the expression of AR, PSA, c-Jun, and Fra-1 in LNCaP cells, and it was c-Jun and c-Fos in case of PC-3 cells, while bicalutamide decreased their expression. In addition, constitutive activation and non-regulation of Fra-1 by bicalutamide in PC-3 cells suggested that Fra-1, probably a key component, involved in transition of aggressive androgen-independent PC-3 cells with poor prognosis.

Cadmium induces oxidative stress and apoptosis in lung epithelial cells.

Cadmium (Cd) is one of the well-known highly toxic environmental and industrial pollutants. Cd first accumulates in the nucleus and later interacts with zinc finger proteins of antiapoptotic genes and inhibit the binding of transcriptional factors and transcription. However, the role of Cd in oxidative stress and apoptosis is less understood. Hence, the present study was undertaken to unveil the mechanism of action. A549 cells were treated with or without Cd and cell viability was measured by MTT assay. Treatment of cells with Cd shows reduced viability in a dose-dependent manner with IC50 of 45 µM concentration. Cd significantly induces the reactive oxygen species (ROS), lipid peroxidation followed by membrane damage with the leakage of lactate dehydrogenase (LDH). Cells with continuous exposure of Cd deplete the antioxidant super oxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Further, analysis of the expression of genes involved in apoptosis show that both the extrinsic and intrinsic apoptotic pathways were involved. Death receptor marker tumor necrosis factor-α (TNF-α), executor caspase-8 and pro-apoptotic gene (Bax) were induced, while antiapoptotic gene (Bcl-2) was decreased in Cd-treated cells. Fluorescence-activated cell sorting (FACS) analysis further confirms the induction of apoptosis in Cd-treated A549 cells.

Interplay of nuclear receptors (ER, PR, and GR) and their steroid hormones in MCF-7 cells.

Steroid hormones and their nuclear receptors play a major role in the development and progression of breast cancer. MCF-7 cells are triple-positive breast cancer cells expressing estrogen receptor (ER), progesterone receptor (PR), and glucocorticoid receptor (GR). However, interaction and their role in expression pattern of activator protein (AP-1) transcription factors (TFs) are not completely understood. Hence, in our study, MCF-7 cells were used as an in vitro model system to study the interplay between the receptors and hormones. MCF-7 cells were treated with estradiol-17ß (E2), progesterone (P4), and dexamethasone (Dex), alone or in combination, to study the proliferation of cells and expression of AP-1 genes. MTT assay results show that E2 or P4 induced the cell proliferation by more than 35 %, and Dex decreased the proliferation by 26 %. E2 and P4 are found to increase ERα by more than twofold and c-Jun, c-Fos, and Fra-1 AP-1 TFs by more than 1.7-fold, while Dex shows opposite effect of E2- or P4-induced effect as well as effect on the expression of nuclear receptors and AP-1 factors. E2 antagonist Fulvestrant (ICI 182,780) found to reduce proliferation and E2-induced expression of AP1-TFs, while P4 or Dex antagonist Mifepristone (RU486) is found to block GR-mediated expression of NRs and AP-1 mRNAs. Results suggest that E2 and P4 act synergistically, and Dex acts as an antagonist of E2 and P4.

Optimization of Parameters to Achieve High Yield and Purity Single-Walled Carbon Nanotube by Thermal and Chemical Oxidation and Its Effect on Conductivity.

Single wall carbon nanotubes due to their unique structural and electronic characteristics have revolutionized the field of nanotechnology and are widely used the field of transistors, drug delivery, and nanocomposities. For improved efficiency of these applications, the utilized tubes must of preeminent purity. Here, we report key parameters that are optimized to achieve their highest purity upto 98 wt%, and yield as high as 50 wt% by thermal and chemical oxidation. The as-produced SWCNT were heated in air at 470 °C, for 90 min, and later subjected to chemical oxidation. The chemical oxidation involved the treatment of thermally treated SWCNT with different concentrations of HCl (4N, 6N, 8N) and 30% H2O2, for different time periods (4 hr, 6 hr). This method does not cause damage to the walls of the tubes, observing no loss of nanotubes. The sheet resistance of as-produced and purified tubes was measured and the conductivity was calculated.

