Changes In Cell Membrane Integrity Research Articles

CHMP3 promotes the progression of hepatocellular carcinoma by inhibiting caspase‑1‑dependent pyroptosis.

Charged multivesicular body protein3 (CHMP3) is an elemental constituent of the endosomal sorting complex required for transport (ESCRT)III, whose function as a tumor susceptibility gene in the development of liver cancer remains unclear. CHMP3 was found to be associated with pyroptosis by bioinformatics analysis of data from patients with hepatocellular carcinoma (HCC) in The Cancer Genome Atlas database. It was aimed to explore the role and potential mechanisms of CHMP3 in the development of liver cancer. The expression of CHMP3 at the tissue level was examined using immunohistochemistry and western blot analysis. Subsequently, HepG2 and Huh‑7cells were transfected with small interfering RNA and overexpression plasmids to change CHMP3 expression. The proliferative capacity of cells was examined using colony formation and Cell Counting Kit‑8 assays. Wound healing and Transwell assays were used to examine the migratory and invasive abilities of the cells. Transmission electron microscopy was used to observe changes in cell morphology. Western blotting was used to examine the expression of caspase‑1 signaling pathway related proteins, a classic pathway of pyroptosis. In addition, a xenograft tumor model was used to examine the tumorigenic ability of CHMP3 invivo. The results demonstrated that CHMP3 expression was upregulated in HCC and was associated with poor prognosis. Knockdown or overexpression of CHMP3 inhibited or promoted the proliferation, migration and invasion of liver cancer cells. Knockdown of Huh‑7 showed changes in cell membrane integrity as well as cytoplasmic leakage. Furthermore, knockdown of CHMP3 may activate the caspase‑1 pyroptosis signaling pathway which in turn inhibits the progression of liver cancer, and this effect can be reversed by the caspase‑1 inhibitor AYC. In conclusion, CHMP3 may affect the development of liver cancer through the caspase‑1‑mediated pyroptosis pathway.

Evaluation of the antibacterial mechanism and biofilm removal effect of eugenol on Vibrio vulnificus and its application in fresh oysters

Eugenol is a natural active substance with high antibacterial activity, but its antibacterial activity against Vibrio vulnificus has not been extensively studied. The purpose of this study was to investigate the antibacterial and biofilm-clearing abilities and potential mechanisms of eugenol against V. vulnificus, and to provide knowledge for the use of eugenol to prevent oyster contamination. It was found that eugenol had an encouraging antibacterial effect on V. vulnificus with a minimum inhibitory concentration (MIC) of 0.2 mg/mL. The accumulation of reactive oxygen species (ROS) and the increase of malondialdehyde (MDA) content suggest that oxidative stress is involved in the bactericidal mechanism. Moreover, cell membrane hyperpolarization, changes in cell membrane integrity and morphology suggest that eugenol can reduce the permeability and integrity of cell membranes in V. vulnificus. In addition, eugenol produced a significant biofilm clearance effect on V. vulnificus, as evidenced by the reduced amount of biofilm and the reduction of polysaccharides and viable cells in the biofilm. Finally, eugenol was able to effectively inhibit the activity of V. vulnificus in artificially contaminated oyster at 4 °C and 25 °C. But sensory analysis showed that 0.10% eugenol was most acceptable to trained panelists. All of these highlight the great promise of eugenol as a natural bacteriostatic agent for the food industry.

Dental resin monomers induce early and potent oxidative damage on human odontoblast-like cells

Resin-based dental materials consist of filler particles and different monomers that are light cured in situ to re-establish dental function and aesthetics. Due to the degree of conversion of adhesive polymers, the monomers triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are released in relatively high amounts and are susceptible to degradation, acting as bioactive compounds and affecting cell and tissues. This study aimed to assess the effect of HEMA and TEGDMA exposure on metabolic activity, membrane integrity, and cell survival of human odontoblast-like cell (hOLCs). Exposure to resin monomers for 24 h induced major changes in cell membrane integrity, metabolic activity, and survival, which were measured by the calcein method and lactate dehydrogenase release. Increased and early reactive oxygen species (ROS) production was observed leading to degradative oxidation of membrane lipids identified as malondialdehyde production. Severe alteration in mitochondria occurred due to transmembrane mitochondrial potential collapse, possibly inducing activation of apoptotic cell death. hOLCs exposure to resin monomers modified the cell redox potential, with consequences on membrane permeability and integrity, including mitochondrial function. Lipid peroxidation appears to be a key phenomenon for the membrane structures oxidation after HEMA and TEGDMA exposure, leading to cell death and cytotoxicity. hOLCs respond early by differential induction of adaptive mechanisms to maintain cell homeostasis. Modulation of oxidative stress-induced response involves the regulation of genes that encode for antioxidant proteins such as catalase and heme oxygenase-1; regulation that functions as a critical protection mechanism against oxidative cell damage induced by HEMA and TEGDMA. Ascorbic acid as an antioxidant substance mitigates the oxidative damage associated with exposure to monomers.

