Reduction In Cellular Viability Research Articles

Reduction of paraquat-induced renal cytotoxicity by manganese and copper complexes of EGTA and EHPG

Superoxide anion generation plays an important role in the development of paraquat toxicity. Although superoxide dismutase mimetics (SODm) have provided protection against organ injury involving generation of superoxide anions, they often suffer problems, e.g., regarding their bioavailability or potential pro-oxidant activity. The aim here was to investigate and compare the therapeutic potential of two novel SODm, manganese(II) and copper(II) complexes of the calcium chelator ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and of the contrast agent ethylenebis(hydroxyphenylglycine) (EHPG), against paraquat-induced renal toxicity in vitro. Incubation of renal NRK-52E cells with paraquat (1 mM) for 24 h produced submaximal, yet significant, reduction in cellular viability and cell death and produced significant increases in superoxide anion and hydroxyl radical generation. Manganese and copper complexes of EGTA (10–100 μM) and EHPG (30–100 μM) reduced paraquat-induced renal cell toxicity and reduced superoxide anion and hydroxyl radical generation significantly. Manganese complexes displayed greater efficacy than copper complexes and, at equivalent concentrations, manganese complexed with EHPG provided the greatest protection. Furthermore, these metal complexes did not interfere with the uptake of [ methyl- 14C]paraquat into NRK-52E cells, suggesting that they provided protection against paraquat cytotoxicity via intracellular mechanisms. These complexes did not display cytotoxicity at the concentrations examined. Together, these results suggest that manganese and copper complexes of EGTA and EHPG, and especially the manganese–EHPG complex, could provide benefit against paraquat nephrotoxicity.

Characterization of chlorpyrifos-induced apoptosis in placental cells

The mechanism by which chlorpyrifos exerts its toxicity in fetal and perinatal animals has yet to be elucidated. Since the placenta is responsible for transport of nutrients and is a major supplier hormone to the fetus, exposure to xenobiotics that alter the function or viability of placenta cells could ostensibly alter the development of the fetus. In this study, JAR cells were used to determine if CPF and the metabolites 3,5,6-trichloro-2-pyridinol (TCP) and chlorpyrifos-oxon (CPO) are toxic to the placenta. Our results indicate that chlorpyrifos (CPF), and its metabolite chlorpyrifos-oxon (CPO) caused a dose-dependent reduction in cellular viability with CPF being more toxic than its metabolites. Chlorpyrifos-induced toxicity was characterized by the loss of mitochondrial potential, the appearance of nuclear condensation and fragmentation, down-regulation of Bcl-2 as well as up-regulation of TNFα and FAS mRNA. Pharmacological inhibition of FAS, nicotinic and TNF-α receptors did not attenuate CPF-induced toxicity. Atropine exhibited minimal ability to reverse toxicity. Furthermore, signal transduction inhibitors PD98059, SP600125, LY294002 and U0126 failed to attenuate toxicity; however, SB202190 (inhibitor of p38α and p38ß MAPK) sensitized cells to CPF-induced toxicity. Pan-caspase inhibitor Q-VD-OPh produced a slight but significant reversal of CPF-induced toxicity indicating that the major caspase pathways are not integral to CPF-induced toxicity. Taken collectively, these results suggest that chlorpyrifos induces apoptosis in placental cells through pathways not dependent on FAS/TNF signaling, activation of caspases or inhibition of cholinesterase. In addition, our data further indicates that activation of p38 MAPK is integral to the protection cells against CPF-induced injury.

Potential pathogenic role of β-amyloid1–42–aluminum complex in Alzheimer's disease

