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PubMed1987

Directed targeting of immunoerythrocytes provides local protection of endothelial cells from damage by hydrogen peroxide.

Red blood cells bearing anti-mouse IgG antibody on their surface (immunoerythrocytes) may provide for local protection of endothelial cells from the action of hydrogen peroxide. Subconfluent cultures of human umbilical vein endothelial cells responded sharply to increasing concentrations of hydrogen peroxide. Permeabilization of cellular membrane occurred at doses of hydrogen peroxide of from 1 to 3 mM, and was assured by incorporation of trypan blue stain immediately after treatment. Latent damage of cells produced by much lower doses of hydrogen peroxide (0.2-0.4 mM) could be observed after 24-hour incubation of treated cells in the normal culture medium with no hydrogen peroxide. The apparently dead cells differed from intact cells in morphology, were poorly attached to the substrate, and were readily incorporated by trypan blue, thus permitting easy visualization. Immunoerythrocytes bound to the antigen-coated surface enzymatically decreased the concentration of hydrogen peroxide in their microenvironment at least fivefold with respect to the total hydrogen peroxide concentration. Erythrocytes deposited on a part of the endothelial monolayer locally protected it from the damage at hydrogen peroxide concentrations ranging from 0.4 to 1.2 mM. Localization of protected zones corresponded precisely to the geometry of the erythrocyte coating. Immunoerythrocytes targeted to the endothelial cells by means of mouse anti-endothelial antiserum did not impair their viability and protected the endothelium from being killed at 0.3-1.2 mM hydrogen peroxide. This approach might be useful for a cell selection in mixed cell populations. The problem of local protection of cells involved in the inflammation focus are discussed.

Animals↗
PubMedDate not supplied

A model of anoxic preconditioning in the isolated rat cardiac myocyte. Importance of adenosine and insulin.

OBJECTIVE: Ischemic or hypoxic preconditioning has been shown, in multicellular preparations, to reduce post-ischemic injury. In the present study, we attempted to develop a model of preconditioning in isolated rat myocytes in order to facilitate investigation into the mechanism of preconditioning. METHODS: The protective effect of a short period (10 min) of anoxia and reoxygenation against a subsequent longer period of anoxia was studied in single, electrically stimulated (0.2 Hz, 37 degrees C) adult rat cardiac myocytes. The control group received only the long period of anoxia. Three protocols were tested: Protocol 1 in which octanoate was the only substrate; Protocol 2 in which only glucose was present during all normoxic phases and during the preconditioning anoxia and octanoate alone during the prolonged period of anoxia and; Protocol 3 in which protocol 2 was repeated with the addition of adenosine (100 microM) and insulin (15 microU/ml) during the prolonged anoxic period. The end-point of assessment was loss of cell morphology, i.e., hypercontracture (death) or relengthening (survival) on reoxygenation following the prolonged anoxic period. Membrane integrity was also examined at the end of each protocol by observing if the cells excluded trypan blue. RESULTS: No protective effect of preconditioning on cell survival was observed in protocols 1 or 2. In contrast, in protocol 3, a significant protection was observed in the preconditioned versus control group (58% vs 27% survival respectively; p < 0.001). However, in the absence of preconditioning, adenosine and insulin provided no additional protection in the control group. No significant differences in trypan blue exclusion were observed between the groups in any protocol. CONCLUSIONS: These results suggest that preconditioning cannot protect against a subsequent period of anoxia where the accumulation of metabolic products, e.g., adenosine is prevented. However, that protection can be re-instated by the presence of adenosine and insulin during the period of prolonged anoxia. Furthermore, this study suggests that the preconditioning by anoxia may induce a change in the A1-receptor or its second messenger system such that adenosine is able to provide protection.

Adenosine↗
PubMed1993

Evaluation of University of Wisconsin cold-storage solution in warm hypoxic perfusion of rat liver: the addition of fructose reduces injury.

