Release Of Lactate Dehydrogenase Activity Research Articles

Silencing Cardiac Troponin I-Interacting Kinase Reduces Lipopolysaccharide-Induced Sepsis-Induced Myocardial Dysfunction in Rat by Regulating Apoptosis-Related Proteins.

The aim of this study was to investigate the effect of cardiac troponin I-interacting kinase (TNNI3K) on sepsis-induced myocardial dysfunction (SIMD) and further explore the underlying molecular mechanisms. In this study, a lipopolysaccharide- (LPS-) induced myocardial injury model was used. qRT-PCR was performed to detect the mRNA expression of TNNI3K. Western blot was conducted to quantitatively detect the expression of TNNI3K and apoptosis-related proteins (Bcl-2, Bax, and caspase-3). ELISA was performed to detect the content of lactate dehydrogenase (LDH) and creatine kinase (CK). TUNEL assay was used to detect the apoptosis of H9C2 cells. In LPS-induced H9C2 cells, TNNI3K was up regulated. Besides, the CK activity, the content of LDH, and the apoptosis of H9C2 cells were significantly increased after treatment with LPS. Silencing TNNI3K decreased the LDH release activity and CK activity and inhibited apoptosis of H9C2 cell. Further research illustrated that si-TNNI3K promoted the protein expression of Bcl-2 and decreased the protein expression of Bax and cleaved caspase-3. The study concluded that TNNI3K was upregulated in LPS-induced H9C2 cells. Importantly, functional research findings indicated that silencing TNNI3K alleviated LPS-induced H9C2 cell injury by regulating apoptosis-related proteins.

Revival of Krebs-Ringer balanced salt solution for the investigation of polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilm interaction.

To study the interaction between aggregating bacteria and polymorphonuclear leukocytes (PMNs) in vitro, the chosen medium must favor both the isolated PMNs and the bacteria. To investigate the best-suited medium for the in vitro survival of isolated unactivated human PMNs, we compared three different mammalian cell media: Krebs-Ringer balanced salt solution (BSS), Hanks' BSS (HBSS) and Roswell Park Memorial Institute (RPMI) 1640. The death of PMNs was estimated by the release of lactate dehydrogenase activity. Furthermore, two types of serum, human (HS) and fetal bovine (FBS), were compared at different concentrations (0%, 2%, 5%, 10%) and at three different time points (2, 4, 20 h). We show that Krebs-Ringer BSS prolonged the survival of PMNs compared to HBSS and RPMI 1640 and that the addition of 10% FBS significantly enhanced the long-term survival (20 h) compared to HS. Furthermore, we observed aggregation of Pseudomonas aeruginosa when grown in the presence of either a mixture of histones, histone H3, arginine or lysine. In this study, we show that the use of Krebs-Ringer BSS is highly relevant for the study of the interaction of bacteria and PMNs in relation to novel treatment strategies of biofilm infections due to the reproduction of bacterial aggregation as seen in chronic bacterial infections.

Mast cell chymase impairs bronchial epithelium integrity by degrading cell junction molecules of epithelial cells.

An increased degree of mast cell (MC) degranulation and damage to the epithelial lining are prominent features of bronchial asthma. In asthmatic airways, it seems likely that epithelial cells will be exposed to increased concentrations of proteases from MC, though their actions on the epithelium are still not very clear. Bronchial rings from human lung tissue or 16HBE cell monolayer were incubated with MC chymase in different doses or various inhibitors. The sections of paraffin-embedded tissue were haematoxylin-eosin stained and computerized by image analysis for epithelial damage-scale-evaluation; the cell viability, proliferation, adhesion and lactate dehydrogenase activity release were assayed; the expressions of gelatinases, cell junction molecules and structure proteins of 16HBE were examined. Mast cell chymase was found to provoke profound changes in the morphology of bronchi epithelial layer. Following incubation with chymase, there was 40% reduction in the length of epithelium that was intact, with detachment of columnar epithelial cells and basal cells. Chymase reduced epithelial cell proliferation and induced cell detachment, which were associated with the changes in secretion and activation of matrix metalloproteinase-2/9. In intact epithelial cell layers, immunocytochemistry study revealed that chymase reduced the expressions of occludin, claudin-4, ZO-1, E-cadherin, focal adhesion kinase and cytokeratin. Overall data of this study indicated that MC chymase can influence tissue remodelling, disrupt epithelial cell junctions, inhibit wound healing and impair the barrier function of epithelium, resulting in dysfunction of airway wall and ECM remodelling in pathogenesis of asthma. Mast cell chymase plays a key role in inducing the damage to bronchial epithelium in asthma.

Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury

Withania somnifera has been used in traditional medicine for a variety of neural disorders. Recently, chronic neurodegenerative conditions have been shown to benefit from treatment with this extract. To evaluate the action of this extract on traumatically injured neurons, the efficacy of W. somnifera root extract as a neuroprotective agent was examined in cultured model neurons exposed to an in vitro injury system designed to mimic mild traumatic brain injury (TBI). Neuronal health was evaluated by staining with annexin V (an early, apoptotic feature) and monitoring released lactate dehydrogenase activity (a terminal cell loss parameter). Potential mechanisms underlying the observed neuroprotection were examined. Additionally, morphological changes were monitored following injury and treatment. Although no differences were found in the expression of the antioxidant transcription factor nuclear factor erythroid 2-like 2 (Nrf2) or other Nrf2-related downstream components, significant changes were seen in apoptotic signaling. Treatment with the extract resulted in an increased length of neurites projecting from the neuronal cell body after injury. W. somnifera extract treatment also resulted in reduced cell death in the model neuron TBI system. The cell death factor Bax was involved (its expression was reduced 2-fold by the treatment) and injury-induced reduction in neurite lengths and numbers was reversed by the treatment. This all indicates that W. somnifera root extract was neuroprotective and could have therapeutic potential to target factors involved in secondary injury and long-term sequelae of mild TBI.

Influence of light-curing distance on degree of conversion and cytotoxicity of etch-and-rinse and self-etch adhesives

BackgroundThe degree of conversion (DC) of resin based materials depends, beside other factors, on the light-intensity applied during light curing. A lower DC might be correlated with an increased cytotoxicity of the respective materials.Therefore, aim of the present study was to investigate the influence of the distance between light-curing tip and adhesives on their cytotoxicity and degree of conversion (DC).MethodsFor the cytotoxicity assay, a total of 98 bovine dentine samples were prepared, distributed to seven groups (G1-G7; n = 14) and treated as follows: G1: untreated; G2-G4: OptiBond FL; G5-G7: OptiBond All-In-One. Adhesives were light-cured (1200 mW/cm2) at 1 mm (G2;G5), 4 mm (G3;G6) or 7 mm (G4;G7) distance. Samples were stored in culture media for 24 h and extracts were added to cell cultures (dental pulp cells and gingival fibroblasts) for a further 24 h. Finally, released lactate dehydrogenase activity (LDH) was photometrically determined, as measure for the cytotoxic effects of the extracts. The cytotoxicity assay was performed three times.Additionally, the DC of the adhesives was determined by FTIR spectroscopy. DC measurements were performed five times.ResultsFor both cell types, no significant difference of LDH release was observed between untreated control group (G1) and treated groups G2-G7 (p > 0.05, respectively), between the groups treated with same adhesive and light-cured at different distance (p > 0.05, respectively), as well as between groups treated with different adhesives and light-cured at the same distance (p > 0.05, respectively). Within the respective adhesive, no significant difference in the DC was observed when light-cured at different distance (p > 0.05, respectively), while OptiBond FL showed significantly higher DCs compared to OptiBond All-In-One when light-cured at same distances (p < 0.05, respectively).ConclusionsThe distance between light-curing tip and adhesive surface does not significantly influence either the cytotoxicity or the DC of the tested adhesives.

