Changes In Membrane Integrity Research Articles

The Toxic Effects of Formate in Dissociated Primary Mouse Neural Cell Cultures

Primary dissociated mouse cerebrocortical cell cultures containing both neurons and glial cells were used as an experimental model to study the neurotoxic effects of formate, the putative toxic metabolite of methanol. Neural cells were isolated and prepared from the cerebral cortex of fetal CD-1 mice on Gestational Day 15. Mature 7- to 15-day-old monolayer cultures were exposed to formate (0 to 240 mM) for 8 hr at 37°C over a range of extracellular pH (6.0 to 7.6). Cytotoxicity was evaluated by histopathology, changes in membrane integrity (lactate dehydrogenase release, LDH; [ 14C]adenine nucleotide leakage), and mitochondrial metabolic activity [reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]. Similar quantitative estimates of cell injury were obtained by LDH release or [ 14C]adenine nucleotide leakage from prelabeled cells. Exposure of neural cells produced time- and concentration-dependent toxic responses. The concentration of formate that resulted in 50% LDH leakage after an 8-hr incubation was estimated to be 45 mM. As determined by light microscopy, formate (20 to 60 mM) was specifically neuronotoxic, primarily affecting large polygonal neurons. Higher concentrations of formate (≥120 mM) induced nonspecific cytotoxicity. MTT reduction appeared to be a more sensitive endpoint by showing significant toxic effects at 20 mM (8-hr incubation), while significant leakage of LDH occurred only at formate concentrations ≥60 mM. Total intracellular ATP concentration was significantly decreased following a 20 or 40 mM formate exposure for 8 hr. These results are consistent with the hypothesis that formate may inhibit mitochondrial function resulting in decreased intracellular ATP and formate-induced neurotoxicity.

Mechanisms of post-traumatic seizures: a quantum pharmacological analysis of the molecular properties of an epileptogenic focus following iron-induced membrane peroxidation

Late post-traumatic epilepsy following severe head trauma has been well documented. While there is increasing evidence suggesting that iron-induced lipid peroxidation of neural membranes may accompany cerebral haemorrhage, the pathogenic processes of post-traumatic epileptogenesis remain unknown. Furthermore, the effective prophylactic use of standard anticonvulsant drugs is unsubstantiated. The rational design of therapeutic agents specific for the prevention and treatment of post-traumatic epilepsy hinges on understanding the molecular membrane events at the epileptogenic focus. This study employs the techniques of theoretical quantum pharmacology to provide a structural analysis of neural phospholipid membranes, investigating changes in membrane integrity at the epileptogenic focus as the molecular basis for seizure activity. Molecular mechanics calculations and molecular dynamics simulations were used to model the biochemical events of the epileptogenic focus. We predict that applications of quantum pharmacological techniques to model biochemical events may provide an understanding of proconvulsive pathogenic mechanisms in post-traumatic epilepsy.

The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis?

DNA breaks can arise from many sources after incubation of cells with toxic agents. Very few agents break DNA directly, rather most breaks occur as a result of metabolic participation by the cell, such as during attempts to repair the damage. It is now realized that many DNA breaks arise as a consequence of steps in the pathway of cell death. Upon reanalyzing the methodology commonly used to detect DNA breaks, it is evident that many studies would not have observed DNA breaks associated with cell death. Frequently experimental conditions have been used that are extremely toxic to cells with the justification that the cells were still viable as measured by their ability to exclude dyes such as trypan blue. However, the DNA digestion associated with cell death by apoptosis occurs prior to changes in membrane integrity. Because the possibility of endogenous endonuclease activity was not realized, many studies may have inaccurately assumed that DNA breaks arose during, for example, inhibition of DNA repair or as intermediates in recombination. In light of the new understanding of apoptosis and the formation of DNA breaks as an early event in cell death, it is important to both reevaluate past conclusions and to ensure that future studies fully consider the breaks derived from the cytotoxicity of every agent under investigation.