BACKGROUND: University of Wisconsin cold-storage solution (UW solution) has markedly improved organ preservation for liver transplantation. However, the efficacy of this solution in preserving hepatocyte viability during warm ischemia is undefined; hence, the effects of UW solution on warm hypoxic injury in the isolated perfused rat liver were examined. METHODS: Livers were perfused using a modified protocol that included a period of hypoxic perfusion with isosmotic Krebs' solution at the end of each experiment. Hepatic injury was evaluated by aspartate aminotransferase (AST) release into the perfusate and the trypan blue perfusion technique. RESULTS: Although UW solution appeared to decrease hepatic injury during hypoxic perfusion, as reflected by low AST release, perfusion with UW solution led to hepatocyte shrinkage and cessation of bile flow even under oxygenated conditions. UW solution did not protect against warm hypoxic injury, as assessed by AST release into the perfusate (182 +/- 15 U/mL, mean +/- SD) or trypan blue staining of the dead hepatocyte nuclei (56% +/- 5%). However, the addition of fructose to UW solution resulted in a significant decrease in AST release (66 +/- 15 U/mL) and parenchymal cell death (39% +/- 7%). CONCLUSIONS: These data suggest that the addition of fructose or other gluconeogenic substrates may complement the overall hepatoprotective effects of UW solution, particularly during periods of warm hypoxia.

Adenosine↗
PubMed2004

Membrane damage thresholds for 1- to 10-MHz pulsed ultrasound exposure of phagocytic cells loaded with contrast agent gas bodies in vitro.

Monolayers of mouse macrophage-like cells provide a model system for the study of bioeffects of pulsed ultrasound (US) activation of contrast agent gas bodies. In this study, the dependence of membrane damage on ultrasonic frequency was examined for gas bodies attached to the cells. The monolayers cultured on the inside of one window of an exposure chamber were incubated with 2% Optison (Amersham Health Inc., Princeton, NJ) and then rinsed to remove unattached gas bodies. The chamber was filled with culture medium plus 20% trypan blue stain solution and was mounted at the 3.8-cm focus of an US transducer in a 37 degrees C water bath. Transducers were used with center frequencies of 1.0, 2.25, 3.5, 5.0, 7.5 and 10.0 MHz. The 1-min pulsed exposures utilized two-cycle excitation with 1% duty cycle. After exposure, cells in the focal zone were scored for trypan blue dye exclusion, with stained nuclei indicative of cell membrane damage. Exposure-response functions were approximated by performing a series of exposures with peak rarefactional pressure amplitudes differing by a factor of radical 2 (i.e., 3 dB apart). Linear regressions were performed on selected data to determine a threshold pressure amplitude at each frequency. Thresholds ranged from 0.066 MPa at 1.0 MHz to 0.62 MPa at 10 MHz and were approximately proportional to the frequency. These thresholds are less than the pressure amplitudes needed for nucleation of inertial cavitation and have a different frequency dependence than the general Mechanical Index.

Albumins↗
PubMed2004

Efficient transfer of chromosome-based DNA constructs into mammalian cells.

Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.

Animals↗
PubMed2004

Hyperammonemia, brain edema and blood-brain barrier alterations in prehepatic portal hypertensive rats and paracetamol intoxication.

AIM: To study the blood-brain barrier integrity, brain edema, animal behavior and ammonia plasma levels in prehepatic portal hypertensive rats with and without acute liver intoxication. METHODS: Adults male Wistar rats were divided into four groups. Group I: sham operation; II: Prehepatic portal hypertension, produced by partial portal vein ligation; III: Acetaminophen intoxication and IV: Prehepatic portal hypertension plus acetaminophen. Acetaminophen was administered to produce acute hepatic injury. Portal pressure, liver serum enzymes and ammonia plasma levels were determined. Brain cortex water content was registered and trypan blue was utilized to study blood brain barrier integrity. Reflexes and behavioral tests were recorded. RESULTS: Portal hypertension was significantly elevated in groups II and IV. Liver enzymes and ammonia plasma levels were increased in groups II, III and IV. Prehepatic portal hypertension (group II), acetaminophen intoxication (group III) and both (group IV) had changes in the blood brain-barrier integrity (trypan blue) and hyperammonemia. Cortical edema was present in rats with acute hepatic injury in groups III and IV. Behavioral test (rota rod) was altered in group IV. CONCLUSION: These results suggest the possibility of another pathway for cortical edema production because blood brain barrier was altered (vasogenic) and hyperammonemia was registered (cytotoxic). Group IV, with behavioral altered test, can be considered as a model for study at an early stage of portal-systemic encephalopathy.

