Selenomethionine protects hematopoietic stem/progenitor cells against cobalt nanoparticles by stimulating antioxidant actions and DNA repair functions.

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) can differentiate into all blood lineages to maintain hematopoiesis, wound healing, and immune functions. Recently, cobalt-chromium alloy casting implants have been used extensively in total hip replacements; however, cobalt nanoparticles (CoNPs) released from the alloy were toxic to HSCs and HPCs. We aimed to investigate the mechanism underlying the toxic effect of CoNPs on HSCs/HPCs and to determine the protective effect of selenomethionine (SeMet) against CoNPs in vitro and in vivo. Human and rat CD34+ HSCs/HPCs were isolated from cord blood and bone marrow, respectively. CoNPs decreased the viability of CD34+ HSCs/HPCs and increased apoptosis. SeMet attenuated the toxicity of CoNPs by enhancing the antioxidant ability of cells. The protective effect of SeMet was not completely abolished after adding H2O2 to abrogate the improvement of the antioxidant capacity by SeMet. SeMet and CoNPs stimulated ATM/ATR DNA damage response signals and inhibited cell proliferation. Unlike CoNPs, SeMet did not damage the DNA, and cell proliferation recovered after removing SeMet. SeMet inhibited the CoNP-induced upregulation of hypoxia inducible factor (HIF)-1α, thereby disrupting the inhibitory effect of HIF-1α on breast cancer type 1 susceptibility protein (BRCA1). Moreover, SeMet promoted BRCA1-mediated ubiquitination of cyclin B by upregulating UBE2K. Thus, SeMet enhanced cell cycle arrest and DNA repair post-CoNP exposure. Overall, SeMet protected CD34+ HSCs/HPCs against CoNPs by stimulating antioxidant activity and DNA repair.

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments.

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 µg/mL; B16: IC50 = 4.6 ± 1.8 µg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 µg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Synthesis and Evaluation of Novel Pyrazole Ethandiamide Compounds as Inhibitors of Human THP-1 Monocytic Cell Neurotoxicity.

Neuroinflammation and microglia-mediated neurotoxicity contribute to the pathogenesis of a broad range of neurodegenerative diseases; therefore, identifying novel compounds that can suppress adverse activation of glia is an important goal. We have previously identified a class of trisubstituted pyrazoles that possess neuroprotective and anti-inflammatory properties. Here, we describe a second generation of pyrazole analogs that were designed to improve their neuroprotective activity toward neurons under inflammatory conditions. Pyrazolyl oxalamide derivatives were designed to explore the effects of steric and electronic factors. Three in vitro assays were performed to evaluate the compounds' anti-neurotoxic, neuroprotective, and cytotoxic activity using human THP-1, PC-3, and SH-SY5Y cells. Five compounds significantly reduced the neurotoxic secretions from immune-stimulated microglia-like human THP-1 monocytic cells. One of these compounds was also found to protect SH-SY5Y neuronal cells when they were exposed to cytotoxic THP-1 cell supernatants. While one of the analogs was discarded due to its interference with the cell viability assay, most compounds were innocuous to the cultured cells at the concentrations used (1-100 µM). The new compounds reported herein provide a design template for the future development of lead candidates as novel inhibitors of neuroinflammation and neuroprotective drugs.

Enhanced ability of plasma-activated lactated Ringer's solution to induce A549 cell injury.

Non-thermal plasma (NTP) is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma-activated medium (PAM), which is prepared by the irradiation of culture medium with NTP, induces cell death in cancer cells. However, difficulties are associated with applying PAM to the clinical phase because culture media cannot be used for medical treatments. The objectives of the present study were to demonstrate the inhibitory effects of plasma-activated lactated Ringer's solution (PAL) on the viability of the A549 cancer cell line and elucidate the underlying mechanisms. The anti-tumor activity of PAL was significantly stronger than that of PAM, whereas their concentrations of H2O2 and nitrite were similar. Lactated Ringer's solution (Lac-R) consists of lactate and three types of inorganic salts. The results showing that NTP irradiation of the lactate solution rather than the inorganic salt solution induced the inactivation of catalase were dependent on the presence or absence of nitrite in these solutions. We detected nitrotyrosine in A549 cells treated with PAM or PAL, and the addition of catalase to PAM rather than to PAL reduced its production. The PAL treatment of A549 cells led to mitochondrial dysfunction with the down-regulation of NF-κB-Bcl2 signaling.