Low‐Pressure Membrane Filtration for Removing Giardia and Microbial Indicators

As part of a project on the use of ultrafiltration (UF) for particle removal, studies were performed to evaluate the use of low‐pressure hollow‐fiber UF as an alternative for complying with Surface Water Treatment Rule (SWTR) requirements for microbial removal and/or inactivation. Pilot studies were conducted on four different untreated source waters, two from northern California and two from Boise, Idaho. Process efficacy was assessed by conducting MS2 virus, total coliform bacteria, and Giardia muris seeding studies, as well as monitoring for naturally occurring bacteria. The study showed that UF was capable of meeting SWTR requirements for alternative filtration technologies without the use of chemical disinfection. Four or more logs of Giardia and more than 6.5 logs of virus were removed from each of the untreated source waters. Differences in water quality or changes in operating parameters did not appear to affect removal capabilities of the process. Maintenance of membrane integrity was critical to assuring process efficacy. When module integrity was compromised, as in fiber breakage, both MS2 virus and G. muris were detected in the permeate. Changes in membrane integrity were not necessarily reflected by changes in permeate turbidity; however, particle counting was an effective method for detecting a compromised membrane module.

Semen characteristics of asymptomatic males affected byTrichomonas vaginalis

Trichomonas vaginalis is a sexually transmitted anaerobic parasite which causes vaginitis and cervicitis. The present study was carried out to determine the incidence of Trichomonas infection in semen samples of asymptomatic men and also to understand the changes of semen characteristics in the infected individuals before treatment and after treatment with metronidazole (Flagyl, 400 mg x 3x a day for 10 days). The semen obtained from 1131 men was examined for different characteristics and Trichomonas were detected in wet smear preparations in 50 cases (4.42%). The characteristics of semen in them was compared with 52 normal semen samples. Statistical analysis showed that the seminal fluid viscosity and percentage particulate debris was increased significantly in the infected group (P less than 0.001). There was no significant change in the pH of semen. Spermatozoan motility and morphologically normal forms were decreased significantly (P less than 0.001), spermatozoan viability was altered, and there was a significant change in membrane integrity (P less than 0.001) as indicated by the hypoosmotic swelling test. The significant improvement in semen characteristics were seen in 25 cases after a single course of treatment. Therefore, it is possible that in some cases, the infertility seen in asymptomatic individuals may be due to infection by Trichomonas.

Increased fragility of neuronal membranes with aging

Changes in membrane integrity have been implicated as a contributing factor in the aging process. We have examined the relative stability of synaptosomal membranes from young and aged mice, in response to chemically induced damage. The basal characteristics of the resting synaptosomes prepared from mice of either age were indistinguishable, as judged by intracellular levels of ionic calcium and by efflux of the fluorescent probe employed, fura-2. However, the response of isolated nerve endings to the presence of a neurotoxic agent, the insecticide chlordecone, was markedly different. Exposure to this chemical invariably elevated free calcium and increased the rate of leakage of fura-2 into the extracellular space. These effects were much more pronounced in preparations derived from aged animals. The implication of these findings is that neural membranes of aged animals are more susceptible to malfunction induced by xenobiotic agents and are less able to maintain homeostasis under such circumstances.

Changes in trichloroethylene-treated rat erythrocyte membranes in vitro.

Trichloroethylene (TCE) is a widely used organic solvent, the most important toxic effect of which is a narcotic central nervous (CNS) effect. In the present study we have used rat erythrocyte membranes as a nerve cell model for studying the changes in membrane integrity caused by TCE treatment. The parameters determined were osmotic resistance and the activities of acetylcholinesterase (AchE) and adenosinetriphosphatase (ATPase), both of which are integral membrane proteins. TCE had a dose-dependent effect on all these parameters. It increased the osmotic resistance at low concentrations and caused a decrease at high concentrations. Enzyme inhibition was only significant at high solvent concentrations. Decrease of temperature potentiated these effects. Our results indicate that changes in membrane proteins may be the initial factor leading to other changes in the membrane, e.g. increased osmotic resistance.

Mechanism of chemical-induced toxicity: I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K + and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.