Anti-Inflammatory Effect of Apigenin on LPS-Induced Pro-Inflammatory Mediators and AP-1 Factors in Human Lung Epithelial Cells.

Apigenin is one of the plant flavonoids present in fruits and vegetables, acting as an important nutraceutical component. It is recognized as a potential antioxidant, antimicrobial, and anti-inflammatory molecule. In the present study, the mechanism of anti-inflammatory action of apigenin on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and activator protein-1 (AP-1) factors in human lung A549 cells was investigated. The anti-inflammatory activity of apigenin on LPS-induced inflammation was determined by analyzing the expression of pro-inflammatory cytokines, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and different AP-1 factors. Apigenin significantly inhibited the LPS-induced expression of iNOS, COX-2, expression of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8, and TNF-α), and AP-1 proteins (c-Jun, c-Fos, and JunB) including nitric oxide production. Study confirms the anti-inflammatory effect of apigenin by inhibiting the expression of inflammatory mediators and AP-1 factors involved in the inflammation and its importance in the treatment of lung inflammatory diseases.

Toxicity of Raw and Purified Single-Walled Carbon Nanotubes in Rat's Lung Epithelial and Cervical Cancer Cells.

The increased applications of carbon nanotubes in the field of drug delivery, bioimaging and biosensors demand nanotubes to be of highest purity, free from metallic impurities and amorphous carbon. All of these sectors require a profound investigation about the toxic effects on human and the environment. Many attempts have been made to purify and surface modify the carbon nanotubes, however a detailed study on the raw and purified material has yet to be conducted. Here we present the toxicity studies of raw and the purified single-walled carbon nanotubes in rat's lung epithelial cell and cervical cancer cells (HeLa). These cells were treated with increasing concentration of 0.5 µg/mL to 50 µg/mL and the various biocompatibility assays were performed. The results showed an increased cell death with purified single-walled carbon nanotubes followed by the depletion of antioxidant levels and activation of the caspase cascade at a rapid rate compared to raw single-walled carbon nanotubes. This suggests that purified single walled carbon nanotubes are more toxic to the cells and exhibit ultra-fine particulate matter like toxicity.

Apigenin inhibits PMA-induced expression of pro-inflammatory cytokines and AP-1 factors in A549 cells.

Acute and chronic alveolar or bronchial inflammation is thought to be central to the pathogenesis of many respiratory disorders. Cytokines and granulocyte macrophage colony-stimulating factors (GM-CSF) play an important role in chronic inflammation. Activator protein-1 (AP-1) the superfamily of transcription factors is involved in proliferation, differentiation, apoptosis, and transformation including inflammation. Understanding the function and regulation of proinflammatory factors involved in inflammation may provide the novel therapeutic strategies in the treatment of inflammatory diseases. Our aim of the present study is to investigate the pro-inflammatory cytokines and pattern of AP-1 factors expressed during activation of lung adenocarcinoma A549 cells by Phorbol-12-myristate-13-acetate (PMA) and to understand the anti-inflammatory effect of apigenin. A549 cells were treated with and without PMA or apigenin, and the cell viability was assessed by MTT assay. Expressions of inflammatory mediators and different AP-1 factors were analyzed by semi-quantitative RT-PCR. IL-6 protein secreted was analyzed by ELISA, and expressions of IL-1ß, c-Jun, and c-Fos proteins were analyzed by Western blotting. Activation of A549 cells by PMA, induced the expression of pro-inflammatory cytokine (IL-1ß, IL-2, IL-6, IL-8, and TNF-α) mRNAs and secretion of IL-6 and the expression of specific AP-1 factors (c-Jun, c-Fos, and Fra-1). Treatment of cells with apigenin, significantly inhibited PMA-stimulated mRNA expression of above pro-inflammatory cytokines, AP-1 factors, cyclooxygenase-2, and secretion of IL-6 protein. Results suggested that the AP-1 factors may be involved in inflammation and apigenin has anti-inflammatory effect, which may be useful for therapeutic management of lung inflammatory diseases.

