Induced Cell Necrosis Research Articles

Molecular Pathways: Targeting Mechanisms of Asbestos and Erionite Carcinogenesis in Mesothelioma

Malignant mesothelioma is an aggressive malignancy related to asbestos and erionite exposure. AP-1 transcriptional activity and the NF-κB signaling pathway have been linked to mesothelial cell transformation and tumor progression. HGF and c-Met are highly expressed in mesotheliomas. Phosphoinositide 3-kinase, AKT, and the downstream mTOR are involved in cell growth and survival, and they are often found to be activated in mesothelioma. p16(INK4a) and p14(ARF) are frequently inactivated in human mesothelioma, and ∼50% of mesotheliomas contain the NF2 mutation. Molecular therapies aimed at interfering with these pathways have not improved the dismal prognosis of mesothelioma, except possibly for a small subset of patients who benefit from certain therapies. Recent studies have shown the importance of asbestos-induced inflammation in the initiation and growth of mesothelioma, and HMGB1 and Nalp3 inflammasome have been identified as key initiators of this process. Asbestos induces cell necrosis, causing the release of HMGB1, which in turn may activate Nalp3 inflammasome, a process that is enhanced by asbestos-induced production of reactive oxygen species. HMGB1 and Nalp3 induce proinflammatory responses and lead to interleukin-1β and TNF-α secretion and NF-κB activity, thereby promoting cell survival and tumor growth. Novel strategies that interfere with asbestos- and erionite-mediated inflammation might prevent or delay the onset of mesothelioma in high-risk cohorts, including genetically predisposed individuals, and/or inhibit tumor growth. The very recent discovery that germline BAP1 mutations cause a new cancer syndrome characterized by mesothelioma, uveal melanoma, and melanocytic tumors provides researchers with a novel target for prevention and early detection.

Effects of quercetin nanoliposomes on C6 glioma cells through induction of type III programmed cell death

BackgroundQuercetin has been shown to induce apoptosis in a number of cancer cell lines, but a quercetin-loaded nanoliposomal formulation with enhanced antitumor activity in C6 glioma cells and its effect on cancer cell death has not been well studied. The aim of this study was to examine if quercetin-loaded liposomes (QUE-NL) has enhanced cytotoxic effects and if such effects involve type III programmed cell death in C6 glioma cells.MethodsC6 glioma cells were treated with QUE-NL and assayed for cell survival, apoptosis, and necrosis. Levels of reactive oxygen species production and loss of mitochondrial membrane potential (ΔΨm) were also determined by flow cytometry assay to assess the effects of QUE-NL. ATP levels and lactate dehydrogenase activity were measured, and Western blotting was used to assay cytochrome C release and caspase expression.ResultsQUE-NL induced type III (necrotic) programmed cell death in C6 glioma cells in a dose-dependent and time-dependent manner. High concentrations of QUE-NL induced cell necrosis, which is distinct from apoptosis and autophagy, whereas liposomes administered alone induced neither significant apoptosis nor necrosis in C6 glioma cells. QUE-NL-induced ΔΨm loss and cytochrome C release had no effect on caspase activation, but decreased ATP levels and increased lactate dehydrogenase activity indicated that QUE-NL stimulated necrotic cell death.ConclusionC6 glioma cells treated with QUE-NL showed a cellular pattern associated with necrosis without apoptosis and was independent of caspase activity. Nonapoptotic cell death induced by high concentrations of QUE-NL for controlling caspase-independent type III programmed cell death may provide the basis for novel therapeutic approaches to overcome avoidance of apoptosis by malignant cells.