Artefacts with ascorbate and other redox-active compounds in cell culture: epigenetic modifications, and cell killing due to hydrogen peroxide generation in cell culture media.

This letter discusses the various artefacts that can arise when ascorbate or other redox-active compounds are added to cell culture media.

In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid.

Inflammation and oxidative stress play main roles in neurodegeneration. Interestingly, different natural compounds may be able to exert neuroprotective actions against inflammation and oxidative stress, protecting from neuronal cell loss. Among these natural sources, Cannabis sativa represents a reservoir of compounds exerting beneficial properties, including cannabigerol (CBG), whose antioxidant properties have already been demonstrated in macrophages. Here, we aimed to evaluate the ability of CBG to protect NSC-34 motor neurons against the toxicity induced from the medium of LPS-stimulated RAW 264.7 macrophages. Using MTT assay, we observed that CBG pre-treatment was able to reduce the loss of cell viability induced by the medium of LPS-stimulated macrophages in NSC-34 cells. Indeed, CBG pre-treatment inhibited apoptosis, as shown by the reduction of caspase 3 activation and Bax expression, while Bcl-2 levels increased. Furthermore, CBG pre-treatment counteracted not only inflammation, as demonstrated by the reduction of IL-1ß, TNF-α, IFN-γ and PPARγ protein levels assessed by immunocytochemistry, but also oxidative stress in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7. Indeed, immunocytochemistry showed that CBG pre-treatment reduced nitrotyrosine, SOD1 and iNOS protein levels and restored Nrf-2 levels. All together, these results indicated the neuroprotective effects of CBG, that may be a potential treatment against neuroinflammation and oxidative stress.

Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.

Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning.

Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet ß-cells: reversal of such metabolic defects by metformin.

Emerging evidence suggests that long-term exposure of insulin-secreting pancreatic ß-cells to hyperglycemic (HG; glucotoxic) conditions promotes oxidative stress, which, in turn, leads to stress kinase activation, mitochondrial dysfunction, loss of nuclear structure and integrity and cell apoptosis. Original observations from our laboratory have proposed that Rac1 plays a key regulatory role in the generation of oxidative stress and downstream signaling events culminating in the onset of dysfunction of pancreatic ß-cells under the duress of metabolic stress. However, precise molecular and cellular mechanisms underlying the metabolic roles of hyperactive Rac1 remain less understood. Using pharmacological and molecular biological approaches, we now report mistargetting of biologically-active Rac1 [GTP-bound conformation] to the nuclear compartment in clonal INS-1 cells, normal rat islets and human islets under HG conditions. Our findings also suggest that such a signaling step is independent of post-translational prenylation of Rac1. Evidence is also presented to highlight novel roles for sustained activation of Rac1 in HG-induced expression of Cluster of Differentiation 36 [CD36], a fatty acid transporter protein, which is implicated in cell apoptosis. Finally, our findings suggest that metformin, a biguanide anti-diabetic drug, at a clinically relevant concentration, prevents ß-cell defects [Rac1 activation, nuclear association, CD36 expression, stress kinase and caspase-3 activation, and loss in metabolic viability] under the duress of glucotoxicity. Potential implications of these findings in the context of novel and direct regulation of islet ß-cell function by metformin are discussed.

Genotoxic Effects of Culture Media on Human Pluripotent Stem Cells.

Culture conditions play an important role in regulating the genomic integrity of Human Pluripotent Stem Cells (HPSCs). We report that HPSCs cultured in Essential 8 (E8) and mTeSR, two widely used media for feeder-free culturing of HPSCs, had many fold higher levels of ROS and higher mitochondrial potential than cells cultured in Knockout Serum Replacement containing media (KSR). HPSCs also exhibited increased levels of 8-hydroxyguanosine, phospho-histone-H2a.X and p53, as well as increased sensitivity to γ-irradiation in these two media. HPSCs in E8 and mTeSR had increased incidence of changes in their DNA sequence, indicating genotoxic stress, in addition to changes in nucleolar morphology and number. Addition of antioxidants to E8 and mTeSR provided only partial rescue. Our results suggest that it is essential to determine cellular ROS levels in addition to currently used criteria i.e. pluripotency markers, differentiation into all three germ layers and normal karyotype through multiple passages, in designing culture media.