Acetaminophen↗
PubMed2003

Effects of nasal saline spray on human neutrophils.

BACKGROUND: Nasal saline spray (NSS) used in the treatment of rhinitis and sinusitis often contains the preservative benzalkonium chloride (BKC). Previous studies have shown that corticosteroid nasal sprays and topical decongestants containing BKC damage respiratory mucosa, decrease mucociliary activity, and inhibit neutrophil functions in vitro. OBJECTIVE: To evaluate the effects of NSS with BKC on human neutrophils. DESIGN: Prospective, basic science observations. METHODS: Human neutrophils were exposed to NSS with BKC or to phosphate-buffered saline (PBS) at varying times and concentrations. The cells were examined for morphologic changes by light microscopy and for viability as determined by trypan blue exclusion. Lactate dehydrogenase levels were measured to quantify neutrophil cell lysis. In vivo morphologic changes were studied in neutrophils obtained from the oral mucosa in human volunteers who rinsed their mouths with NSS or PBS. RESULTS: Neutrophils exposed to NSS concentrations as low as 15% showed near-total cell lysis, and neutrophils exposed to 20% NSS demonstrated no cell viability by trypan blue staining. Phosphate-buffered saline-exposed cells were unaffected. The release of lactate dehydrogenase from lysed neutrophils increased sharply at NSS concentrations higher than 10% but remained stable in PBS-exposed cells. All neutrophils isolated from NSS oral rinses were lysed, while a mean of 78% of neutrophils from PBS rinses showed normal morphologic structure. CONCLUSIONS: Nasal saline spray with BKC is toxic to human neutrophils even at concentrations far lower than those found in commercially available preparations. Saline solutions without BKC appear to be safer alternatives, and additional studies are needed to determine the clinical significance of these findings.

Administration, Intranasal↗
PubMed1990

Hepatotoxicity due to clofibrate is oxygen-dependent in the perfused rat liver.

Toxicity of clofibrate, a hypolipidemic drug, was assessed in livers from fasted rats perfused in both the anterograde and the retrograde directions. Oxygen uptake decreased steadily following infusion of clofibrate (15 mM) and was diminished by about 40% in 15 min. Cell damage, assessed by the appearance of lactate dehydrogenase (LDH) in the effluent perfusate, began within 20 min. Maximal values for LDH release into perfusate were around 250 U/g/hr after perfusion with clofibrate for 40 min. Inhibition of oxygen uptake and release of LDH into the perfusate was dose-dependent (half-maximal effect = ca. 12 mM clofibrate). Nearly 90% of hepatocytes in oxygen-rich, periportal regions but only about 30% in oxygen-poor, pericentral areas took up trypan blue, an indicator of irreversible cell death, following perfusion with clofibrate in the anterograde direction. In contrast, when livers were perfused in the retrograde direction, 85% of cells in upstream, oxygen-rich pericentral regions were damaged whereas only about 30% in downstream areas were stained. When local oxygen tension was lowered by reducing the flow rate to one-quarter of normal, trypan blue uptake in periportal areas was diminished nearly completely (ca. 5% of cells were stained). Incubation in vitro of isolated cylinders of periportal and pericentral tissue with clofibrate at 800 or 200 microM oxygen led to about three times greater LDH release in incubations carried out at high than at low oxygen tension. This experiment led us to rule out the involvement of clofibrate delivery in the mechanism of zone-specific toxicity. Subsequently, local rates of oxygen uptake were measured using miniature oxygen electrodes placed on the liver surface. Clofibrate decreased oxygen uptake about 30% in oxygen-rich, periportal regions of the liver lobule, yet had no effect on respiration in downstream, pericentral areas. These phenomena can best be explained by a direct effect of clofibrate on active mitochondria in periportal regions of the liver lobule where oxygen uptake predominates, since state 3 but not state 4 rates of respiration were inhibited by clofibrate in isolated mitochondria (half-maximal effect = ca. 1.8 mM clofibrate). Thus, toxicity of clofibrate in upstream, periportal areas of the liver lobule is dependent on local oxygen tension and affects actively respiring mitochondria. This may lead to local cell death and be responsible for initiating a sequence of events leading to the well-known carcinogenic effects of this compound.

