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PubMedDate not supplied

Antibiotics directly induce apoptosis in B cell lymphoma cells derived from BALB/c mice.

BACKGROUND: The effects of bacterial eradication therapy cannot be fully explained simply by elimination of the target bacteria, if one considers the effects of eradication therapy in H. pylori-negative cases of low-grade malignancy MALTomas of the rectum. The present study was undertaken to examine the possibility and mechanism of direct induction of apoptosis of the tumor cells by the antibiotics used for bacterial eradication therapy. MATERIALS AND METHODS: A B cell lymphoma cell line (300-19) derived from BALB/c mice was co-cultured with amoxicillin or clarithromycin, and amoxicillin and clarithromycin at concentrations equal to or 1/10 x MIC of either drug. Cells co-cultured with 1/100 x MIC of the standard anti-tumor agents, adriamycin, vincristine and cyclophosphamide, served as positive controls. Cells cultured without any antibiotic or anti-tumor agent served as controls. In each group, the following analyses were performed: (i) the time-course of changes in the cellular morphology, (ii) the time-course of occurrence of DNA fragmentation, (iii) the appearance of apoptotic changes as evaluated by trypan blue staining, (iv) Bcl-2 expression as examined by immunoblotting; and (v) the expression of TNFR1, Fas, FasL and caspase-3, -8 and -9, as evaluated by immunoblotting. RESULTS: Cells treated with amoxicillin and clarithromycin showed the formation of apoptotic bodies, and degeneration and detachment of the cells in a dose-dependent manner. DNA fragmentation was induced in these cells to a degree similar to that seen in cells treated with the anti-tumor agents. Trypan blue staining also demonstrated apoptosis of the cells and loss of cell viability. Bcl-2 expression was seen only in the control group and FasL was never seen, while the expression of TNFR1, Fas and caspase-3, -8 and -9 was seen in the amoxicillin-treated group, clarithromycin-treated group, amoxicillin and clarithromycin-treated group and the positive control group. CONCLUSION: Antibiotics used for the eradication of H. pylori can also directly induce apoptosis in mouse B cell lymphoma cells, an action which involves the TNF system.

Amoxicillin↗
PubMed2001

Possible role of prostaglandin E2 in human amniotic epithelial cell death: an in vitro study.

OBJECTIVE AND DESIGN: To understand the optimal conditions necessary for successful transplantation of normal human amniotic epithelial (HAE) cells in vivo, the factors which influence the cell death were investigated. In this study, the role of prostaglandin E2 (PGE2) in HAE cells in vitro was examined. MATERIAL OR SUBJECTS: Normal HAE cells. TREATMENT: The HAE cells were incubated with PGE2 100 nM, 500 nM or 1,000 nM for 8 h. METHODS: The PGE2 treated and untreated cells were collected at 2 h, 4 h and 8 h intervals and stained using trypan blue and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) methods to detect necrosis and apoptosis respectively. RESULTS: Prostaglandin E2 induced more HAE cell death at 8 h treatment. The percentages of cell death induced by PGE2 100 nM, 500 nM and 1,000 nM at 8 h were 14.0 +/- 0.35 (n=9), 14.9 +/- 0.64 (n=9) and 18.3 +/- 0.65 (n=9) respectively compared to the control (7.0 +/- 0.35; n = 9) (P < 0.001) determined using trypan blue staining method. The apoptotic cell death induced by the above doses of PGE2 at 8 h were 5.7 +/- 0.6 (n=9), 7.14 +/- 0.84 (n=9) and 8.6 +/- 0.67 (n=9) respectively compared to the control (0.86 +/- 0.05; n=9) (P<0.001) determined using TUNEL method. The PGE2 induced high level cell death was via necrosis rather than apoptosis. CONCLUSIONS: These results indicate that PGE2 is an important mediator in HAE cell death.