Utilization of highly purified single wall carbon nanotubes dispersed in polymer thin films for an improved performance of an electrochemical glucose sensor.

In this work we report the improved performance an electrochemical glucose sensor based on a glassy carbon electrode (GCE) that has been modified with highly purified single wall carbon nanotubes (SWCNTs) dispersed in polyethyleneimine (PEI), polyethylene glycol (PEG) and polypyrrole (PPy). The single wall carbon nanotubes were purified by both thermal and chemical oxidation to achieve maximum purity of ~98% with no damage to the tubes. The SWCNTs were then dispersed by sonication in three different organic polymers (1.0mg/ml SWCNT in 1.0mg/ml of organic polymer). The stable suspension was coated onto the GCE and electrochemical characterization was performed by Cyclic Voltammetry (CV) and Amperometry. The electroactive enzyme glucose oxidase (GOx) was immobilized on the surface of the GCE/(organic polymer-SWCNT) electrode. The amperometric detection of glucose was carried out at 0.7 V versus Ag/AgCl. The GCE/(SWCNT-PEI, PEG, PPY) gave a detection limit of 0.2,633 µM, 0.434 µM, and 0.9,617 µM, and sensitivities of 0.2411 ± 0.0033 µA mM(-1), r(2)=0.9984, 0.08164 ± 0.001129 µA mM(-1), r(2)=0.9975, 0.04189 ± 0.00087 µA mM(-1), and r(2)=0.9944 respectively and a response time of less than 5s. The use of purified SWCNTs has several advantages, including fast electron transfer rate and stability in the immobilized enzyme. The significant enhancement of the SWCNT modified electrode as a glucose sensor can be attributed to the superior conductivity and large surface area of the well dispersed purified SWCNTs.

Distinctive microRNA expression signatures in proton-irradiated mice.

Proton particles comprise the most abundant ionizing radiation (IR) in outer space. These high energy particles are known to cause frequent double- and single-stranded DNA lesions that can lead to cancer and tumor formation. Understanding the mechanism of cellular response to proton-derived IR is vital for determining health risks to astronauts during space missions. Our understanding of the consequences of these high energy charged particles on microRNA (miRNA) regulation is still in infancy. miRNAs are non-coding, single-stranded RNAs of ~22 nucleotides that constitute a novel class of gene regulators. They regulate diverse biological processes, and each miRNA can control hundreds of gene targets. To investigate the effect of proton radiation on these master regulators, we examined the miRNA expression in selected mice organs that had been exposed to whole-body proton irradiation (2 Gy), and compared this to control mice (0 Gy exposure). RNA was isolated from three tissues (testis, brain, and liver) from treated and control mice and subjected to high-throughput small RNA sequencing. Bioinformatics analysis of small RNA sequencing data revealed dysregulation of (p < 0.05; 20 up- and 10 down-regulated) 14 mouse testis, 8 liver, and 8 brain miRNAs. The statistically significant and unique miRNA expression pattern found among three different proton-treated mouse tissues indicates a tissue-specific response to proton radiation. In addition to known miRNAs, sequencing revealed differential expression of 11 miRNAs in proton-irradiated mice that have not been previously reported in association with radiation exposure and cancer. The dysregulation of miRNAs on exposure to proton radiation suggest a possible mechanism of proton particles involvement in the onset of cell tumorgenesis. In summary, we have established that specific miRNAs are vulnerable to proton radiation, that such differential expression profile may depend upon the tissue, and that there are more miRNAs affected by proton radiation than have been previously observed.

Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation.

The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17ß (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation.

Optimization of process parameters of polymer solution mediated growth of calcium carbonate nanoparticles.