Influence of freezing temperature before freeze-drying on the viability of various Lactobacillus plantarum strains

Although freeze-drying is an excellent method for preserving microorganisms, it inevitably reduces cell activity and function. Moreover, probiotic strains differ in terms of their sensitivity to the freeze-drying process. Therefore, it is necessary to optimize the variables relevant to this process. The pre-freezing temperature is a critical parameter of the freeze-drying process, but it remains unclear whether the optimal pre-freezing temperature differs among strains and protectants. This study explored the effects of 4 different pre-freezing temperatures on the survival rates of different Lactobacillus plantarum strains after freeze-drying in the presence of different protectants. Using phosphate-buffered saline solution and sorbitol as protectants, pre-freezing at -196°C, -40°C, and -20°C ensured the highest survival rates after freeze-drying for AR113, AR307, and WCFS1, respectively. Using trehalose, pre-freezing at -20°C ensured the best survival rate for AR113, and -60°C was the best pre-freezing temperature for AR307 and WCFS1. These results indicate that the pre-freezing temperature can be changed to improve the survival rate of L. plantarum, and that this effect is strain-specific. Further studies have demonstrated that pre-freezing temperature affected viability via changes in cell membrane integrity, membrane permeability, and lactate dehydrogenase activity. In summary, pre-freezing temperature is a crucial factor in L. plantarum survival after freeze-drying, and the choice of pre-freezing temperature depends on the strain and the protectant.

Hyperthermic therapy using warm sterile water enhances cytocidal effects on bladder cancer cells

Objectives: To investigate whether warm sterile water enhances the cytocidal effect of hypotonic shock on bladder cancer cells that show resistance to sterile water.Methods: Four bladder cancer cell lines of varying grades (T24, RT4, J82, and RT112) were exposed to sterile water, and morphological changes were closely observed under microscopy. Changes in cell membrane integrity and cell viability after water exposure were measured to determine the effects of water-induced hypotonic shock. Additionally, the effects of warm sterile water were analyzed.Results: T24, RT4, and J82 cells started swelling immediately upon exposure to water, followed by rupture within five minutes. RT112 cells demonstrated limited hypotonic swelling with significantly less cell rupture after 10 min. The percentages of viable cells at 10 min were 1.6 ± 0.8%, 3.5 ± 3.5%, 5.0 ± 3.2%, and 22.0 ± 10.3% for T24, RT4, J82, and RT112, respectively. The percentage of viable cells with 48 °C sterile water at one minute was 0% for RT112 cells.Conclusions: These findings support the efficacy of sterile water against bladder cancer cells and reveal that warm sterile water enhances the cytocidal effects of hypotonic shock, potentially avoiding the need for radical surgery.

Antifungal activity of silver salts of Keggin-type heteropolyacids against Sporothrix spp.

Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats, and in humans, the symptoms are restricted to skin in immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts (Ag-HPA salts) such as Ag3[PW12O40], Ag6[SiW10V2O40], Ag4[SiW12O40] and Ag3[PMo12O40] and interactions with the antifungal drugs itraconazole (ITC), terbinafine (TBF) and amphotericin B (AMB) on the yeast and mycelia forms of Sporothrix spp. Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 μg/mL. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.

Miniature Pigs Carrying <i>DMD</i> Exon51 Deletion Suffered from Severe Myocardial Damage, Accelerated Muscle Atrophy and Malabsorption