The corky root rot pathogen Pyrenochaeta lycopersici manipulates tomato roots with molecules secreted early during their interaction

Corky root rot is a ubiquitous soil-borne disease of tomato caused by the pathogen Pyrenochaeta lycopersici. This filamentous fungus is found on the roots of many crops and can persist in the soil up to 15 years as microsclerotia. High prevalence of corky root rot can be partly explained by the endurance and the broad host range of P. lycopersici, but how this fungus can gain access to host roots is still poorly understood, as its competitive saprophytic ability is very low. We have combined microscopy and reporter gene techniques to investigate the tomato–P. lycopersici interaction in vitro, and discovered the pathogen secretes molecules that change the direction of root growth and induce cell necrosis specifically in the apical part of the root of tomato (apex, elongation zone and beginning of the root hair zone). Moreover, we found that the fungus preferentially infects immature root cells that are sensitive to these secreted fungal molecules, whereas infection is blocked in mature and insensitive parts of the root. Our study sheds light on novel and important features of the biology of this pathogen, which could contribute to its fitness in the rhizosphere.

Forkhead Transcription Factor FOXO3a Protects Alveolar Epithelial Cells from Oxidative Stress

Acute lung injury (ALI) is a devastating clinical problem involving the key events of inflammation and alveolar epithelial cell death. The fundamental mechanism of this serious condition evolves from an imbalance between pro- inflammatory and anti-inflammatory cytokines finally resulting in oxidative stress induced cell death. FOXO transcription factors are important regulators of cell survival in response to a variety of stress stimuli, among which are oxidative stress, DNA damage, and nutrient deprivation. However, the role of FOXO3a in acute lung injury and epithelial cell death is not known. It was hypothesized that FOXO3a protects alveolar epithelial cells from H2O2 induced cell death. To test this hypothesis, the effects of H2O2 on subconfluent human alveolar epithelial cell (A549) cultures were tested. A549 cells were transfected with vector containing wild type FOXO3a cDNA (FOXO3a- WT) or vector containing mutant FOXO3a cDNA (FOXO3a-M) and treated with or without H2O2 (1hour) to induce oxidative stress. The results showed that H2O2 induced a significant alveolar epithelial cell necrosis and apoptosis. In contrast epithelial cells transfected with FOXO3a –WT inhibited H2O2 induced cell necrosis and apoptosis. However cells transfected with a mutant, FOXO3a-M did not alter H2O2 mediated epithelial cell apoptosis and necrosis. Together these results suggest that FOXO3a protects alveolar epithelial cells from oxidative stress induced cell death. This work was supported by T32-HL007874 and American Heart Association National Scientist Development Grant 09SDG2260957 to N.K.

Effect of composition, morphology and size of nanozeolite on its in vitro cytotoxicity

The extensive applications of nanoparticle materials in biomedical and biotechnological fields trigger the rapid development of nanotoxicology, because nanoparticles are reported to cause more damage than larger ones when human exposure to them. In the present manuscript, we prepared a series of zeolite nanocrystals with different frameworks, sizes, compositions and shapes, and provided the first report on their toxic difference. As our results, the toxicities of zeolite nanoparticles depend on their size, composition and shape when they are exposed to HeLa cells. The pure-silica nanozeolite silicalite-1 displays nontoxicity, but aluminum-containing nanozeolites, such as ZSM-5, LTL, and LTA, show a dose-dependent toxic manner. The different shapes of nanozeolites can lead to different cytotoxicities, while the influences of the surface charge differences of various nanozeolites on their toxicities are unconspicuous. More importantly, caspase-3 activity and LDH released assays showed that the toxic nanozeolites seem to induce cell necrosis rather than cell apoptosis by the damnification for the cell membranes. These results are expected to direct the applications of nanozeolites with different structures and shapes in biomedicine and clinic science.

Efek Ekstrak Sambiloto (Andrographis paniculata Nees) pada Ekspresi Telomerase dari Kanker Payudara Tikus yang Diinduksi dengan DMBA