Adult↗
PubMed1985

Effects of the lipidperoxidation product 4-hydroxynonenal and related aldehydes on proliferation and viability of cultured Ehrlich ascites tumor cells.

The mechanism by which the lipid peroxidation product 4-hydroxynonenal and several other homologous, yet non biogenic aldehydes inhibit proliferation of cultured Ehrlich ascites tumor cells has been studied. Incubation of cells (5 X 10(-4)/ml) in a minimum essential medium supplemented with 10 or 20 microM 4-hydroxynonenal reduces the 36-hr cell count to 65 and 30% of the control value. The reduced growth rate is most likely due to a blockage of the DNA synthesis. Cells labelled by a [3H]-thymidine pulse prior to exposure to 4-hydroxynonenal (20 microM, 8 hr) showed no change of the specific radioactivity of the DNA, indicating that no de novo synthesis occurred in the presence of the aldehyde. In the absence of the aldehyde the specific radioactivity of the DNA decreased by 25%. A 2-hr incubation in the presence of 10 or 20 microM of 4-hydroxynonenal reduced [3H]-thymidine incorporation into the HClO4 insoluble fraction to 85 and 50% of the controls, but had no effect of the [3H]-thymidine and 86Rb uptake. Moreover, examination of the cell cultures by the Trypan Blue exclusion technique revealed that 20 microM 4-hydroxynonenal does not cause cell death. The high reactivity of 4-hydroxynonenal towards sulfhydryl groups suggests that the aldehyde inhibits DNA synthesis by interacting with a functional SH group of DNA polymerase. The specific action on DNA synthesis is abolished at an aldehyde concentration of 50 microM, which leads to 30% (6 hr exposure) and 95% (36 hr exposure) of dead cells. The cytostatic index (CI), i.e. concentration at 50% Trypan Blue positive cells/concentration at 50% inhibition of cell growth deducted from the dose effect curves is 3.0 for 4-hydroxynonenal. The other homologous 4-hydroxyalkenals with chain length of 5, 6, 7, 8, 10 and 11 carbon atoms also inhibit cell growth. The CI varied from 1.20 to 1.94, indicating that these non biogenic 4-hydroxyalkenals have a distinctively lower specific effect on proliferation than the biogenic 4-hydroxynonenal. The Michael adducts of 4-hydroxynonenal with glutathione and cysteine were nearly one order of magnitude less toxic than the free aldehyde, the CI (2.41 cysteine adduct, 2.06 glutathione adduct), however, were not improved since the growth inhibitory action was also reduced.

Aldehydes↗
PubMed1995

Ontogenic expression of the erythroid-type glucose transporter (Glut 1) in the telencephalon of the mouse: correlation to the tightening of the blood-brain barrier.

Since Glut 1 was shown to be highly abundant in brain microvessels, its distribution during early developmental stages seems of importance in respect to the timing of blood-brain barrier (bbb) formation in the developing CNS. Here we have followed the temporal expression of the erythroid-type glucose transporter Glut 1 in the telencephalon of the embryonic and newborn mouse, beginning at the 9th intrauterine day. Glut 1 immunofluorescence staining was done on cryosections using a rabbit polyclonal antiserum to purified human erythrocyte glucose transporter. Endothelial cells resp. capillaries were detected by staining with a rhodamin-coupled Bandeiraea simplicifolia lectin (BSL). In parallel, the developmental tightening of the embryonic bbb was assessed by perfusion of mouse embryos with Trypan blue and horse radish-peroxidase. At E9, prior to the onset of intraneural neovascularization, strong Glut 1 immunoreactivity was found in the whole neuroectoderm but only minor staining was seen in the perineural domain. Glut 1 expression remained uniformly distributed in the intraneural tissue at E10, the beginning of intraneural neovascularization in the mouse. From E11 onwards, Glut 1 immunoreactivity was invisible in neuroepithelial cells, but appeared tightly associated with intraneural capillaries. Perfusion of E12 embryos using trypan blue solution and HRP revealed that most parts of the CNS and spinal cord were impermeable to the tracer substances at that stage. Thus, we suggest that the bbb is established very early in CNS development, probably in the course of intraneural neovascularization. In addition, our data indicate that the restriction of Glut 1 expression to the intraneural capillaries reflects the onset of bbb function in the mouse embryo.