All-trans-retinoic acid inhibition of transforming growth factor-β-induced collagen gel contraction mediated by human Tenon fibroblasts: role of matrix metalloproteinases

Background/aimsScarring and contraction of the conjunctiva are common complications of many ocular diseases. We investigated the effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon's capsule fibroblasts (HTFs)...

Synthesis and structure-activity relationships of lapacho analogues. 2. Modification of the basic naphtho[2,3-b]furan-4,9-dione, redox activation, and suppression of human keratinocyte hyperproliferation by 8-hydroxynaphtho[2,3-b]thiophene-4,9-diones.

The basic structure of linearly anellated lapacho quinones, naphtho[2,3-b]furan-4,9-dione (7), was modified in the search for novel agents against keratinocyte hyperproliferation. The synthesis and structure-activity relationships of several heterocycle-fused naphthoquinones as well as a full range of 2- and 7-substituted derivatives of one of these, 8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a), are described. Out of a total of 71 analogues, particularly 2-thenoyl-substituted 26l, 2-nicotinoyl-substituted 26m, and 2-oxadiazole-substituted 35a compared favorably with the antipsoriatic agent anthralin. Their potency for suppression of keratinocyte hyperproliferation, which was evaluated using HaCaT cells as a model, was combined with comparably low membrane-damaging effects toward keratinocytes, as established by the release of lactate dehydrogenase activity from the cytoplasm of the cells. With respect to the mechanism of action, redox activation of lapacho quinones by one- and two-electron reduction in isolated enzymatic assays was studied, and their potential to generate superoxide was confirmed in the keratinocyte-based hyperproliferation assay.

Cytotoxic activity of extracts from Hypochaeris radicata

Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 μg/mL) and repin (30 μg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

THE CYTOTOXIC EFFECTS OF LOW INTENSITY VISIBLE AND INFRARED LIGHT ON HUMAN BREAST CANCER (MCF7) CELLS

A concept of using low intensity light therapy (LILT) as an alternative approach to cancer treatment is at early stages of development; while the therapeutic effects of LILT as a non-invasive treatment modality for localized joint and soft tissue wound healing are widely corroborated. The LEDs-based exposure system was designed and constructed to irradiate the selected cancer and normal cells and evaluate the biological effects induced by light exposures in visible and infrared light range. In this study, human breast cancer (MCF7) cells and human epidermal melanocytes (HEM) cells (control) were exposed to selected far infrared light (3400nm, 3600nm, 3800nm, 3900nm, 4100nm and 4300nm) and visible and near infrared wavelengths (466nm, 585nm, 626nm, 810nm, 850nm and 950nm). The optical intensities of LEDs used for exposures were in the range of 15µW to 30µW. Cellular morphological changes of exposed and sham-exposed cells were evaluated using light microscopy. The cytotoxic effects of these low intensity light exposures on human cancer and normal cell lines were quantitatively determined by Lactate dehydrogenase (LDH) cytotoxic activity and PrestoBlueTM cell viability assays. Findings reveal that far-infrared exposures were able to reduce cell viability of MCF7 cells as measured by increased LDH release activity and PrestoBlueTM assays. Further investigation of the effects of light irradiation on different types of cancer cells, study of possible signaling pathways affected by electromagnetic radiation (EMR) and in vivo experimentation are required in order to draw a firm conclusion about the efficacy of low intensity light as an alternative non-invasive cancer treatment.

Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion

Cells from primary islets and beta-cell lines form pseudoislets (PIs) in static cultures. Interestingly, MIN6 beta-cells with aberrant regulation of proliferation form PIs which cease to grow after a week in culture. This growth arrest is attributed to a pro-apoptotic and anti-proliferative PI environment. We hypothesized that cell necrosis due to poor nutrient transport in dishes rather than apoptosis effects the observed PI size restriction. Formation of beta-cell PIs was explored in stirred-suspension bioreactors with enhanced mass transfer. Cells in stirred-suspension proliferated continuously and the PI size increased for two weeks. Bioreactor PIs displayed regulated basal insulin secretion and enhanced responsivity to glucose and incretins. Compared to dishes, cell viability in the bioreactor was higher with lower released lactate dehydrogenase activity. Similar expression of p21 and p27 in monolayers and PIs did not suggest an anti-proliferative PI milieu. Caspase-2, -8 and -9 activities were comparable in dish and bioreactor PIs, and the latter continued to grow after one week of culture. Thus, apoptosis is not sufficient to explain the differences in PI size between dishes and bioreactor. Moreover, the bioreactor method described here may be used to generate PIs with increased cell viability and function for research and clinical applications.