The chemotherapy of rodent malaria, XXXIX

Ultrastructural changes were followed in Plasmodium berghei after the treatment of the mouse host with a single 10 mg kg-1 dose of artemisinine (qinghaosu). After 30 minutes, changes in the limiting and other membranes of the parasite were seen, together with alterations in ribosomal organization and endoplasmic reticulum. No changes were noted in digestive vacuoles or pigment, but nuclear membrane blebbing developed after one hour and segregation of the nucleoplasm after three hours. Further degenerative changes with disorganization and death occurred from eight hours onwards. The morphological changes in ribosomes and endoplasmic reticulum correlate in time with the depression in protein synthesis observed in P. falciparum in vitro. Similarly, the onset of nucleoplasmic segregation correlates with the development of nucleic acid synthesis inhibition. Tritiated reduced drug was shown to be localized in parasite membranes, indicating that changes in membrane integrity might precede the early depression of protein synthesis. Membrane association of artemisinine may be related to its amphipathic characteristics and similarity in some respects to a sterol.

Early membrane damage during coronary reperfusion in dogs. Detection by radiolabeled anticardiac myosin (Fab')2.

There is currently great interest in acute coronary reperfusion as a therapeutic modality for severe myocardial ischemia. While some studies have demonstrated a reduction in the overall extent of necrosis by early reperfusion, other studies have identified potentially deleterious effects produced by reflow. Because membrane disruption may be an important mechanism of irreversible cell injury, we measured changes in cell membrane integrity early during reperfusion using radiolabeled anticardiac myosin (Fab')2 antibody fragments in dogs. Our method involved brief periods of exposure to the (Fab')2 so that the levels of (Fab')2 binding indicated the degree of membrane disruption at discrete times during the progression of cell injury. In the first protocol (Fab')2 fragments labeled with either 125I and 131I were injected into the left circumflex coronary artery at the onset of reflow and at 45 min of reflow after a 1-h circumflex artery occlusion. Coronary sinus flow was diverted for 5 min following each injection to prevent recirculation. The (Fab')2 binding ratio (ischemic/control) increased during the first 45 min of reflow in each of eight experiments (mean increase 170%, P less than 0.01). No significant increase in (Fab')2 binding was observed in five additional experiments in which nonspecific (Fab')2 was injected. This indicates that the increase in binding seen with antimyosin-specific (Fab')2 was due to changes in specific binding rather than to alterations in (Fab')2 delivery produced by changes in blood flow distribution. The increase in membrane damage during reflow was confirmed by a second protocol in which each animal received only a single left atrial injection of (Fab')2 followed by rapid excision of the heart. The (Fab')2 binding ratio was 1.7 +/- 0.3 (SEM) in the group that received (Fab')2 at the onset of reflow and 3.7 +/- 0.6 (SEM) (P less than 0.05) in the group that received (Fab')2 after 45 min of reflow. In a third set of experiments in which hyperosmotic mannitol was infused during reflow the mean increase in (Fab')2 binding using the first protocol was only 80 +/- 40 vs. 170 +/- 30% without mannitol (P less than 0.05). Thus, membrane damage develops early during coronary reperfusion following 1 h of circumflex coronary artery occlusion, and part of this membrane damage can be prevented by altering the conditions of reflow. A method involving brief exposure of the myocardium to antimyosin (Fab')2 is promising for detecting changes in membrane integrity during evolving ischemic injury.

EFFECTS OF BORON DEFICIENCY ON MITOSIS AND INCORPORATION OF TRITIATED THYMIDINE INTO NUCLEI OF SUNFLOWER ROOT TIPS

Boron deprivation has multiple effects upon root growth within 6 hr after this essential micronutrient is withheld. Root elongation is inhibited and this response has been attributed to a cessation of mitosis and DNA synthesis. Our preliminary results using an autoradiographic analysis of sunflower roots labeled with [3H]‐thymidine demonstrated no difference in label distribution between +/‐B root tips. We found that mitosis in inhibited in ‐B roots but does not completely cease. Scintillation counting of whole root tips shows that boron‐deficient roots up to 72 hr of treatment incorporate radioactive label at a level comparable to that of the controls. Because mitosis and presumably DNA synthesis are affected by prolonged boron deficiency, these results may be brought about by a change in membrane integrity or permeability. We propose that effects of boron deprivation on DNA synthesis and mitosis in sunflower are secondary and that primary events involve alterations in cellular membranes.