Glucose-Induced Oxidative Stress Reduces Proliferation in Embryonic Stem Cells via FOXO3A/ß-Catenin-Dependent Transcription of p21(cip1).

Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted ß-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.

Functional impairment of endothelial cells by the antimycotic amphotericin B.

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.

Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas.

BACKGROUND/AIMS: In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model. METHODS: Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3. RESULTS: In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells. CONCLUSIONS: Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.

Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines.

Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R (2) between 0.4519 and 0.6171; p<0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.

Excitotoxicity triggered by Neurobasal culture medium.

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12-15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca²âº influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.

Atividade antimicrobiana de produtos naturais frente a bactérias formadoras do biofilme dentário / Antimicrobial activity of herbal products against dental biofilm forming bacteria

Objeti vo: Verifi car, in vitro, o efeito anti microbiano do pólen e dos extratos alcoólico e aquoso da própolis em suas formas pura e diluídas sobre cepas de referência Streptococcus mutans ATCC25175, Streptococcus salivarius ATCC 7073, Streptococcus mitis ATCC 903 e Lactobacillus casei ATCC 9595 pela determinação da Diluição Inibitória Máxima (DIM). Método: Uti lizou-se a clorexidina como controle positivo e água desti lada e álcool de cereais 70% como controles negativos. Efetuou-se a diluição das soluções de 1:1 até 1:64dos extratos alcoólico e aquoso da própolis diluídos em álcool 70% e água desti lada, respecti vamente. O pólen foi diluído em álcool, por ser uma substancia apolar, nas concentrações de 5% (quanti dade presente na composição química da própolis) e 50%. Cada linhagem bacteriana foi reati vada em caldo nutritivo Brain Heart Infusion (BHI) e semeadas as placas com auxílio de swabs, procedendo-se com testes de susceti bilidade, em duplicata, por meio do método da difusão em ágar e técnicado ágar recortado. Em seguida, foram incubadas a 37°C, em microaerofilia, por 48h. Resultados: Constatou-se que todas as diluições da própolis alcoólica inibiram o crescimento bacteriano enquanto a própolis aquosa mostrou os menores resultados tendo efeitoapenas sobre S. miti s na forma pura e nas diluições de 1:1 até 1:4. O pólen a 5% foi efi ciente sobre todas as bactérias, porém o pólen a 50% teve ação apenas sobre S. mitis. Os controlesnegati vos não apresentaram ati vidade. Conclusão: Apesar da própolis e do pólen apresentarem atividade anti microbiana contra as cepas de referência superiorà do placebo, esta, porém, foi inferior à da clorexidina.

Objective: To evaluate, in vitro, the antimicrobial effect of pollen and alcoholic and aqueous propolis extracts in their pure and diluted forms against reference strains Streptococcus mutans ATCC 25175, Streptococcus salivarius ATCC 7073, Streptococcus mitis ATCC 903 and Lactobacillus casei ATCC 9595, by determination of Maximum Inhibitory Dilution (MID). Methods: Chlorhexidine was used as a positive control and distilled water and 70% grain alcohol as negative controls. The alcoholic and aqueous propolis extracts were subjected to dilutions from 1:1 to 1:64 in 70% alcohol and distilled water, respectively. For being an apolar substance, pollen was diluted in alcohol at the concentrations of 5% (amount present in the chemical composition of propolis) and 50%. Each bacterial strain was reactivated in Brain Heart Infusion (BHI) broth and seeded onto plates with swabs, and the susceptibility tests were performed in duplicate by the agar diffusion method using the agar well technique. Next, the plates were incubated at 37øC in microaerophilia during 48 hours. Results: All dilutions of alcoholic propolis extract inhibited the bacterial growth while the aqueous propolis extract showed less efficient results, being effective only against S. mitis in its pure form and in the 1:1 to 1:4 dilutions. Pollen at 5% was efficient against all bacteria, but pollen at 5% had action only against S. mitis. The negative controls did not present antimicrobial activity. Conclusion: The antimicrobial activity of propolis and pollen against the reference strains was higher than that of placebo but lower than that of chlorhexidine.