Endotoxin Promotes Adverse Effects of Amorphous Silica Nanoparticles on Lung Epithelial Cells in Vitro

Amorphous silica engineered nanoparticles (ENP) are used for drug delivery and food additive under current regulations. Although the adverse effects of amorphous silica ENP may be negligible, contamination by bacterium products may enhance the toxic potential of these so-called safe products. Lipopolysaccharide (LPS), an endotoxin component generated by gram-negative bacteria, is a potential contaminant of amorphous silica ENP due to its ubiquitous presence in the environment. The combined effects of amorphous silica ENP and LPS are therefore of particular concern. In this study, A549 cells were exposed to amorphous silica ENP in combination with LPS for comparison with the cells treated with ENP. Measurements of MTT assay and lactate dehydrogenase (LDH) activity indicated that the toxicity of amorphous silica ENP was low but co-treatment of the cells with LPS significantly enhanced this toxicity. Decreased cell viability and increased LDH activity release occurred earlier and at lower concentration levels in co-treated cells. Co-treatment of LPS with amorphous silica ENP might also enhance the increase in oxidative stress produced by amorphous silica ENP. However, there were no detectable changes in nitric oxide generation and 8-hydroxy-2-deoxy guanosine formation in the cells treated with either ENP or ENP plus LPS, indicating low effect on oxidative DNA damage. These results showed that LPS may enhance the oxidative stress induced by amorphous silica ENP to initiate cytotoxicity of these engineered nanoparticles.

Magnetometric evaluation of toxicities of chemicals to the lungs and cells

Because the lungs are exposed to airborne hazardous materials, alveolar macrophages (AMs) play a major role in defending against the exposure to various noxious chemical substances. In this study, we reviewed magnetometric investigations of the effects of various chemicals on the lungs and AMs. Magnetometry, using magnetite as an indicator, was used to evaluate the effects of certain chemicals on the lung and AMs. A rapid decrease of the remanent magnetic field after the cessation of external magnetization, a phenomenon called relaxation, was impaired when the lungs and macrophages were exposed to toxic substances. The delayed in vivo relaxation observed in the lungs exposed to magnetite and gallium arsenide was almost identical to the in vitro relaxation observed in the AMs exposed to the same materials. Delayed relaxation was observed in the AMs exposed to silica dust; various fibers, such as chrysotile and some man-made mineral fibers; and toxic arsenic and cadmium compounds. The extracellular release of lactate dehydrogenase activity was found in the AMs exposed to the chemicals. Relaxation is attributed to the cytoskeleton-driven rotation of phagosomes containing magnetite. While the exact mechanism of delayed relaxation due to exposure to harmful chemicals remains to be clarified, cell magnetometry appears to be useful for the safety screening of chemical substances.

Evaluation of cytotoxic concentration–time response in A549 cells exposed to respirable α‐quartz

