apoptosis

/Tag:apoptosis

Apoptosis Versus Cytotoxicity in HeLa Cells Exposed to Paracetamol

Katarína Ruppová, Miroslava Urbancíková, Ladislava Wsólová and
Darina Slamenová

Apoptosis is a programmed form of cell death which occurs in response to specific stimuli. It is distinguished from necrotic or accidental cell death by unique events, including the degradation of chromatin and a loss of cellular volume. In contrast to necrotic cell death, cell membrane integrity and mitochondrial function are thought to be maintained until the apoptotic process is well advanced. One of the novel assays for detecting apoptosis is flow cytometry. In our experiments, we used a flow cytometric assay to detect DNA changes in a human cell line (HeLa) exposed to paracetamol, by measuring propidium iodide binding. We were able to detect the apoptotic process in cells exposed to paracetamol. Apoptosis did not correlate with cytotoxicity, and was only found in samples exposed to 4–5mg/ml paracetamol for 8 hours in minimum essential medium and incubated in fresh medium without paracetamol for 14–19 hours. The greatest effect was noted 18 hours after paracetamol exposure. These results were confirmed by studying cell morphology and chromatin condensation by fluorescent microscopy with the fluorochromes acridine orange and ethidium bromide. Our results support the hypothesis that, in cultured cells, apoptosis is induced by a relatively narrow range of chemical concentrations which are known to inhibit the cell cycle, and that apoptosis and inhibition of cell proliferation coincide to some degree.
You need to register (for free) to download this article. Please log in/register here.

Cellular Effects of Electromagnetic Fields

Jonne Naarala, Anne Höytö and Ari Markkanen

Studies at the cellular level are needed to reveal the cellular and molecular biological mechanisms underlying the biological effects and possible health implications of non-ionising radiation, such as extremely low frequency (ELF) magnetic fields (MFs) and radiofrequency (RF) fields. Our research group has studied the effects of 50Hz ELF MFs (caused by power lines and electric devices) and 872MHz or 900MHz RFs (emitted by mobile phones and their base stations) on cellular ornithine decarboxylase activity, cell cycle kinetics, cell proliferation, and necrotic or apoptotic cell death. For RFs, pulse-modulated (217Hz modulation frequency corresponding a global system for mobile communication-type signal) or continuous wave (unmodulated) signals were used. To expose
the cell cultures to MFs or RFs, specially developed exposure systems were used, where levels of electromagnetic field exposure and the conditions of cell culture could be precisely controlled. A coexposure approach was used in many studies, i.e. the cell cultures were exposed to other stressors in addition to MFs or RFs. Ultraviolet radiation, serum deprivation, or fresh medium addition, were used as co-exposures. The results presented in this short review show that the effects of mere MFs or RF on cell culture models are quite minor, but that various co-exposure approaches warrant additional study.
You need to register (for free) to download this article. Please log in/register here.

Modelling of Normal and Premalignant Oral Tissue by using the Immortalised Cell Line, SVpgC2a: A Review of the Value of the Model

Claudia A. Staab, Martin Vondracek, Hipolito Custodio, Katarina Johansson, Jan Anders Nilsson, Peter Morgan, Jan-Olov Höög, Ian Cotgreave and Roland C. Grafström

Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, nontumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in longterm cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols — for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium
You need to register (for free) to download this article. Please log in/register here.

Cytotoxic and Apoptotic Effects on Hepatocytes of Secondary Metabolites Obtained from Lichens

Estela Raquel Correché, Ricardo Daniel Enriz, Marisa Piovano, Juan Garbarino and María José Gómez-Lechón

There are a large number of species of Antarctic lichens, and several studies describing the secondary metabolites present in these lichens, as well as the advances in understanding the chemistry of these metabolites, have been reported. In addition, some derivatives displaying interesting antibacterial effects have been described. The cytotoxic and apoptotic effects of 15 secondary metabolites (depsides, depsidones and usnic acid) obtained from Continental (Chilean) and Antarctic lichens were evaluated in primary cultures of rat hepatocytes. Intracellular lactate dehydrogenase release, caspase 3 activation and DNA fragmentation were measured. In this study, we have evaluated a set of markers associated with pivotal steps in the execution phase of apoptosis, in order to detect compounds with apoptotic effects on hepatocytes before significant necrosis takes place. Flow cytometric analysis of DNA fragmentation revealed an increase in apoptotic nuclei with sub-diploid DNA content after the exposure of hepatocytes to sub-cytotoxic concentrations of the compounds. Among these, salazinic acid, stictic acid and psoromic acid displayed significant apoptotic activities. Divaricatic acid showed only moderate apoptotic effects at sub-cytotoxic concentrations.
You need to register (for free) to download this article. Please log in/register here.

Psi-Screen, an In Vitro Toxicity Test System: Applications in the Bioassay of Perfumes and Fragrance Chemicals

David E. Griffiths

The effects of 65 perfume formulations (perfume oils, perfumes, eau de parfum, eau de toilette) on mitochondrial membrane potential (ΔΨm) and mitochondrial respiration have been investigated using a mitochondria-based assay for ΔΨm, termed Psi-Screen. All the perfume formulations tested are highly active in the Psi-Screen assay, and the major site of inhibition in all cases is NADH-ubiquinone reductase (Complex I). This is confirmed in studies on the inhibition of NADH oxidase and NADH-ubiquinone reductase. Some formulations also inhibit succinate oxidation at either Complex II or Complex III. Evidence for the inhibition of mitochondrial ATPase is presented, as well as for the induction of reactive oxygen species production by perfume inhibition of Complex I. Thus, perfume formulations are multiple inhibitor mixtures which inhibit multiple bioenergetic functions at high dilutions (103 to 7 × 104). The implications of these findings are discussed with respect to cell toxicity via necrosis and/or apoptosis. Twenty candidate fragrance chemicals were investigated and all inhibited Complex I (5 at < 35μM). Mass screening strategies and high-throughput screening assays are discussed.
You need to register (for free) to download this article. Please log in/register here.

Cyclosporin A Potentiates the Cytotoxic Effects of Methyl Methanesulphonate in HL-60 and K562 Cells

Petr Mlejnek, Ivo Frydrych and Petr Dolez∨el

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hall marks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P–glycoprotein.
You need to register (for free) to download this article. Please log in/register here.

Insulin and IGF-1 Mediated Inhibition of Apoptosis in CHO Cells Grown in Suspension in a Protein-free Medium

Lars Adamson and Erik Walum

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl- 2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death — at 1.0μg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.
You need to register (for free) to download this article. Please log in/register here.