Animals↗
PubMed1999

Dissemination of melanoma cells within electrocautery plume.

BACKGROUND: The observed occurrence of port site recurrence in laparoscopic surgery for malignant disease has stimulated interest in the dissemination of tumor cells during surgery. Study of electrocautery smoke has revealed the presence of large particles and viable viruses. The purpose of this study was to determine if viable malignant cells are present in suspension within the electrocautery plume. METHODS: Pellets of B16-F0 mouse melanoma cells were cauterized and the plume collected into culture medium. In part 1 of this study, the trypan blue assay was used to assess cell viability immediately after collection and 7 days later. A cautery current of 30 W was applied for 5 minutes. In part 2, the tetrazolium (MTT) viability assay was used to assess cell viability after cauterization of tumor pellets at 10, 20, and 30 W for 5 seconds. RESULTS: Although intact melanoma cells were identified with the trypan blue assay immediately after plume collection, no viable cells were seen at 7 days using this assay. In part 2, viable melanoma cells were present in the culture wells at 7 days. Lower fulguration currents appeared to yield higher cell counts: 2,250 cells/well at 10 W, 2,100 cells/well at 20 W, and 1,800 cells/well at 30 W. CONCLUSIONS: Results of this study confirm that application of electrocautery to a pellet of melanoma cells releases these cells into the plume. These cells are viable and may be grown in culture. This release of malignant cells may explain the appearance of port metastases at sites that are remote from the surgical dissection or that were never in direct contact with the tumor.

Animals↗
PubMed2003

Development of the peritoneal lymphatic stomata and lymphatic vessels of the diaphragm in mice.

The generation and development of the peritoneal lymphatic stomata (PLS) and lymphatic vessels of the diaphragm were studied in mice at gestational ages from the embryonic to the postnatal period with TEM, SEM and enzyme histochemistry and the PLS data were quantitatively analyzed with computer-assisted image processing technology (Elescope image analysis software). The results showed that the diaphragmatic mesothelium was covered only by flattened mesothelial cells (FMC) at the 13th embryonic day (ED 13). At ED 15, some cuboidal mesothelial cells (CMC) and immature lymphatic stomata (NLS) were found scattered on the diaphragmatic mesothelium. The sub-peritoneal lymphatic capillaries did not appear until ED 18. However, no absorptive function was observed in NLS when trypan blue granules were injected into the peritoneal cavity. At postnatal day 1 (PND 1), the endothelial cytoplasm processes of the diaphragm lymphatic capillaries span the connective tissue fibers and the basal membrane of CMC to form the subperitoneal channels. These channels were connected with NLS and serve as the absorptive route between the peritoneal cavity and the sub-peritoneal lymphatic vessels. The trypan blue absorption test demonstrated that postnatal PLS possessed an absorptive function and had transformed to mature lymphatic stomata (MLS) by PND 1. Thus, NLS were renamed of MLS. At PND 5, the cuboidal mesothelial cell ridge (CMCR) appeared with increased CMC areas. At PND 10, CMCR were fused to form the band-like CMC area with much more MLS distributed in the muscular portion of the diaphragm. With distribution area and density of PLS increasing and growth of lymphatic vessels, an increased absorptive function from the peritoneal cavity was observed in the experiment.