ABSTRACT
 
 It has been well documented that chemical carcinogen, 7.12 dimethylbenz(a)anthracene (DMBA), plays a role in the incidence and growth of mammary cancer. Present study was designed to investigate the influence of Andrographis paniculata extract on telomerase activities on DMBA induced breast cancer in the female rat Sprague Dawley strain. DMBA-induced mammary cancer is a useful model to investigate the changes of epithelial cells that occur during mammary cancer progression. Mammary cancer model was induced 10 times twice a week by oral DMBA 20 mg/kg body weight. Mammary cancer occurred in 75 % animals nine weeks after oral administration of DMBA, it was represented with nodule on the mammary gland and the increasing of mammary gland volume compare with normal control F(1.8) = 731.711; p < 0.001. This study was also designed to investigate the effect of Andrographis paniculata extract mammary carcinoma induced by DMBA. Administration of three different dose of Andrographis paniculata (100 mg/kg, 300 mg/kg and 1000 mg/kg) had statistically different with mammary gland volume of DMBA treated rat F (4.17) = 92.777; p<0.05. So, Andrographis paniculata has significant effect on the treatment of DMBA-induced mammary carcinoma. The Epithelial cells were harvested on day 90 and stained with routine histology staining, hematoxylineosin, for morphological qualitative analysis, immunohistochemical examination. The lesions observed from the removed samples ranged widely from benign to malignant. The results showed that DMBA induce cell proliferation, nuclear irregularities, and numerous mitoses and induced cell necrosis. The effect of Andrographis paniculata inhibits cell proliferation and induces apoptosis in cancer cells. On immunohistochemical examination, it shows that Andrographis paniculata can stimulate of telomerase enzyme.
 
 Key word: Andrographis paniculata, DMBA, mammary cancer, cell proliferation
 
 
 ABSTRAK
 
 Telah dilakukan penelitian efek ekstrak sambiloto (Andrographis paniculata Nees) pada ekspresi telomerase terhadap kanker payudara tikus betina (Sprague dawley) yang diinduksi dengan 7,12 dimethylbenz(a)anthracene (DMBA) menggunakan metode imunohistokimia. Diketahui model kanker payudara dengan induksi DMBA untuk menginvestigasi perubahan dari sel epitel yang terjadi selama prorses karsinogenesis kanker payudara. Pemberian ekstrak sambiloto pada tikus yang mengalami kanker payudara menyebabkan penurunan volume tumor dan ditinjau dari aspek hispatologi dan imunohistokimia adanya ekstrak sambiloto menyebabkan penghambatan proliferasi sel, penurunan ekspresi telomerase dan meningkatkan apoptosis.
 
 Kata Kunci : Andrographis paniculata,DMBA, kanker payudara, proliferasi sel

Simultaneous Imaging and Chemical Attack of a Single Living Cell within a Confluent Cell Monolayer by Means of Scanning Electrochemical Microscopy

Epithelial cell monolayers from rat kidney were imaged by scanning electrochemical microscopy (SECM) with sub-micrometer resolution in both lateral and vertical direction. Platinum disk ultra-microelectrodes (UMEs) with effective electrode radii between 200 and 600 nm were operated in the constant-height mode. The quality of the recorded SECM images compare favorably with those of phase contrast and confocal laser scanning microscopy. Besides the acquisition of SECM images, the UME was used to selectively attack a single living cell within the monolayer ensemble. Hydroxide ions were locally generated in the vicinity of a single target cell by the UME. The increase in pH induced cell necrosis that was subsequently imaged by SECM. It could be clearly demonstrated that the single target cell was selectively affected, whereas the adjacent reference cells remained unchanged.

A Proapoptotic Peptide Derived from Reovirus Outer Capsid Protein μ1 Has Membrane-Destabilizing Activity

The reovirus outer capsid protein μ1 is responsible for cell membrane penetration during virus entry and contains determinants necessary for virus-induced apoptosis. Residues 582 to 611 of μ1 are necessary and sufficient for reovirus-induced apoptosis, and residues 594 and 595 independently regulate the efficiency of viral entry and reovirus-induced cell apoptosis, respectively. Two of three α-helices within this region, helix 1 (residues 582 to 611) and helix 3 (residues 644 to 675), play a role in reovirus-induced apoptosis. Here, we chemically synthesized peptides representing helix 1 (H1), H1:K594D, H1:I595K, and helix 3 (H3) and examined their biological properties. We found that H1, but not H3, was able to cause concentration- and size-dependent leakage of molecules from small unilamellar liposomes. We further found that direct application of H1, but not H1:K594D, H1:I595K, or H3, to cells resulted in cytotoxicity. Application of the H1 peptide to L929 cells caused rapid elevations in intracellular calcium concentration that were independent of phospholipase C activation. Cytotoxicity of H1 was not restricted to eukaryotic cells, as the H1 peptide also had bactericidal activity. Based on these findings, we propose that the proapoptotic function of the H1 region of μ1 is dependent on its capacity to destabilize cellular membranes and cause release of molecules from intracellular organelles that ultimately induces cell necrosis or apoptosis, depending on the dose.

Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c Proteins Participate in the Suppression of Macrophage Apoptosis

BackgroundInhibition of macrophage apoptosis by Mycobacterium tuberculosis has been proposed as one of the virulence mechanisms whereby the pathogen avoids the host defense. The mechanisms by which M. tuberculosis H37Rv strain suppress apoptosis and escapes human macrophage killing was investigated.Methodology/Principal FindingsThe screening of a transposon mutant bank identified several mutants, which, in contrast to the wild-type bacterium, had impaired ability to inhibit apoptosis of macrophages. Among the identified genes, Rv3659c (31G12 mutant) belongs to an operon reminiscent of type IV pili. The Rv3654c and Rv3655c putative proteins in a seven-gene operon are secreted into the macrophage cytoplasm and suppress apoptosis by blocking the extrinsic pathway. The operon is highly expressed when the bacterium is within macrophages, compared to the expression level in the extracellular environment. Rv3654c recognizes the polypyrimidine tract binding Protein-associated Splicing Factor (PSF) and cleaves it, diminishing the availability of caspase-8. While M. tuberculosis inhibits apoptosis by the extrinsic pathway, the pathogen does not appear to affect the intrinsic pathway. Inactivation of the intrinsic pathway by pharmacologic agents afftects M. tuberculosis and induces cell necrosis. Likewise, inactivation of PSF by siRNA significantly decreased the level of caspase-8 in macrophages.ConclusionWhile M. tuberculosis inhibits the extrinsic pathway of apoptosis, it appears to activate the intrinsic pathway leading to macrophage necrosis as a potential exit strategy.

Morpho-Functional Features of In-Vitro Cell Death Induced by Physical Agents

Based on the study of the recent literature, the aim of this report is to present and discuss in vitro cell death, and, more specifically, apoptosis appearing after exposure to physical conditions such as hyperthermia, hypothermia, UVB radiation and static magnetic fields. Hyperthermia (i.e. variable exposures to 42 degrees C) as well as hypothermia conditions (i.e. variable exposures to 0 degrees C) were considered mild and were both followed by incubation at physiological conditions. Heat exposure can be considered as a powerful apoptotic inducer in a variety of cells, where it induces classical apoptotic changes and well known biochemical pathways. The effect of hyperthermia has been described in adherent human tumour cells that undergo cell rounding and progressively detach from the substrate, due to the concomitant down-regulation of adhesion molecules. Hypothermia instead, as a cold treatment, only occasionally triggers apoptosis, but more frequently induces cell necrosis. UVB radiation induces oxidative stress, leading, in most experimental models, to apoptotic cell death, generally through the intrinsic apoptotic pathway, even if the extrinsic pathway cannot be excluded. Interestingly, UVB radiation appears effective also on cell systems that are normally apoptosis-resistant, such as muscle cells. Most cell alterations induced by static magnetic fields result from alterations at the plasma membrane and microvilli; in this case, apoptotic cell death is rarely detected. Therefore, cell death can be induced with physical agents in dependence on the treatment and cell model employed. In particular, hyperthermia and UVB can be considered a reliable and reproducible apoptotic trigger.