The etiopathogenesis of Alzheimer's disease is far from being clearly understood. However, the involvement of metal ions as a potential key factor towards conformational modifications and aggregation of amyloid is widely recognized. The aim of the present study is to shed some light on the relationship between metal ions, amyloid conformation/aggregation, and their potential relationship with the conformational aspects of AD. We compare the effects of β-amyloid1–42 and its various metal complexes (β-amyloid–Al, β-amyloid–Zn, β-amyloid–Cu, β-amyloid–Fe) in human neuroblastoma cells in terms of cell viability, membrane structure properties, and cell morphology. No significant toxic effects were observed in neuroblastoma cells after 24h treatment both with β-amyloid and β-amyloid–metals (β-amyloid–Zn, β-amyloid–Cu, β-amyloid–Fe); on the other hand, there was a marked reduction of cellular viability after treatment with β-amyloid–Al complex. In addition, treatment with β-amyloid–Al increased membrane fluidity much more than other β-amyloid–metal complexes, whose contribution was negligible. Furthermore, the cellular morphology, as observed by electron microscopy, was deeply altered by β-amyloid–Al. Importantly, β-amyloid–Al toxicity is closely and significantly associated with a great difference in the structure/aggregation of this complex with respect to that of β-amyloid alone and other β-amyloid–metal complexes. In addition, β-amyloid, as a consequence of Al binding, becomes strongly hydrophobic in character. These findings show a significant involvement of Al, compared to the other metal ions used in our experiments, in promoting a specific amyloid1–42 aggregation, which is able to produce marked toxic effects on neuroblastoma cells, as clearly demonstrated for the first time in this study.

Viabilidade celular de Saccharomyces cerevisiae cultivada em associação com bactérias contaminantes da fermentação alcoólica

The aim of this project was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products to decrease the cellular viability of the yeast Saccharomyces cerevisiae. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast S. cerevisiae (strain Y-904) for 72 hours at 32 oC under agitation. The cellular viability, budding rate and population of S. Cerevisiae and the total acidity, volatile acidity and pH of culture medium were determined at 0, 24, 48 and 72 hours of incubation of the mixed culture. The bacteria cultures were treated by heat sterilization, antibacterial agent and irradiation. The results showed that only the more acidified culture medium, contaminated with active bacteria L. fermentum and B. subtilis, caused a reduction in the yeast cellular viability. Except for the bacteria B. subtilis treated for radiation, the other bacteria treated by the different procedures (heat, radiation and antibacterial) did not cause a reduction in the cellular viability of S. cerevisiae, indicating that the isolated presence of the cellular metabolic of these bacteria was not enough to reduce the percentage of the living yeast cells.

Effects of superoxide dismutase and polyclonal tumor necrosis factor-alpha antibodies on chloroacetate-induced cellular death and superoxide anion production by J774.A1 macrophages

Dichloroacetate (DCA) and trichloroacetate (TCA) are by-products that are formed during the process of water chlorination and have been previously shown to induce superoxide anion (SA) production and cellular death when added to J774.A1 macrophage cultures. In this study, the effects of superoxide dismutase (SOD) and polyclonal tumor necrosis factor-alpha (TNF-α) antibodies on DCA- and TCA-induced SA production and cellular death have been tested on the J774.A1 macrophage cultures. TCA and DCA were added to different cultures either alone, each at a concentration of 16 mM, or in combination with SOD (2–12 units/ml), or with TNF-α antibodies (10 and 25 units/ml). Cells were incubated for 48 h, after which cellular death/viability, lactate dehydrognase (LDH) leakage by the cells, and SA production by the cells were determined. While TCA and DCA caused significant cellular toxicity, indicated by reduction in cellular viability and increases in LDH leakage and SA production, SOD addition resulted in significant reduction of the effects induced by the compounds. On the other hand, addition of TNF-α antibodies to the DCA- and TCA-treated cultures resulted in significant reduction of DCA- but not TCA-induced cellular death and SA production by the cells. Although these results suggest a significant role for SA in DCA- and TCA-induced cellular death, they may also suggest two different mechanisms for the chloroacetate-induced SA production by the cells.

Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells.

To investigate the biological effects of indocyanine green (ICG) and acute illumination on human retinal pigment epithelial (RPE) cells. Three concentrations (0, 0.25, and 2.5 mg/mL) of ICG were applied to ARPE19 cells for 1 minute. After isotonic rinsing, the cells were irradiated with a light beam with a wavelength spectrum from 400 to 800 nm and an output of 1850 lumens for 15 minutes. The cells were collected at timed intervals for the investigation of cell death and expression of stress-response genes by reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis. After ICG incubation, photoreactive changes were observed in the RPE cells. A reduction in cellular viability and considerable shrinkage of the cells were observed. The expressions of the apoptosis-related genes p53 and bax and the cell cycle arrest protein p21 were upregulated in cells treated with both ICG and light. Of the early-response genes, the expression of c-fos was specifically enhanced by light, with additive effects from the presence of ICG. Such stimulatory effects on these gene expressions were greater at 2.5 mg/mL than at 0.25 mg/mL ICG. ICG in the presence of acute illumination can elicit cell-cycle arrest and even apoptosis in RPE cells. The establishment of a safety level in the application of ICG in the region of 0.25 mg/mL is recommended.