Animals↗
PubMedDate not supplied

Edible mushroom (Agaricus bisporus) lectin inhibits human retinal pigment epithelial cell proliferation in vitro.

The retinal pigment epithelium (RPE) plays a major role in the development of the anomalous retinal scarring response termed proliferative vitreoretinopathy. The present study was undertaken to investigate whether agaricus bisporus lectin inhibited human RPE proliferation in vitro. Fluorescein isothiocyanate-labeled agaricus bisporus lectin was used to study binding of lectin to cultured human RPE. The effect of a 24-hour exposure of agaricus bisporus lectin on RPE proliferation was measured using (methyl-3H)-thymidine incorporation into DNA. Toxicity studies were assessed using morphologic evaluation, trypan blue exclusion, and a cell viability assay. Agaricus bisporus lectin bound to RPE cells and was inhibited by preincubation of lectin with asialomucin. Agaricus bisporus lectin caused a dose-dependent inhibition of RPE proliferation (one-way ANOVA, F = 94.470, p < 0.001) that was partially reversible on removal of the lectin. Compared with controls, cells remained viable and no morphological changes or trypan blue staining was noted in RPE exposed to agaricus bisporus lectin. Human RPE binds agaricus bisporus lectin and inhibits proliferation without apparent cytotoxicity. It therefore merits consideration as a potential antiproliferative agent in the prevention and treatment of proliferative vitreoretinopathy and other nonocular anomalous wound healing processes.

Cell Division↗
PubMed1989

Evidence that graft survival is not related to parenchymal cell viability in rat liver transplantation. The importance of nonparenchymal cells.

Injury to parenchymal and nonparenchymal cells of livers stored in cold Euro-Collins solution was assessed following reperfusion and compared with graft survival following orthotopic rat liver transplantation. Parenchymal cells maintained their viability nearly completely after up to 24 hr of cold storage as assessed by trypan blue exclusion (97% of cells) and LDH release (4% of total) from livers reperfused for 20 min following storage. Furthermore, hepatic glycolysis (rates of lactate plus pyruvate production), oxygen uptake and NADH redox state (lactate:pyruvate ratio) were in the normal range at all time points studied up to 24 hr of cold storage. In contrast, nonparenchymal cells lost viability as assessed from trypan blue staining beginning after 8 hr of storage: 40% were nonviable after 24 hr of storage. Since injury to nonparenchymal cells occurs only upon reperfusion, oxygen radicals may be involved. Accordingly, xanthine and hypoxanthine, substrates for oxygen radical formation, were measured in perfusate upon reperfusion. Both purines accumulated (up to 80 microM) with time of storage and were washed out rapidly (less than 10 min) upon reperfusion. Although parenchymal cell function was in the normal range in livers stored in the cold for 24 hr, liver grafts stored for 6 hr and longer in Euro-Collins solution could not be transplanted successfully. Thus, we conclude that viability of parenchymal cells in liver grafts prior to transplantation is a poor parameter to predict the outcome of transplantation. Therefore, assessment of parenchymal cell energy state (e.g., with 31P NMR and other methods) most likely will not predict survival reliably. On the other hand, nonparenchymal cells lose their viability significantly earlier following storage and reperfusion. These data suggest that preservation of nonparenchymal cell viability is critical for successful liver transplantation.

Animals↗
PubMed1987

Histochemical and biochemical observations on the cytotoxicity of paracetamol and its effects on glycogen metabolism in rat liver.