A causal pathway between quartz, silicosis and lung cancer has been postulated. The aim of our study was to assess cytotoxic effects induced in a human lung epithelial cell line (A549) by exposure to alpha-quartz. Cells were exposed to respirable alpha-quartz (SRM1878a, NIST) at 25, 50 or 100 microg ml(-1 )for 24 h and at 50 or 100 microg ml(-1) for 48 h. Cytotoxic effects were analyzed by scanning electron microscopy (SEM), apoptotic morphology analysis with Hoechst staining and lactate dehydrogenase (LDH) release assay. In cells exposed to alpha-quartz for 24 h, a concentration-dependent bleb development and in particular the localization of blebs at the cell edge at higher concentrations were observed. The blebbing phenomenon was more evident after 48 h of exposure to 50 or to 100 microg ml(-1) of alpha-quartz and large blebs were localized at the cell edge. At the same concentrations surface smoothing was also observed. Moreover the presence of holes and tears was detected at the highest concentration both at 24 and 48 h. Results of morphological analysis with Hoechst stain evidenced an increase concentration-time dependent of apoptotic cell percentage that was more marked after 48 h exposure to 100 microg ml(-1) and a prevalence of late apoptosis stage with the increase of exposure time and concentration. Cells exposed to 50 or 100 microg ml(-1) of alpha-quartz for 24 and 48 h produced a significant increase in LDH release. The concentration-time-dependent bleb induction evidenced by SEM correlates with the increase of apoptotic cells and LDH activity release, demonstrating the onset of cytotoxic effects in human lung cells exposed to alpha-quartz.

No evidence for protective erythropoietin alpha signalling in rat hepatocytes

BackgroundRecombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved.MethodsLoss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting.ResultsIn contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA.ConclusionTogether, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.

Intramuscular Immunization with a Plasmid DNA Vaccine Encoding prM-E Protein from Japanese Encephalitis Virus: Enhanced Immunogenicity by Co-Administration of GM-CSF Gene and Genetic Fusions of prM-E Protein and GM-CSF

Objective: To investigate the immune responses elicited by pJME with or without various forms of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene. Methods: The changes of the T lymphocyte subsets and the levels of Th cell intracellular cytokines IFN-γ and IL-4 were evaluated by flow cytometric analysis. The cytotoxic T lymphocyte kill activity was assessed by lactate dehydrogenase activity release test. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. Results: We demonstrated that simultaneous administration of pJME plus plasmid-ecoded GM-CSF (pGM-CSF) activated Th1 immune responses similar to those found by injecting pGM-CSF i.m. into mice 3 days before pJME vaccination, and enhancement of Th2 immunity predominated when the pGM-CSF was injected 3 days after pJME vaccination. Furthermore, the immunization with DNA vaccine encoding precursor membrane envelope/GM-CSF fusion protein was more effective in generating immune responses than that induced by immunization with pJME alone or in combination with pGM-CSF. Conclusions: These observations support the potential of GM-CSF DNA adjuvant for the Th1/Th2 balance and the enhancement of immune responses by showing that the timing of the administration of pGM-CSF and the application of different forms of GM-CSF gene influence the outcome of the resultant immune responses.

Mechanisms of attenuation of ischemic heart injury by means of a modified reperfusion

Mechanisms of attenuation of membrane injury and metabolic disturbances in postischemic cardiomyocytes have been studied on a model of ischemic and reperfusion stress of rat heart using a modified early reperfusion. Optimization of reperfusion infusate composition augmented cardiac pump and contractile function recovery. This was accompanied by a reduced release of lactate dehydrogenase activity and systems generating short-lived reactive oxygen species into myocardial effluent and was associated with more efficient oxidative metabolism recovery and decreased losses of intracellular total creatine and amino acids pools. The results indicate availability of postischemic functional and metabolic myocardial injury correction by means of a controlled reperfusion.

Immunologic Analysis Induced by DNA Vaccine Encoding E Protein of Beijing-1 Strain Derived from Japanese Encephalitis Virus