Effects of a phytotherapeutic agent, PC-SPES, on prostate cancer: a preliminary investigation on human cell lines and patients.

To evaluate the in vitro activity of PC-SPES, a complex phytotherapeutic agent, against prostate cancer cell lines, and to assess its activity in suppressing serum prostate specific antigen (PSA) level in patients with prostate cancer. Four variant prostate cancer cell lines (LNCaP and an apoptosis-resistant derivative, LNCaP-bcl-2, PC3 and DU145) were exposed to three different concentrations of PC-SPES extract. Cell viability was measured at 3, 4 and 5 days of exposure using a colorimetric assay and was compared with control cultures receiving aliquots of the ethanolic extraction medium alone. Clinically, a prospective study was initiated in patients with prostate cancer who refused conventional therapy or who had failed previous cryosurgery, radiation therapy and/or hormonal therapy. The patients were treated with PC-SPES (three capsules of 320 mg/day). The serum PSA responses and side-effects were evaluated. All cultured prostate cancer cell lines showed a significant dose-dependent reduction in cellular viability (compared with control cultures) by exposure to 4 and 6 microL of PC-SPES extract/mL of culture medium (P<0.001). In contrast to the hormone-insensitive cell lines tested (LNCaP-bcl-2, PC-3 and DU-145), only the hormone-sensitive cell line LNCaP was affected by the lowest dose of PC-SPES extract tested (2 microL/mL medium). In the prospective clinical trial of 33 patients, with a mean (range) follow-up of 6.8 (2-24) months after initiating PC-SPES therapy, serum PSA levels were lower in 87% at 2 months and in 78% at 6 months (n=18, P=0.026). The side-effects in these patients were nipple tenderness in two (6%) and leg clots requiring heparinization in two (6%). No gynaecomastia or hot flashes were observed in this group and the treatment was well tolerated. In this preliminary study, an extract of the phytotherapeutic agent PC-SPES was active in suppressing the growth of cultured hormone-sensitive and -insensitive prostate cancer cell lines. In the small clinical study, PC-SPES therapy decreased serum PSA levels in most patients. However, a longer follow-up and more patients will be required to evaluate the long-term efficacy of this new phytotherapy.

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O−2), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24–48 hours. Approximately twofold increases in the production of O−2 were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36–48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1–10 ng/mL after 24 and 36–48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1–10 ng/mL of ricin for 36–48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10–100, 10, and 1–10 ng/mL, after incubation for 24, 36, and 48 hours, respectively. © 1999 John Wiley & Sons, Inc. J Biochem Toxicol 13: 179–185, 1999

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells.

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O2-), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24-48 hours. Approximately twofold increases in the production of O2- were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36-48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1-10 ng/mL after 24 and 36-48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1-10 ng/mL of ricin for 36-48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10-100, 10, and 1-10 ng/mL, after incubation for 24, 36, and 48 hours, respectively.

Retinoic acid reduces staurosporine-induced apoptotic damage in chick embryonic neurons by suppressing reactive oxygen species production

The effect of all-trans retinoic acid (RA), which is known as a regulator of cell growth and differentiation, was studied during neuronal apoptosis. Apoptosis was induced in primary cultures of chick embryonic neurons by treatment with staurosporine (200 nM) for 24 h which led to a reduction of cellular viability to 40% compared to 83% in untreated cultures as well as to an increase in the number of apoptotic neurons (determined by nuclear staining with Hoechst 33258) to 60% compared to 15% in untreated cultures. RA (1 nM–10 μM) reduced the number of non-viable and apoptotic cells in a concentration-dependent manner and the maximal response was seen at 1 μM RA with 60% cellular viability and 38% apoptotic neurons. The production of mitochondrial reactive oxygen species (ROS, determined by the fluorescent indicator dihydrorhodamine) was elevated 4.4-fold after 4 h of staurosporine-treatment which was reduced to a 2-fold increase in the presence of 10 μM RA. The results indicate that RA was able to reduce apoptotic damage in staurosporine-treated chick embryonic neurons by suppressing the production of ROS.