The effects of paracetamol overdose on glycogen metabolism in rat liver have been investigated and related to its cytotoxicity. Paracetamol was administered to male rats by gavaging after a 24-h fast and refeeding was not permitted. An early (9-12-h) increase in histochemically demonstrable glycogen phosphorylase alpha activity in perivenous hepatocytes preceded major loss of membrane integrity as assessed by serum glutamate-pyruvate transaminase (SGPT) activity and uptake of trypan blue during perfusion. These changes occurred only after a decrease in the concentration of reduced glutathione, which is generally observed about 4 h after paracetamol treatment. The activation of glycogen phosphorylase in perivenous hepatocytes occurred concurrently with an increase in glycogen content of periportal hepatocytes, indicating a clear heterogeneity in the response of the two-cell populations to the hepatotoxin. The use of trypan blue perfusion together with histochemical techniques allowed changes in glycogen content and phosphorylase alpha activity of individual hepatocytes to be assessed with reference to the extent of membrane damage evident. The relevance of the results to possible mechanisms of hepatotoxicity is discussed.

Acetaminophen↗
PubMed2003

[Cortical neuron injury occurs following recurrent epileptiform discharges induced by magnesium-free treatment in vitro].

OBJECTIVE: To study cortical neuron injury following recurrent epileptiform discharges induced by magnesium-free treatment in vitro. METHODS: Cultured embryo cortical neurons were exposed to magnesium-free media for 3 h, then they were returned to regular media containing normal level magnesium. At different time after Mg(2+)-free treatment, trypan blue staining and determination of LDH activity were used to determine the cell viability, flow cytometry was applied to measure neuronal apoptosis, and MTT assay to study metabolic rate. RESULTS: (1) Neuronal morphology on light microscopy following Mg(2+)-free treatment showed that there were no prominent alterations. (2) At different time (6, 12, 72 h) after Mg(2+)-free treatment, neuronal viability by trypan blue staining and LDH activity showed modest changes compared with time-matched control in different culture days (6, 12, 17 d) (P > 0.05). (3) Cell apoptosis increased mildly at different time after Mg(2+)-free treatment in neurons cultured for different days, but the increase was not significant (P > 0.05). (4) Metabolic rate decreased at 6 h after Mg(2+)-free treatment (P < 0.05) in neurons cultured for 6 d, and was 86.4% of that of the control; while the rate at 24 h in neurons cultured for 12 d and 17 d also decreased (P < 0.05), being 78.7% and 70.9%, respectively, of that of the control. CONCLUSIONS: These findings demonstrated that the injury occurred on cultured cortical neurons caused by magnesium-free-treatment-induced recurrent epileptiform discharges was mainly functional and relatively mature neurons displayed more severe and much later mitochondrial function impairment than immature neurons.

Animals↗
PubMed2004

Evidence for injurious effect of cocaethylene in human microvascular endothelial cells.

BACKGROUND: Cocaethylene (CE) is a conjugate of cocaine and ethanol that may contribute to the pathogenesis of systemic vascular diseases. This study was conducted to investigate the effect of CE on human microvascular endothelial cells (HMEC-1) in culture. METHODS: Proliferating and confluent monolayers of HMEC-1 were used for assessing growth kinetics, viability, cytotoxicity, and morphologic/barrier alterations after CE treatment (0-1 mmol/l) for up to 7 days. The Trypan blue exclusion, lactate dehydrogenase (LDH) release assay, manual cell counts, and silver nitrate staining technique were used. RESULTS: The doubling times of 30.0 and 31.4 h for the 0.5 and 1.0 mmol/l CE-treated HMEC-1, respectively, were significantly longer than the 28.6 h for the control group (p < 0.05). The viabilities of 90.4 +/- 3.8% (control) and 93.1 +/- 1.9% (CE-treated) from the Trypan blue exclusion-staining experiments indicated non-lethality of CE. LDH activities of 173 +/- 33 U/l (control) and 157 +/- 43 U/l (CE-treated) confirmed the absence of CE cytotoxicity. Silver staining results indicated increased monolayer permeability as demonstrated by the formation of intercellular gaps after 1 h of exposure. CONCLUSIONS: HMEC-1 exposure to CE induced cellular injury that could affect the permeability of small blood vessels. These cellular changes could in part be the pivotal point for studies to explain the edema and inflammation in surrounding tissues of individuals exposed to CE.