Vitrification of human ovarian tissue: effect of different solutions and procedures.

OBJECTIVE: To test the effect of different vitrification solutions and procedures on the morphology of human preantral follicles. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from nine women aged 22-35 years. INTERVENTION(S): Ovarian tissue fragments were subjected to [1] different vitrification solutions to test their toxicity or [2] different vitrification methods using plastic straws, medium droplets, or solid-surface vitrification before in vitro culture. MAIN OUTCOME MEASURE(S): Number of morphologically normal follicles after toxicity testing or vitrification with the different treatments determined by histologic analysis. RESULT(S): In the toxicity tests, only VS3 showed similar results to fresh tissue before and after in vitro culture (fresh controls 1 and 2). In addition, this was the only solution able to completely vitrify. In all vitrification procedures, the percentage of normal follicles was lower than in controls. However, of the three protocols, the droplet method yielded a significantly higher proportion of normal follicles. CONCLUSION(S): Our experiments showed VS3 to have no deleterious effect on follicular morphology and to be able to completely vitrify, although vitrification procedures were found to affect human follicles. Nevertheless, the droplet method resulted in a higher percentage of morphologically normal follicles.

Washing mineral oil reduces contaminants and embryotoxicity.

OBJECTIVE: To determine if washing improves the quality of mineral oil used for embryo culture. DESIGN: A 2 × 3 factorial experimental study. SETTING: University hospital-based infertility center. ANIMAL(S): Mice. INTERVENTION(S): The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined. MAIN OUTCOME MEASURE(S): The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media. RESULT(S): Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%. CONCLUSION(S): Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil.

l-Glutamate in Middlebrook 7H9 culture medium upregulates matrix metalloproteinase-2 secretion from human astrocytoma cells.

Matrix metalloproteinases (MMPs) are implicated in the pathology of CNS tuberculosis. Whilst investigating the secretion of MMP-2 from human U373-MG astrocytoma cells, we observed elevated MMP-2 secretion in response to Middlebrook 7H9 media but not to Mycobacterium tuberculosis itself. Middlebrook 7H9 media did not stimulate MMP-1 or MMP-9 secretion from astrocytoma cells. The excitatory neurotransmitter l-glutamate, at concentrations found in Middlebrook 7H9 media, induced significant astrocytoma MMP-2 secretion (p<0.05). l-Glutamate-induced MMP-2 activity may contribute to neuropathology in various CNS diseases and may generate misleading data in pathogen studies where Middlebrook 7H9 is the culture medium.

Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells.

The ability of two types of single walled carbon nanotubes (SWCNT), namely Arc Discharge (AD) and HiPco single walled carbon nanotubes, to induce an indirect cytotoxicity in A549 lung cells by means of medium depletion was investigated. The nanotubes were dispersed in a commercial cell culture medium and subsequently removed by centrifugation and filtration. Spectroscopic analysis confirmed the removal of the nanotubes and showed differing degrees of alteration of the composition of the medium upon the removal of the nanotubes. The ability to induce an indirect cytotoxic effect by altering the medium was evaluated using two endpoints, namely the Alamar Blue (AB) and the Clonogenic assay. Exposure of the A549 cells to the depleted medium which had previously contained carbonaceous nanoparticles, revealed significant cytotoxicity for both endpoints employed. The results presented demonstrate that single walled carbon nanotubes can induce an indirect cytotoxicity by alteration of cell culture medium (in which they have previously been dispersed) which potentially results in a false positive toxic effect being observed in cytotoxicity studies.

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line.

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA-type-MnSOD. This LSA-type-SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA-type-SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA-type-SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L-rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA-type-SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb-C-FR(III) mice. Animals subcutaneously injected with L-rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA-type-SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.