Induction of lethal mutations in experimental tumours after single and fractionated irradiations in vivo

Purpose: To investigate the prolonged reduction in cellular viability (lethal mutations) of surviving cells following irradiation of tumours in vivo and to test the effects of fractionation on the expression of lethal mutations. Materials and methods: A mouse mammary carcinoma (CaNT) was treated with single dose or fractionated X-ray treatments in vivo and survival quantified with an in vitro excision assay soon after irradiation and at various times up to 35 days after in vitro propagation of the surviving cells. Results: A dose-dependent reduction in the plating efficiency was observed in cells isolated from irradiated tumours up to 35 days and many cell generations after irradiation. Considerable heterogeneity in plating efficiency was observed in clonal cell lines isolated from individual colonies grown from irradiated tumours. Delayed expression of lethal damage was observed after fractionated irradiation, although recovery of cellular fitness was greater than after irradiation with single doses (reported previously) suggesting that this form of damage is affected by inter-fraction repair. At equi-toxic doses, delayed expression of lethal damage was similar after three compared with two fractions of radiation per day (reported previously). Conclusions: These effects indicate that conventional excision assays of tumour cell viability under-estimate the total lethal damage caused by irradiation and have implications for modelling of the response of tumours to radiotherapy. The effect of fractionation on expression of this type of damage implies the involvement of repair processes. Therefore the repair proficiency may affect the balance between the immediate and delayed reduction of viability in irradiated cells.

The effect of basic fibroblast growth factor on glutamate-injured neuroarchitecture and arachidonic acid release in adult hippocampal neurons

During development in culture, basic fibroblast growth factor (bFGF) protected immature primary hippocampal neurons against glutamate-induced neurotoxicity. We investigated the effects of bFGF on mature, differentiated rat hippocampal neurons cultured for 10–12 days after an 8-min exposure to 500 μM glutamate. Seven days post-injury, hippocampal cells demonstrated severe reductions in cellular viability and axonal and dendritic outgrowth, which were accompanied by a marked increase in [ 3H]arachidonic acid (ARA) release from prelabelled neurons. bFGF applied post-injury attenuated cell death and cytoarchitectural destruction at all concentrations used (500 pg/ml, 1, 10, 20 ng/ml). However, neurite elongation and branching processes were only significantly protected by 10 ng/ml bFGF. [ 3H]ARA release decreased in a dose-related fashion within a concentration range of 1–10 ng/ml bFGF. 20 ng/ml bFGF was not superior to 10 ng/ml bFGF. Therefore, bFGF's neurotropic actions appear to be concentration-dependent. Our data suggest that bFGF applied post-injury may have a neuroprotective potential for mature, differentiated, completely polarized hippocampal neurons.

The Anoxic Fibroblast Response Is an Early-Stage Wound Healing Program

When fibroblasts experience anoxia, a cellular program is initiated which results in stepwise activation of an endogenous SVL30 retroelement, metabolic adaptation to reliance on glycolysis, protease secretion, and endonuclease induction. This response is reversible and occurs with no reduction in cellular viability. Examination of experimental wounds reveals that this same response is physiologically activated during the early phase of wound healing when near-anoxic conditions prevail and wound debridement predominates. Procathepsin D has been identified as one of three major anoxia-inducible secretory proteins; its secretion is the result of altered routing within the cell, with no apparent change in the amount of the protein synthesized. A delayed cellular contractile response occurs approximately 1 week after fibroblasts experience as little as 16 hr of anoxia; this response appears to contribute to wound contraction.

Nitric oxide synthase induction and intestinal epithelial cell viability in rats.