Animals↗
PubMed2001

Systemic steroid pretreatment improves cerebral protection after circulatory arrest.

BACKGROUND: This study evaluates whether systemic steroid pretreatment enhances neuroprotection during deep hypothermic circulatory arrest (DHCA) compared with steroid in cardiopulmonary bypass (CPB) prime. METHODS: Four-week-old piglets randomly placed into two groups (n = 5 per group) were given methylprednisolone (30 mg/kg) into the pump prime (group PP), or pretreated intravenously 4 hours before CPB (group PT). All animals underwent 100 minutes of DHCA (15 degrees C), were weaned off CPB, and were sacrificed 6 hours later. Postoperative changes in body weight, bioimpedance, and colloid oncotic pressure (COP) were measured. Cerebral trypan blue content, immunohistochemical evaluation of transforming growth factor-beta1 (TGF-beta1) expression, and caspase-3 activity were performed. RESULTS: Percentage weight gain (group PP 25.0% +/- 10.4% versus group PT 12.5% +/- 4.0%; p = 0.036), and percentage decrease in bioimpedance (PP 37.2% +/- 14.5% versus PT 15.6% +/- 7.9%; p = 0.019) were significantly lower, whereas postoperative COP was significantly higher in group PT versus group PP (PT 15.3 +/- 1.8 mm Hg versus PP 11.6 +/- 0.8 mm Hg; p = 0.003). Cerebral trypan blue (ng/g dry tissue) was significantly lower in group PT (PT 5.6 x 10(-3) +/- 1.1 x 10(-3) versus PP 9.1 x 10(-3) +/- 5.7 x 10(-4); p = 0.001). Increased TGF-beta1 expression and decreased caspase-3 activity were shown in group PT. CONCLUSIONS: Systemic steroid pretreatment significantly reduced total body edema and cerebral vascular leak and was associated with better immunohistochemical indices of neuroprotection after DHCA.

Animals↗
PubMedDate not supplied

Kinetics of apoptosis and secondary necrosis in cultured rat thymocytes and S.49 mouse lymphoma and CEM human leukemia cells.

Cell culture systems are widely used to study metabolic changes during apoptosis. In cell culture, unlike in vivo, apoptotic cells are not phagocytosed and eventually lyse (secondary necrosis). This is of practical importance because metabolic changes seen in cultures may be due to the transition from apoptosis to necrosis, rather than to the induction of apoptosis itself. In the present study, we followed the kinetics of the occurrence of several indicators of cell death in rat thymocytes and mouse lymphoma (S.49), and human leukemia (CEM) cell cultures after dexamethasone treatment (10(-6) M). The presence of apoptosis and secondary necrosis was demonstrated by electron microscopy. Nuclear condensation and fragmentation, which are considered to reflect early stages of apoptosis, were visualized with Hoechst fluorescent dye H 33258 for quantitative determination by light microscopy. In S.49 and CEM cultures their incidence increased after glucocorticoid treatment, but remained at relatively low levels not exceeding 6-9% until 36 h (S.49) or 3-4% until 92 h (CEM). The trypan blue positive cells, however, increased steadily to about 60%. Furthermore, flow cytometry (single parameter DNA analysis after propidium iodide staining) revealed the occurrence of cells with reduced DNA fluorescence. Morphological and biochemical (internucleosomal DNA cleavage) analysis of FACS-sorted cells showed that early after dexamethasone the majority of them were apoptotic. In S.49 and CEM cell cultures no clear-cut time lag between increase in cells with reduced DNA fluorescence, chromatin condensation/fragmentation, and the uptake of trypan blue could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Successful nonfreezing, subzero preservation of rat liver with 2,3-butanediol and type I antifreeze protein.