With the advent of nanotechnology, many methods of synthesis of nanoparticles have come into practice and the 'polymer mediated growth' technique is among them. In this route, ions of one of the reactants are allowed to diffuse from an external solution into a polymer matrix where the other reactant is complexed and bound. The exact role of ionic diffusion in the formation of nanoparticles was investigated in the current study by studying the patterns of kinetics of nanoparticle formation using UV vis spectroscopy. Typically, calcium carbonate nanoparticles were formed by the aforementioned technique using polyethylene glycol solution. The particle size was calculated using Scherrer's formula on x-ray diffraction plots and was reconfirmed with field emission scanning electron microscope and transmission electron microscope images. Energy-dispersive x-ray analysis was used to study the composition and purity of the nanoparticles formed. The reactant to polymer ratio, reaction temperature and molecular weight of polyethylene glycol affected the size of the particles formed. Through this knowledge we optimized these parameters to obtain particles as small as 20 nm and confirmed that this technique can be used to control the size of nanoparticles.

Magnetite induces oxidative stress and apoptosis in lung epithelial cells.

There is an ongoing concern regarding the biocompatibility of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. In this study, we investigated the toxic properties of magnetite stabilized with polyacrylate sodium. The magnetite was characterized by X-ray powder diffraction analysis, and the mean particle diameter was calculated using the Scherrer formula and was found to be 9.3 nm. In this study, we treated lung epithelial cells with different concentrations of magnetite and investigated their effects on oxidative stress and cell proliferation. Our data showed an inhibition of cell proliferation in magnetite-treated cells with a significant dose-dependent activation and induction of reactive oxygen species. Also, we observed a depletion of antioxidants, glutathione, and superoxide dismutase, respectively, as compared with control cells. In addition, apoptotic-related protease/enzyme such as caspase-3 and -8 activities, were increased in a dose-dependent manner with corresponding increased levels of DNA fragmentation in magnetite-treated cells compared to than control cells. Together, the present study reveals that magnetite exposure induces oxidative stress and depletes antioxidant levels in the cells to stimulate apoptotic pathway for cell death.

Reactive oxygen species mediated tissue damage in high energy proton irradiated mouse brain.

Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. We recently reported that the high energy protons induce cell death through activation of apoptotic signaling genes; caspase 3 and 8 (Baluchamy et al. J Biol Chem 285:24769-24774, 2010). In this study, we investigated the effect of different doses of protons in in vivo mouse system, particularly, brain tissues. A significant dose-dependent induction of reactive oxygen species and lipid peroxidation and reduction of antioxidants; glutathione and superoxide dismutase were observed in proton irradiated mouse brain as compared to control brain. Furthermore, histopathology studies on proton irradiated mouse brain showed significant tissue damage as compared to control brain. Together, our in vitro and in vivo results suggest that proton irradiation alters oxidant and antioxidant levels in the cells to cause proton mediated DNA/tissue damage followed by apoptotic cell death.

Calcium carbonate nanoparticles: synthesis, characterization and biocompatibility.

The synthesis of nanoparticles and their functionalization to effectively utilize them in biological applications including drug delivery is currently a challenge. Calcium carbonate among many other inorganic nanosized particles offers promising results for such applications. We have synthesized calcium carbonate nanoparticles using polymer mediated growth technique, where one of the ions bound within polymer matrix and the other diffuses and reacts to form desired compound. The synthesized nanoparticles are characterized using X-ray diffraction, Scanning Electron Microscopy and spectroscopic techniques such as Fourier-Transform Infra-red spectroscopy and UV-Vis spectroscopy. The diameter of the calcium carbonate nanoparticles is estimated to be 39.8 nm and their biocompatibility studies showed no significant induction of oxidative stress or cell death even at higher concentrations (50 microg) upon exposure to HeLa and LE cells. Here, we report that the synthesized calcium carbonate nanosized particles using polymer mediated growth technique are biocompatible and can be safely used for biomedical applications.