The present study determined the presence of constitutive and inducible nitric oxide (NO) synthase activities in intestinal isolated epithelial cells and the effects of NO induction on intestinal epithelial cell viability. Epithelial cells were isolated from rat proximal small intestine by dispersion using citrate and EDTA. Constitutive NO synthase activity, determined by [14C]arginine conversion to citrulline that was inhibited by in vitro incubation with the arginine analogue NG-monomethyl-L-arginine (L-NMMA; 300 microM) or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 1 mM), was observed in these cells. Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg iv) significantly augmented NO synthase activity (determined 4 h later), which was inhibited in vitro by incubation with L-NMMA but not by EGTA. The highest level of constitutive and inducible NO synthase activity occurred in intestinal villus cells, and the lowest was in crypt cells. Induction of NO synthase activity was associated with a decrease in cellular viability as assessed by a decrease in trypan blue exclusion. Dexamethasone pretreatment (1 mg/kg iv 2 h before LPS administration) significantly reduced both induction of NO synthase activity and the reduction in cellular viability. Likewise concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg sc) ameliorated the reduction in cell viability induced by LPS administration, an effect abolished by pretreatment with the NO substrate L-arginine (350 mg/kg sc). Whereas constitutively formed NO may have a physiological role in these cells, the results in this study suggest that induction of NO synthase in epithelial cells may represent a mechanism of local intestinal damage.

表在性膀胱腫瘍に対する膀胱内注入療法をモデルとした dye exclusion assay による制癌剤感受性試験

A chemosensitivity test was carried out on superficial bladder cancers using the trypan blue dye exclusion assay for the purpose of screening chemosensitive drugs for intravesical chemotherapy. Transplantable murine bladder tumor cells (MBT-2) were incubated, in vitro, in the presence of adriamycin (4, 40, 400, 1000 micrograms/ml) as well as verapamil (3, 30, 100, 500 micrograms/ml) at 5% CO2, 37 degrees C for two hours. After cellular viabilities were calculated, MBT-2 cells were inoculated into the hind limbs of mice. The cellular viability was correlated well with the ratio of tumor appearance, tumor growth inhibition and prolongation of survival, and was dose dependent in the adriamycin treated groups. On the other hand, a reduction of cellular viability, tumor growth inhibition and prolongation of survival were seen in the high dose verapamil (100, 500 micrograms/ml) treated groups. Human superficial bladder cancer cells were incubated in the presence of adriamycin, 4'-0-tetrahydropyranyladriamycin, mitomycin C and pepleomycin (1000 micrograms/ml) and/or verapamil (500 micrograms/ml). The reduction rates of cellular viability markedly varied with the kind of anticancer drugs. A reduction of cellular viability of human tumor cells as well as MBT-2 cells was seen in the verapamil treated groups. This rapid and handy assay seems to be useful for the purpose of screening chemosensitive drugs for intravesical chemotherapy.

Human PC‐3 prostate cell line DNA synthesis is suppressed by eicosatetraynoic acid, an in vitro inhibitor of arachidonic acid metabolism

When human PC-3 cells derived from a metastatic prostatic adenocarcinoma were incubated for 15 min to 4 h with the in vitro inhibitor of eicosanoid biosynthesis, eicosatetraynoic acid (ETYA) at 10-80 microM, DNA synthesis was suppressed. No reduction in cellular viability occurred, as judged by exclusion of trypan blue or unaltered release of 51Cr-labeled proteins, and the inhibition was partially reversible. Indomethacin (to 12.5 micrograms/ml) did not inhibit DNA synthesis or alter the suppression of DNA synthesis by ETYA, suggesting a role for a lipoxygenase product in this effect. Addition of leukotriene B4 (LTB4) at 10(-8) M did not reverse the inhibition of DNA synthesis produced by ETYA, nor did arachidonic acid (10(-5) - 10(-9) M) incubated with control cells mimic the effect of that agent. 3H-arachidonic acid incubated with PC-3 cells was rapidly incorporated into phospholipids and this labeling was differentially inhibited by ETYA. Positive modulation of PC-3 cellular DNA synthesis by lipoxygenase products and inhibition of their synthesis by ETYA is one attractive hypothesis with which to account for these results. Other consequences of producing a selective deficiency of arachidonic acid in cellular membrane phospholipids and even the probable substitution of ETYA for arachidonic acid could also contribute to the inhibition of DNA synthesis by ETYA.