BACKGROUND: Organ cryopreservation is hindered by ice inflicted damage. Nonfreezing preservation of livers at subzero temperatures might offer advantages over current preservation. METHODS: Sprague-Dawley rats were divided into three groups. UW livers (n = 6) were stored in University of Wisconsin (UW) solution at +4 degrees C. UWB livers (n = 6) were perfused ex vivo with UW + 10% 2,3-butanediol at < or =7 degrees C and stored at -4 degrees C. AFP livers (n = 4) were preserved identical to UWB livers, except for addition of 1 mg/ml of type I antifreeze protein. After 24 h livers were perfused with Krebs-Henseleit buffer (37 degrees C) for 60 min. Bile production, O(2) consumption (O(2)C), taurocholate extraction, and lactate dehydrogenase (LDH) release during perfusion and liver adenine nucleotide content and energy charge at the end of perfusion were measured. Cell membrane integrity was determined by trypan blue infusion. RESULTS: Ice formation was prevented in all livers stored at -4 degrees C. Bile production, O(2)C, and taurocholate extraction were similar among three groups. Livers stored at -4 degrees C contained significantly more adenine nucleotides than livers stored at +4 degrees C but the energy charge was similar. LDH release was significantly greater (P < 0.05) in the AFP group vs UWB and UW (63 vs 28 and 21 mU/min/g liver, respectively). Hepatocyte and sinusoidal cell trypan blue uptake was similar in all three groups. CONCLUSIONS: Butanediol with or without AFP was effective in preventing ice formation up to 24 h in rat livers stored at -4 degrees C. Although as effective as current +4 degrees C protocols, subzero preservation for longer periods needs to be achieved prior to clinical application.

Adenosine Diphosphate↗
PubMed2002

[Influence on human keratocyte proliferation by cationic liposome mediated transfer of p21WAF1].

OBJECTIVE: The aim of this study is to investigate the safety and efficiency of transducing human keratocytes with beta-galactosidase gene through cationic liposome and evaluate the influence of p21WAF1 gene transduction mediated by cationic liposome on proliferation of keratocyte. METHODS: (1) Cationic liposome mediated plasmid DNA bearing beta-galactosidase gene was transferred into cultured human keratocyte, and transduction efficiency was evaluated by enzymatic color reaction using X-gal as a substrate. (2) By immunohistochemistry and MTT, the effect of p21WAF1 gene transduction mediated by cationic liposome on cultured keratocyte proliferation was evaluated. (3) Cellular viability was detected by 0.5% trypan blue dye. RESULTS: (1) Lipofectaminetrade mark reagent could mediate a foreign gene into a cultured human keratocyte, and the highest transduction efficiency was 40.5% when DNA/lipofectamine ratio was 1:8 and the transduction time was 24 hr. (2) p21WAF1 gene transduction mediated by lipofectaminetrade mark reagent could increase the p21 protein expression in the keratocyte, and inhibit the keratocyte proliferation during 2 - 10 days after transduction. And rates of cellular viability in all groups were greater than 90% by trypan blue dye examination. CONCLUSION: Cationic liposome can mediate foreign gene into cultured human keratocytes safely and effectively; p21WAF1 gene transduction mediated by cationic liposome can inhibit the proliferation of human keratocyte.

Cell Division↗
PubMed1986

Effect of gamma radiation on resting B lymphocytes. I. Oxygen-dependent damage to the plasma membrane results in increased permeability and cell enlargement.