Hormones of thyroid gland in sera of rats treated with different dose of concentrated potassium iodine solutions

Potassium iodine (KI) is used as a drug therapy for treating numerous diseases such as small-vessel vasculitis, erythema nodosum, vasculitis nodularis, Sweet's syndrome, tuberculosis and granulomatosis, and for iodized salt. At the same time, KI can be harmful. Iodine intake may increase the frequency of thyroiditis in humans, and may induce the occurrence of experimental thyroiditis (ET) in animals. Investigations on an experimental model for the examination of thyroiditis in Wistar rats have clearly showed morphological changes in the rat thyroid evoked by KI administration. The purpose of this study was to compare the effects of low and high doses of KI on the thyroid gland of Wistar rats and determine the effect on hormone status (T4, T3 and TSH) in this rat strain. Two groups of rats from the Wistar strain were treated with a low iodine dose (225 microg/g BW) and with a high iodine dose (675 microg/g BW) of KI solutions. Untreated nonimmunized animals served as controls. The solution was administrated daily intraperitoneally during the period of 26 consecutive days. Monitoring hormone status (TSH, T3 and T4) and morphological changes it was found that therapeutic doses of KI applied in treatment induced the occurrence of experimental thyroiditis (chronic destructive Hashimoto's thyroiditis in humans) and cell necrosis in animals not carrying a genetic susceptibility. Significant inflammatory changes were observed in rats treated with a high iodine dose. The early iodine induced cell necrosis and inflammation in the nonimmunized animals without genetic susceptibility is a new experimental model of thyroiditis.

Chemotactic and Cytotoxic Effects of Minocycline on Human Retinal Pigment Epithelial Cells

To reveal the effects of minocycline, an anti-inflammatory and neuroprotective agent, on the viability and physiological properties of retinal pigment epithelial (RPE) cells and to compare the effects with those of triamcinolone acetonide. The proliferation of human RPE cells in vitro was investigated with a bromodeoxyuridine immunoassay; chemotaxis was examined with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. The gene expression of growth factors and MMP-9 was determined with real-time RT-PCR, and the secretion of VEGF was examined with ELISA. The phosphorylation of p38 MAPK and ERK1/2 proteins was determined with Western blot analysis. Minocycline at low concentrations (50 nM-20 microM) stimulated chemotaxis and decreased the proliferation of RPE cells. Minocycline at high concentrations (above 5 microM) decreased the viability of RPE cells through the induction of cell necrosis. The chemotactic effect of minocycline was mediated by the stimulation of autocrine PDGF signaling and the activation of p38 MAPK. Minocycline promoted the expression of PDGF-B, HGF, VEGF, and MMP-9 and increased the amounts of phosphorylated p38 and ERK1/2 proteins in RPE cells. Triamcinolone reduced PDGF-evoked chemotaxis and VEGF expression and secretion and had no significant effects on cell viability and proliferation. Triamcinolone did not reverse the effects of minocycline on cell proliferation, chemotaxis, or viability or the expression of VEGF. Low-dose minocycline induces the activation of RPE cells, as indicated by the activation of p38 and ERK1/2 and by enhanced chemotaxis mediated by autocrine PDGF signaling. High-dose minocycline induces RPE cell degeneration.

Cytometric assessment of cytostatic and cytotoxic effects of topical glaucoma medications on human epithelial corneal line cells

The long-term-treatment of glaucoma with topical medications is associated with side effects involving cornea damage. We examined the effect of glaucoma topical medications (bimatoprost, travoprost, latanoprost, timolol, betaxolol, dorzolamide, brinzolamide, brimonidine) on growth of cells of three human epithelial corneal lines. The cells were cultured in 8-chamber slides, treated with different concentrations of the medications, and fixed at 24, 48, and 72 h. Cell number on slides to estimate viability and growth curves, frequency of apoptosis (FLICA and caspase-3 activation probes), and proliferation (BrdU incorporation assay) were measured by laser scanning cytometry (LSC). Depending on concentration all examined medications induced cell necrosis or apoptosis and suppressed proliferation. Significant variability in proliferation and apoptosis was observed within the same cultures depending on local cell density, with cells in high density areas being more resistant. The data indicate that commonly used topical medications exert cytostatic and cytotoxic effects in cultures of corneal cells and suggest that caution should be exercised in their use, particularly, when the corneal diseases are accompanied by cell proliferation and regeneration, in long-term-treatment. The present approach of using LSC makes it possible to assess and compare cytostatic and cytotoxic effects of different topical medications on the respective target cells.