Objective: We have compared the gene expression and DNA immunization efficacy encoding prME and E proteins of a different strain (JaGAr-01) derived from Japanese encephalitis virus. This study aimed to construct a recombinant encoding E protein of the Beijing-1 strain derived from Japanese encephalitis virus and analyze the humoral, cellular and protective immunity induced by the above recombinant. Methods: The recombinant pJBE containing E (1,500 bps) gene from the Beijing-1 strain of Japanese encephalitis virus was constructed and then transfected into the HepG2 cell line by liposome fusion. The expression of E (about 53 kD) protein in transfected cells was analyzed by Western blot using a specific anti-JEV-E antibody. BALB/c mice were vaccinated with 3 µg of pJBE by the gene-gun technique. JaGAr-01 and Beijing-1 strains (10⁵ PFU/100 µl) of Japanese encephalitis virus were given to BALB/c mice by intraperitoneal injection 3 weeks after double DNA immunization with a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. A lactate dehydrogenase activity release test was used to examine cytotoxic T lymphocyte activity after double DNA immunization. Results: The expression of about 53 kD protein associated with pJBE was determined in transfected HepG2 cells with specific anti-JEV-E antibody. A higher level of neutralization antibodies and the cytotoxicity effect were induced with pJBE immunization using the gene-gun technique, and were similar to those induced with inactivated vaccine derive from the Beijing-1 strain of Japanese encephalitis virus. Balb/c mice immunized with pJBE survived the challenge with the different strains of Japanese encephalitis virus; however, Balb/c mice immunized with inactivated vaccine did not survive the challenge with the JaGAr-01 strain of Japanese encephalitis virus at all. Conclusions: DNA vaccine containing the E protein gene derived from Japanese encephalitis virus can provide not only better efficacy including humoral and cellular immunity, but also cross-protection against infection with homologous and heterologous Japanese encephalitis virus.

Pepsin-Digested Bovine Lactoferrin Induces Apoptotic Cell Death With JNK/SAPK Activation in Oral Cancer Cells

Lactoferrin, a member of the transferrin family, is iron-binding and a strongly cationic 76 kDa glycoprotein. In breast milk it is secreted in high concentrations from glandular epithelia and is also present in other exocrine fluids including saliva. In the present study, we examined the biological mechanisms of apoptosis induced by pepsin-digested-lactoferrin peptide (Lfn-p) in the human oral squamous cell carcinoma cell line SAS. We found that treatment with Lfn-p induced cell death with apoptotic nuclear changes, preceded by the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in the apoptotic cells. Treatment with Lfn-p induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), a member of the MAP kinase family, at early stages of apoptosis. Another MAP kinase, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), was also phosphorylated by treatment with Lfn-p. Pretreatment of SAS cells with SP600125, a JNK/SAPK inhibitor, diminished Lfn-induced apoptosis, as assessed by determining released lactate dehydrogenase activity. On the other hand, the MEK1 inhibitors PD98059 or U0126 showed no effect on repression of cell death, but rather an increase. These results suggest that JNK/SAPK activation may play an important role in Lfn-p-induced apoptotic cell death of human oral squamous cell carcinoma cells.

Magnetometric Evaluation of Cadmium Oxide--Induced Toxicity to Pulmonary Alveolar Macrophages of Syrian Golden Hamsters

Since alveolar macrophages play an important role in the clearance of inhaled dust from airways, these cells have been used as a target for various toxic chemicals. Alveolar macrophages obtained from bronchoalveolar lavage of Syrian golden hamsters were concurrently exposed in vitro to Fe 3 O 4 , as an indicator for magnetometry, and various concentrations of cadmium oxide (CdO) in this study. A rapid decrease of the remnant magnetic field, called relaxation, was observed after the cessation of an external magnetic field stimulus in macrophages concurrently exposed to phosphate-buffered saline or CdO at 0.1 w g/ml, while relaxation was delayed in those concurrently exposed to 1, 25, or 50 w g/ml CdO. Therefore, the concentration of CdO affecting relaxation in vitro was estimated at between 0.1 and 1 w g/ml. Release of LDH activity from CdO-exposed macrophages into the medium significantly increased at levels of 25 and 50 w g/ml CdO. Apoptosis was not detected in macrophages exposed to CdO by the DNA ladder detection method or morphological observations. Electron-microscopic examination revealed severe membrane damage and vacuolar changes in macrophages exposed to CdO. Since delayed relaxation is thought to occur by (1) disrupted cytoskeleton-driven random rotation of phagosomes containing iron oxide particles, (2) significant lactate dehydrogenase (LDH) activity release, and (3) detachment of cell membranes, CdO is considered to affect macrophage functions.

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.