Although the susceptibility of resting B lymphocytes to radiation-induced interphase death is well known, the mechanism by which this occurs is not understood. In this report, we use three measures of plasma membrane integrity (increase in cell volume, uptake of trypan blue, and release of 51Cr) to assess the effect of radiation on the resting B cell plasma membrane. The delivery of 500 to 1000 rad caused the majority of resting B cells to enlarge slightly, whereas 3000 rad caused virtually all of the cells to approximately double in size within 3 to 4 hr. Measurement of the release of 51Cr from resting B cells revealed a similar relationship between the dose of radiation and the loss of radioactive label. Trypan blue exclusion was also found to diminish as a function of radiation dose. An analysis of a variety of lymphoid cells suggested that sensitivity to the membrane damaging effects of gamma radiation was in the order of resting B cells greater than resting T cells greater than a long-term L3T4+ T cell clone greater than a B cell lymphoma. LPS-induced B cell blasts treated with 3000 rad were equivalent to 1000 rad-treated resting B cells. The effects of the gamma radiation could be ameliorated by excluding oxygen (a diradical molecule that can potentially enhance the generation and propagation of highly reactive free radicals) at the time of irradiation, or by adding the free radical scavenging agent cysteamine. These data are compatible with the hypothesis that gamma radiation results in damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species. This damage is reflected in a rapid increase in plasma membrane permeability and swelling of the cells, and may play a major role in causing interphase death.

Animals↗
PubMed1984

Ultrastructure and membrane permeability of cultured pancreatic beta-cells exposed to alloxan or 6-hydroxydopamine.

Stereological techniques on electron microscopy micrographs were used to evaluate the morphological changes of cultured islet beta cells that had been exposed to alloxan or 6-hydroxydopamine. Trypan Blue exclusion by cells cultured for 3 days indicated that the cells were 100% viable. Electron microscopy revealed that nearly all of the surviving cultured cells were beta cells. Exposure to 5 mmol/l alloxan or 1-5 mmol/l 6-hydroxydopamine for 10 or 30 min caused a general swelling of the cultured cells with a concomitant swelling of mitochondria and nuclei. The size of the secretory granules was not affected by the drugs. Only 3-10% of the cells excluded Trypan Blue after exposure to 5 mmol/l alloxan or 6-hydroxydopamine. The data conform with the hypothesis that a primary action of alloxan and 6-hydroxydopamine is at the plasma membrane level of beta cells.

Alloxan↗
PubMed1975

Endothelial survival in corneal tissue stored in M-K medium.

Cat corneas were stored at refrigerator temperatures in M-K medium (TC-199, 5% dextran), modified M-K medium (TC-199, 1% chondroitin sulfate), or on the intact globe in moist chambers for intervals of one to nine days. Less than 5% of the endothelial cells stained with trypan blue in corneas stored one to four days, regardless of the method of storage. In contrast, after five days, 20% or more of the cells took up the stain in 17 to 21 corneas stored in M-K medium. Although stain uptake indicated nonviability of cells, most of the endothelial cells remained structurally intact when observed by scanning and transmission electron microscopy unless the corneas were incubated in fresh TC-199 for two hours at 35 degrees C before trypan blue staining. In that case, the percentages of disrupted cells observed at each time interval correlated with the percentages of cells that had stained in each cornea. These results indicated that storage in M-K medium did not extend the time donor coreas can be stored before transplantation beyond four days.

Animals↗
PubMed2004

[A novel bcl-2 antisense oligodeoxynucleotide F951 increases sensitivity of HL-60 cells to Ara-C].

To investigate whether F951, a novel bcl-2 antisense oligodeoxynucleotide, increases the sensitivity of HL-60 cells to Ara-C, HL-60 cells were cultured with F951 in different doses alone or with F951 combined with low-dose Ara-C; the proliferation of HL-60 cells was assayed by MTT and trypan blue exclusion test; expression of Bcl-2 protein and its mRNA were measured by FACS and RT-PCR, respectively; the apoptotic cells were detected by DNA ladder and TUNEL assay. The results showed that F951 combined with low dose Ara-C revealed stronger effects in the aspects of inhibiting the HL-60 cells proliferation than in different doses of F951 alone or Ara-C alone. HL-60 cells treated with F951 + Ara-C had significantly lower trypan blue exclusion rate than that treated with Ara-C alone. The inhibition rates of HL-60 cells treated with FNS, Ara-C, F951 and F951 + Ara-C were -2.8%, 27.63%, 37.66%, 57.24%, respectively. F951 significantly down-regulated the expression of bcl-2 mRNA and protein in HL-60 cells. HL-60 cells treated with F951 + Ara-C showed more apparent DNA ladder and more apoptotic cells. It is concluded that F951 can inhibit bcl-2 gene expression and enhance the cytotoxicity of Ara-C through promoting apoptosis in HL-60 cells, hence increases the antitumor effect of Ara-C.