Antigens, Polyomavirus Transforming↗
PubMed2006

Synthesis and in vitro cytotoxicity of 1,2,3,4-tetrahydroisoquinoline derivatives.

Several 1-alkyl-1,2,3,4-tetrahydroisoquinoline (TIQ) derivatives, which may play a role in Parkinson's disease, have been synthesized via Pummerer-type cyclization of the sulfonium ion formed in situ from N-formyl sulfoxide. Using an in vitro trypan blue exclusion assay, high concentrations of TIQ derivatives possessing bulky alkyl group substituents such as 1-cyclobutyl-, 1-cyclohexyl-, 1-phenyl- or 1-benzyl- at the C-1 position were found to significantly affect the viability of PC12 cells. Moreover, TIQ derivatives that moderately or strongly induced apoptosis (e.g., 1-phenyl-TIQ and 1-cyclohexyl-TIQ, respectively) paralleled the results obtained using the trypan blue exclusion assay. These results suggest that the size and electron-donating properties of functional groups may affect the cytotoxicity of TIQ derivatives.

Animals↗
PubMed1996

The potential for reintroduction of tumor cells during intraoperative blood salvage: reduction of risk with use of the RC-400 leukocyte depletion filter.

OBJECTIVES: Intraoperative autotransfusion of shed blood is widely utilized in surgery. However, several studies have raised concern about the transmission of tumor cells during oncologic procedures. We compared the ability of a leukocyte depletion filter (RC-400; LDF) to a standard red blood cell filter (SBF) to remove tumor cells derived from urologic malignancies. METHODS: Cells were suspended in media and passed through a SBF or a LDF. The filtrate was evaluated for the presence of viable cells utilizing the trypan blue exclusion method as well as cell culture. In a second experiment, cells were suspended in fresh bovine blood and processed through a cell saver apparatus followed by filtration with either a SBF or a LDF. Aliquots were cultured after admixture with blood, after processing, and after filtration. RESULTS: The LDF was able to remove tumor cells completely, as demonstrated by both counting with the trypan blue exclusion test and by cell culture. In contrast, admixture with blood processing through the cell saver apparatus nor a standard red blood cell filter removed these cells. CONCLUSIONS: Tumor cells derived from urologic malignancies are easily removed with a LDF but not with a SBF. Filtration of blood salvaged at the time of uro-oncologic surgery with a LDF but not with a SBF reduces the potential for reinfusion of viable tumor cells.

Blood Transfusion, Autologous↗
PubMed1993

Anoxia-tolerant hepatocytes: model system for study of reversible metabolic suppression.

Chrysemys picta bellii is well known for its ability to survive extended anoxic periods and has been widely used as a model system to study anoxic metabolism. Described here is a method for the isolation of anoxia-tolerant hepatocytes from this species. Freshly isolated hepatocytes were determined to be viable based on trypan blue exclusion, gluconeogenic capacity from [14C]lactate, responsiveness to epinephrine and glucagon, and maintenance of cellular adenylate concentrations. Under anoxic conditions for 10 h there was no significant increase in cell staining and no decrease in cellular ATP concentration. Furthermore, the addition of cyanide at the 5-h mark did not result in any significant differences in these parameters; however, iodoacetate added at this time caused trypan blue staining to increase and ATP concentrations to fall. The rate of glucose production from the cells was threefold greater under anoxic than normoxic conditions, underscoring the important role of the liver in supplying substrate during anoxia. From the rate of O2 consumption and rate of lactate production under anaerobic conditions, ATP turnover rates were calculated to be 68.4 +/- 7.2 and 6.5 +/- 0.43 mumol ATP.g-1.h-1, respectively; this corresponds to a 90% decrease in metabolic rate during anoxia. Within a cellular system such as this the more complex regulatory mechanisms involved in a large coordinated reduction in metabolism can be probed.

Adaptation, Physiological↗