Effects of Boron Deficiency on Mitosis and Incorporation of Tritiated Thymidine into Nuclei of Sunflower Root Tips

Boron deprivation has multiple effects upon root growth within 6 hr after this essential micronutrient is withheld. Root elongation is inhibited and this response has been attributed to a cessation of mitosis and DNA synthesis. Our preliminary results using an autoradiographic analysis of sunflower roots labeled with [3H]-thymidine demonstrated no difference in label distribution between +/-B root tips. We found that mitosis in inhibited in -B roots but does not completely cease. Scintillation counting of whole root tips shows that boron-deficient roots up to 72 hr of treatment incorporate radioactive label at a level comparable to that of the controls. Because mitosis and presumably DNA synthesis are affected by prolonged boron deficiency, these results may be brought about by a change in membrane integrity or permeability. We propose that effects of boron deprivation on DNA synthesis and mitosis in sunflower are secondary and that primary events involve alterations in cellular membranes.

Measurement of functional metabolic activity as a sensitive parameter of cytotoxicity in cultured hepatocytes

Although standard in vitro toxicity parameters, such as cell viability and enzyme leakage, are useful, it is also important to develop sensitive indices of toxicity which evaluate cell injury other than by measuring changes in membrane integrity. We describe two early, sensitive indices of cytotoxicity involving differentiated liver function: maintenance of cellular urea levels and lactate to pyruvate ratio.

Solute Leakage Resulting from Leaf Desiccation

The leakage of solutes from foliar tissue is utilized as a dynamic measure of apparent changes in membrane integrity in response to desiccation. It is found that rehydrating leaf discs of cowpea (Vigna sinensis [L.] Endl.) show increasing leakiness in proportion to the extent of prior desiccation, whereas Selaginella lepidophylla Spring., a resurrection plant, does not. The elevated leakage rate of cowpea after desiccation recovers with time, and the passage of time in the stressed condition results in reduced subsequent leakiness. These characteristics are interpreted as suggesting that the leakage of solute reflects the condition of cellular membranes, and that desiccation stress leads to lesions in the membranes. The kinetics of solute leakage is suggested as a simple means of following changes in membrane lesions and associated features of membrane repair and hardening.

Alterations in Integrity of Goldfish Membrane Induced by Edetate Disodium

The effect of the chelating agent edetate disodium on the integrity of the goldfish membrane was examined. The time to produce death in goldfish exposed to secobarbital sodium was used as a reflection of membrane integrity. Although a minimum edetate disodium concentration was necessary to induce alterations in integrity, no direct relationship between the effect and concentration of the chelating agent was evident. The chelating agent's effect appeared to be an enhancement of the transport of the ionized drug form. The change in membrane integrity existed at least 24 hr after theoretical exposure to edetate disodium, but cyclic alterations in integrity could not be ruled out. The effect on integrity was also demonstrated to be nonpermanent, and the apparent loss in integrity was partially restored by calcium but not by magnesium.

Chapter 21 Studies of Isolated Secretion Granules of the Rabbit Parotid Gland: Ion-Induced Changes in Membrane Integrity

Publisher Summary This chapter discusses the possible involvement of phospholipase activities in membrane–membrane interactions. The tissue used for the experiment reported in the chapter was the parotid salivary gland of the rabbit. The parotid salivary gland of the rabbit comprises a homogeneous population of secretory cells, each containing numerous apically located secretion granules (diameter 1.0–1.2 μm) that collectively occupy ∼35% of the cellular volume. In the experiment, secretion granules were isolated and then the purified fractions were obtained after discontinuous sucrose gradient centrifugation in 2 M sucrose buffered with 40 mM potassium phosphate containing 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.2. The sucrose concentration was subsequently reduced to 0.4 M by the continuous addition (for ≥1 hour at 4°C using a peristaltic pump) of 0.25 M buffered sucrose. The diluted suspension was subjected to low-speed centrifugation at 2,000gav for 25 minutes, and the pelleted granules were gently resuspended for further studies in fresh 0.4 M buffered sucrose. The intactness of the granule suspension at this point was determined by measuring the amount (expressed as percentage of total) of α-amylase, a secretory protein normally packaged in the granule content, which is sedimentable during centrifugation of ≤60 μL suspension for 1.5 minutes at 8,000gav (Eppendorf 3,200 centrifuge). The latter conditions ensure complete granule sedimentation, and suspensions were found to be 82–92% intact at this stage.