Antimetabolites, Antineoplastic↗
PubMed1976

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.

Animals↗
PubMed2005

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL.

The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 microg/L TRAIL was 41.4% +/- 7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.

Apoptosis↗
PubMed2005

Mesalamine promotes intestinal epithelial wound healing in vitro through a TGF-beta-independent mechanism.

OBJECTIVE: Treatment with 5-aminosalicylic acid (5-ASA) derivatives is one of the main principles in the therapy of uncomplicated mild to moderate inflammatory bowel diseases (IBD). The beneficial effect of 5-ASA in the treatment of IBD is attributed to its anti-inflammatory and anti-oxidant properties within the inflamed gut. The aim of this study was to investigate whether 5-ASA also modulates intestinal epithelial wound repair in vitro. MATERIAL AND METHODS: The effects of 5-ASA on cell migration and proliferation, two key processes in mucosal healing, were studied in the non-transformed small-intestinal epithelial cell line IEC-6 using an in vitro wounding model and colorimetric MTT assays. Furthermore, the effects of 5-ASA on epithelial cell viability were determined by Trypan blue exclusion and flow cytometry-based cell cycle analysis. RESULTS: Clinically relevant concentrations of 5-ASA caused a significant dose-dependent enhancement of epithelial cell migration and proliferation in vitro. An about 2-fold enhancement of intestinal epithelial cell proliferation and migration was observed for pharmacological doses of 100 microg/ml 5-ASA. Neutralizing antibodies against TGFbeta did not modulate 5-ASA effects on IEC-6 cell proliferation and migration, indicating that the effects of 5-ASA were TGFbeta independent. Trypan blue viability tests and cell cycle analysis did not reveal any toxic or apoptotic effects of pharmacological 5-ASA concentrations on IEC-6 cells. CONCLUSIONS: 5-ASA promotes the rapid re-establishment of mucosal integrity in vitro by enhancing epithelial restitution and proliferation, suggesting that 5-ASA in addition to the well-characterized effects on the intestinal inflammatory cascade may also directly stimulate epithelial wound healing.

Animals↗
PubMed1994

Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischaemia.

OBJECTIVE: The aim was to test for "ischaemic" preconditioning in monolayer cultures of quiescent human ventricular cardiomyocytes. METHODS: Stabilised cardiomyocytes (n = 8 plates per group) were preconditioned with varying periods of simulated ischaemia and reperfusion, followed in all groups by 90 min of sustained "ischaemia" with or without 30 min of reperfusion. Cellular injury was assessed by trypan blue exclusion and survival was assessed by culturing the cells for 24 h postintervention. In addition, separate groups of cell plates (n = 8 per group) which had first been preconditioned with 20 min ischaemia and 20 min reperfusion were exposed to either 30, 60, or 90 min sustained ischaemia or 90 min sustained ischaemia with 30 min reperfusion. The supernatants and/or cell homogenates were analysed for hydrogen ion, lactate, lactate dehydrogenase (LDH), and adenine nucleotides and degradation products. RESULTS: Preconditioning (PC) decreased trypan blue uptake following subsequent sustained ischaemia, with the 20 min ischaemia/20 min reperfusion (20/20) regimen having the most profound effect [control ischaemia: 37.0(SEM 2.1); 10/10: 23.9(1.5); 20/20: 15.4(1.4); 30/30: 25.8(2.1) percent blue stained cells, p < 0.05 by ANOVA/Duncan]. The 20/20 preconditioning regimen resulted in less hydrogen ion [control: 2.1(0.4); PC: 1.4(0.1) mmol.g-1 protein, p < 0.05] and less LDH release [control: 20.7(3.1); PC: 11.9(4.2) units.g-1 protein, p < 0.05]. At 90 min of sustained ischaemia, the control group had produced significantly greater lactate [intracellular: control 1.55(0.62); PC 0.54(0.23) mol.g-1 DNA, p < 0.05; extracellular: control 0.47(0.09); PC 0.33(0.07) mol.g-1 DNA, p < 0.05]. There were no differences in ATP depletion in the two groups. CONCLUSIONS: Ischaemic preconditioning can be induced in human cardiomyocytes independent of other cell types. The effect can be established in human cell cultures.