Apoptotic effect of sonodynamic therapy mediated by hematoporphyrin monomethyl ether on C6 glioma cells in vitro

Sonodynamic therapy (SDT) is a new method for treating cancer by inducing cell necrosis and/or apoptosis. However, the molecular mechanisms of cell apoptosis after SDT treatment remain unclear. In this study we investigated the apoptotic effect and mechanisms of SDT mediated by hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. The time of ultrasound irradiation was optimized by MTT. The apoptotic rate, generation of ROS (reactive oxygen species), and mitochondrial membrane potential (MMP) were detected by flow cytometry, and expressions of apoptosis proteins were measured by Western blot after SDT treatment. The results displayed that treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis, which was associated with the production of ROS and loss of MMP. The results also showed that protein expression of Bax, caspase-9, and caspase-3 significantly increased, protein expression of Bcl-2 and Fas-L significantly decreased, and Fas protein expression was unchanged after SDT treatment. Our results suggested that the mitochondrial signal pathway may play a pivotal role in the apoptosis induced by SDT in C6 glioma cells.

The ubiquitin extension protein S27a is differentially expressed in developing flower organs of Thompson seedless versus Thompson seeded grape isogenic clones

In Vitis vinifera L. cv. Thompson Seedless, fertilization occurs but seeds abort, a type of stenospermocarpy. To clone transcripts with differential expression during flower development, suppressive subtractive hybridization was carried out using two isogenic clones 'Thompson seedless' and 'Thompson seeded', at three stages of inflorescence development (from bud break to ~20 days prior to anthesis). Differential screening and sequencing of a forward and reverse subtractive cDNA library yielded several singleton ESTs. One differentially expressed clone in 'Thompson' seeded versus seedless isogenic clones was the ubiquitin extension protein S27a. In situ hybridization demonstrated its significantly higher expression in the carpel and ovaries of 'Thompson' seedless versus seeded isogenic clones during flower development. Overexpression of this gene resulted in abnormal plant regeneration and inhibited shoot development compared to controls; its silencing in embryogenic callus induced cell necrosis and callus death, evidencing tight regulation of this gene in developing organs of grape. S27a overexpression in carpels and integuments of the seedless flower may interfere with normal development of these organs, leading to embryo abortion and seedlessness.

Association of fliR gene in Leptospira interrogans with adhesion and pathogenicity to host cells

To investigate the pathogenicity of Leptospira interrogans fliR gene to J774A.1 cells. fliR gene from L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and kana gene from plasmid pET42a were amplified by PCR. Suicide plasmid of fliR gene was constructed; and specific siRNA for fliR gene was designed and synthesized. fliR gene mutants were constructed by gene knock-out with suicide plasmid (56601fliR-Kana) and gene silencing with siRNA (56601siRNA-R2). The mutants were identified by PCR, sequencing and semi-quantitative RT-PCR. Adhesion to mouse mononuclear-macrophage J774A.1 and induction of cell necrosis and apoptosis by 56601fliR-Kana and 56601siRNA-R2 were examined by adhesion test and flow cytometry, respectively. The nucleotide and putative amino acid sequences of cloned fliR gene had 99.9% and 100% similarities to those of reported sequences in GenBank. The nucleotide sequence of the cloned kana gene was identical to the corresponding sequence in pET42a map. The results of PCR and sequencing confirmed that kana gene was inserted in the sequence of 56601fliR-Kana fliR gene. The mRNA level of fliR gene in 56601fliR-Kana was remarkably decreased (P<0.01) while the mRNA level of fliR gene in 56601siRNA-R2 was much lower than that in wild strain 56601 (P<0.05). 56601fliR-Kana and 56601siRNA-R2 lost the ability to adhere J774A.1 cells; and their ability to induce cell necrosis and apoptosis was markedly weakened (P<0.01). fliR is a virulence-associated gene of L. interrogans and the function of the gene is closely related to adhesion, induction of cell necrosis and apoptosis of the microbe.