Dose-dependent toxicity of CCl 4 in isolated rat hepatocytes and the effects of hepatoprotective treatments

The temporal and dose-response relationships of the effects of CCl 4 were studied in isolated rat hepatocytes. CCl 4 decreased cell membrane integrity, as measured by the loss of alanine aminotransferase to the medium and the ability to retain intracellular K +. CCl 4 altered metabolic activity, as measured by the rate of urea synthesis and the lactate/pyruvate ratio. Addition of the following chemicals—SKF-525A, promethazine, dibenamine (0.1 m m); reduced glutathione (0.25 m m); hexobarbital methionine, cysteine (1 m m)—to the medium did not protect against the toxic effects of CCl 4, although these chemicals have been reported to be hepatoprotective in other experimental models. Incubation at 4°C did protect against CCl 4-induced changes in membrane integrity. These findings suggest that the isolated hepatocyte preparation is a suitable model for demonstrating time and dose-dependent toxic effects. However, they also suggest that there may be differences in mechanism compared with other experimental models.

Membrane Lipids in Senescing Flower Tissue of Ipomoea tricolor

Rib segments excised from flower buds of Ipomoea tricolor Cav. pass through the same phases of senescence as the respective tissue on the intact plant. Such segments were used to correlate changes in lipid content with known symptoms of aging, such as rolling up of the ribs and ethylene formation. It was found that the level of phospholipid had already started to decline before visible signs of senescence were evident. As the segments began to roll up and to produce ethylene, the rate of phospholipid loss accelerated sharply. During the same period, the level of fatty acids esterified to phospholipids also fell by 40%. No qualitative changes in any lipid component could be detected during senescence. Labeling experiments using (33)P as marker showed that the rate at which radioactivity was lost from phospholipids during aging was parallel to the rate at which the level of total phospholipids declined. Exogenously applied ethylene accelerated the loss of phospholipid and the senescence of rib segments while benzyladenine retarded both of these processes.Ag(+), which counteracts the effect of ethylene in many plants, inhibited rolling up of the rib segments but did not affect either spontaneous and ethylene-induced ethylene generation, or phospholipid loss. In contrast, Co(2+), a purported inhibitor of ethylene synthesis, reduced ethylene production, rolling up, and phospholipid loss. The inhibition of ethylene-induced rolling up by Co(2+) could not be overcome with exogenous ethylene, however.Our results indicate that phospholipid loss is a marker for membrane degradation in rib segments. Changes in membrane integrity and in cellular compartmentation may be the basis for ethylene synthesis during aging of flower tissue.

Structure and activity of chloroplasts of sunflower leaves having various water potentials

Changes in membrane integrity, conformation and configuration, and in photosystem II (PS II) activity (measured as dichloroindophenol photoreduction) of sunflower (Helianthus annuus L.) chloroplasts were studied after leaf tissue had been desiccated to various water potentials (ψ w ). Fixatives for electron microscopy were adjusted osmotically to within 1 bar of the ψ w of the tissue to prevent rehydration during fixation. PS II activity decreased to 50% of the control activity at a ψ w of-26 bar. At this ψ w , leaf viability was being lost but there was virtually no loss of integrity of the thylakoid lamellar system. Even at extreme ψ w (below-100 bar), thylakoids retained much structural detail but were less stained. At-26 bar, intrathylakoid spacing (configuration) and lamellar thickness (conformation) were decreased in vivo. Upon isolation of the plastids, the differences in configuration disappeared but the differences in conformation remained. The decreases in membrane conformation and PS II activity both, in vivo and in vitro suggest that alterations in conformation may cause decreases in chloroplast activity at ψ w as low as-26 bar. Since structural detail was maintained, however, previous observations of altered membrane integrity, which involved tissue fixed without osmotic support, may have been affected by tissue rehydration during fixation.

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.