Cells, Cultured↗
PubMed1992

Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury.

Both reactive oxygen metabolites and calcium have been implicated in ischemic and toxic renal tubular cell injury. However, the role of calcium in oxidant injury to renal tubular cells has not been previously examined. In the present study we examined the role of intracellular free Ca2+ ([Ca2+]i) in H2O2-mediated injury to LLC-PK1 cells, a renal tubular epithelial cell line. H2O2 induced a significant rise in [Ca2+]i within 1 min after exposure of cells to 5 mM H2O2, with a sustained rise in [Ca2+]i during the course of experiments, reaching a value of 1.3 microM at 60 min (n = 10). The rise in [Ca2+]i preceded sublethal cell injury as measured by [3H]adenine release or irreversible cell injury as determined by trypan blue exclusion. Buffering [Ca2+]i with quin-2 (50 microM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) was highly protective against the H2O2-induced cell injury. For example, at 120 min after exposure of cells to 5 mM H2O2, irreversible cell injury was reduced from 45 +/- 8 to 9 +/- 1% (n = 3) by quin-2. The acetoxymethyl ester of quin-2 (quin-2/AM) and BAPTA/AM did not interfere with the trypan blue exclusion assay or scavenge H2O2. Preventing mobilization of Ca2+ from intracellular storage sites using 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 10(-4) M) significantly reduced the rise in [Ca2+]i and thus prevented H2O2-mediated cytotoxicity to LLC-PK1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed1998

Pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production.

There are currently no effective, long-term drug therapies for the treatment of leiomyomas. Pirfenidone (Marnac, Inc.) is an antifibrotic agent that is being tested for use in patients with pulmonary fibrosis. Because leiomyomas are characterized also by increased cell proliferation and tissue fibrosis, we examined the effects of pirfenidone on cell proliferation and collagen expression in cultured myometrial and leiomyoma smooth muscle cells. Effects of pirfenidone on proliferation of myometrial and leiomyoma cells were measured using tritiated thymidine incorporation assays and changes in actual cell numbers. Possible cytotoxic effects were examined using lactate dehydrogenase assays and trypan blue exclusion. Effects on collagen type I and type III production were assessed by Northern blotting. Doses of pirfenidone tested were: 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. Serum-stimulated increases in DNA synthesis and cell proliferation by myometrial and leiomyoma cells were significantly inhibited in a dose-dependent manner by pirfenidone. Densitometric analysis of Northern blots showed significantly decreased expression of collagen type I and type III messenger RNAs in both leiomyoma and myometrial cells. Lactate dehydrogenase assays and trypan blue exclusion measurements showed no cytotoxic effect of pirfenidone at concentrations that inhibited cell proliferation and collagen production. Pirfenidone is an effective inhibitor of myometrial and leiomyoma cell proliferation in vitro and reduces the messenger RNA levels of collagen types I and III in a dose-dependent manner. This compound may prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas.

Antineoplastic Agents↗