Crystal structure mediates mode of cell death in TiO2 nanotoxicity

Certain properties that nanoparticles possess differentiate them from their bulk counterparts, and these characteristics must be evaluated prior to nanoparticle studies and include: size, shape, dispersion, physical and chemical properties, surface area, and surface chemistry. Early nanotoxicity studies evaluating TiO2 have yielded conflicting data which identify either size or crystal structure as the mediating property for nano-TiO2 toxicity. However, it is important to note that none of these studies examined size with the crystal structure composition controlled for or examined crystal structure while controlling the nanoparticle size. The goal of this study was to evaluate the role of size and crystal structure in TiO2 nanotoxicity while controlling for as many other nanoproperties as possible using the HEL-30 mouse keratinocyte cell line as a model for dermal exposure. In the size-dependent studies, all the nanoparticles are 100% anatase, and aggregate sizes were determined in order to take into account the effect of agglomeration on size-dependent toxicity. In addition, varying crystal structures were assessed while the size of the nanoparticles was controlled. We were able to identify that both size and crystal structure contribute to cytotoxicity and that the mechanism of cell death varies based on crystal structure. The 100% anatase TiO2 nanoparticles, regardless of size, induced cell necrosis, while the rutile TiO2 nanoparticles initiated apoptosis through formation of reactive oxygen species (ROS).

Controlled Delivery of 5-Chlorouracil Using Poly(Ortho Esters) in Filtering Surgery for Glaucoma

To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

Toxicity studies of nonylphenol on rosy barb ( Puntius conchonious): A biochemical and histopathological evaluation

Acute and subacute toxicity of the nonylphenol (NP) on fish was investigated in laboratory toxicity tests with rosy barb ( Puntius conchonious). The acute toxicity of NP to rosy barb was determined in semi-static bioassays. Median lethal concentration (LC 50) for 96 h was 1.72 μM. The effects of sublethal concentrations of NP (0.17, 0.34 and 0.68 μM) on the structures and biochemical parameters [alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanin aminotransferase (ALT)] in gills, liver and kidney of rosy barb were studied after 14 days. The results showed that NP caused alteration of the structure in organs, as evidenced by the hyperplasia of epithelium and the fusion of secondary lamellae in the gills, the disappearance of the cell membrane and the cell necrosis in the liver as well as haemorrhages in the kidney. In addition, the functional enzyme activities were also changed. The increase trend in ALP activity in organs of fish treated with NP was recorded. The levels of AST and ALT in gills, liver and kidney were stimulated to rise at the lower concentration and fall at the higher concentration NP treatment compared to controls. This study suggests that NP can alter of the structures and biochemical parameters within non-endocrine tissue of fish and these changes may be mediated via destroying membrane structure and inducing cell necrosis.

390 POSTER Proliferation of human lung cancer in orthotopic transplantation model, comparing with in subcutaneous transplantation model of nude mice

The toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with four different ligands (MPA, NAC, GSH and dBSA) to HEK293 and HeLa cells were investigated using flow cytometry, spectroscopic and microscopic methods. The results showed that the cytotoxicity of QDs increased in a dose- and time-dependent manner. No appreciable fraction of cells with sub-G1 DNA content, the loss of membrane integrity, and the swelling of nuclei clearly indicated that CdTe QDs could lead to necrotic cell death in HEK293 cells. JC-1 staining and TEM images confirmed that QDs induced MPT, which resulted in mitochondrial swelling, collapse of the membrane potential. MPT is an important step in QDs-induced necrosis. Moreover, QDs induced MPT through the elevation of ROS. The fluorimetric assay and theoretical analysis demonstrated ROS production has been associated with the internalization of QDs with cells. Due to large surface/volume ratios of QDs, when QDs added in the culture medium, serum proteins in the culture medium will be adsorbed on the surface of QDs. This adsorption of serum protein will change the surface properties and size, and then mediate the cellular uptake of QDs via the clathrin-mediated endocytic pathway. After entering into cells, the translocation of QDs in cells is usually via endosomal or lysosomal vesicles. The rapid degradation of QDs in lysosome and the lysosomal destabilization induce cell necrosis. This study provides a basis for understanding the cytotoxicity mechanism of CdTe QDs, and valuable information for safe use of QDs in the future.