Duchenne muscular dystrophy (DMD), inherited in an X-linked recessive manner, is a lethal disease affecting about 1/3600 males at birth. Mutations in the DMD gene have been shown to be the key determinant of the occurrence of DMD disease. Nowadays, removal of DMD exon 51 has been used as a common therapeutic intervention in disease animal models. Despite all these efforts, the clinical significance of DMD exon 51 has hardly been explored in large animals. Our study aimed to investigate the role of DMD exon 51 in the pathogenesis of DMD disease. We generated cell clones and miniature pigs lacking DMD exon 51 by CRISPR-Cas9 system. Significant changes in cell membrane integrity and apoptosis were found in genetically modified cell clones. All DMD pigs showed the accelerated pathological process of Duchenne muscular dystrophy. In addition, the ability of the small intestine of DMD pigs to digest and absorb nutrients was significantly reduced, compared to wild-type littermates. To conclude, this research could assist in our understanding of the pathological process of Duchenne muscular dystrophy. The findings of our investigation complemented those of earlier studies and might provide new insights into slow down the progression of this disease. Funding: This work was supported by grants from National Natural Science Foundation of China (No. 31572345 and 31472053), the Program for JLU Science and Technology Innovative Research Team (2017TD-28), the Fundamental Research Funds for the Central Universities, the Humanized IAPP gene preparation of human diabetes miniature swine model. Jilin Province Science and Technology Development Plan Project (No.20170623035TC) and the Program for Changjiang Scholars and Innovative Research Team in University in China (No. IRT16R32). Declaration of Interest: The authors declare no competing interest. Ethical Approval: All animal studies were approved by the Animal Welfare and Research Ethics Committee at Jilin University, and all procedures were conducted strictly in accordance with the Guide for the Care and Use of Laboratory Animals. All surgeries were performed under anesthesia, and every effort was made to minimize animal suffering.

A bioimpedance analysis of head-and-neck cancer patients undergoing radiotherapy.

Malnutrition is a frequent manifestation in patients with head-and-neck cancer undergoing radiation therapy and a major contributor to morbidity and mortality. Thus, body composition is an important component of an overall evaluation of nutrition in cancer patients. Malnutrition is characterized by weight loss, loss of muscle mass, changes in cell membrane integrity, and alterations in fluid balance. Bioelectrical impedance analysis is a method to analyze body composition and includes parameters such as intracellular water content, extracellular water content, and cell membrane integrity in the form of a phase angle (Φ). Bioelectrical impedance analysis has consistently been shown to have prognostic value with respect to mortality and morbidity in patients undergoing chemotherapy. The goal of the present study was to evaluate the relationship between Φ, time, intracellular water content, and weight for head-and-neck cancer patients undergoing radiotherapy. The results demonstrate that Φ decreases with time and increases with intracellular water content and weight.

Anticancer activity of some polyamine derivatives on human prostate and breast cancer cell lines.

The aim of this study was to expand our knowledge about anticancer activity of some polyamine derivatives with quinoline or chromane as terminal moieties. Tested compounds were evaluated in vitro towards metastatic human prostate adenocarcinoma (PC3), human carcinoma (DU145) and mammary gland adenocarcinoma (MCF7) cell lines. Cell viability was estimated on the basis of mitochondrial metabolic activity using water-soluble tetrazolium WST1 to establish effective concentrations of the tested compounds under experimental conditions. Cytotoxic potential of polyamine derivatives was determined by the measurement of lactate dehydrogenase activity released from damaged cells, changes in mitochondrial membrane potential, the cell cycle distribution analysis and apoptosis assay. It was revealed that the tested polyamine derivatives differed markedly in their antiproliferative activity. Bischromane derivative 5a exhibited a rather cytostatic than cytotoxic effect on the tested cells, whereas quinoline derivative 3a caused changes in cell membrane integrity, inhibited cell cycle progression, as well as induced apoptosis of prostate and breast cancer cells which suggest its potential application in cancer therapy.

Antimicrobial photodynamic therapy (aPDT) induction of biofilm matrix architectural and bioadhesive modifications

BackgroundDental implants are commonly used today for the treatment of partially and fully edentulous patients. Despite the high success rate they are not resistant to complications and failure due to a variety of problems including peri-implantitis or peri-mucositis due to bacterial biofilm formation on the implant surface. The use of non-surgical and surgical treatment procedure to promote healing in cases with peri-implantitis have limited efficacy. Here we studied the ability of photodynamic therapy to destroy a known bacterial pathogen and the extracellular matrix architecture of biofilm attached to titanium plates and germanium prisms. MethodsTitanium plates or germanium prisms were incubated for 24h with Fusobacterium nucleatum a fusiform, gram-negative bacterium was used to enable biofilm formation. Photodynamic therapy was carried out by incubating the biofilm samples on each substrata with porfimer sodium. Treatment was carried out using a diode laser at 630nm, 150mW/cm2 for light doses ranging from 25–100J/cm2. Evaluation of killing efficacy was done by counting colony forming units compared to controls. Multiple attenuated internal reflection-infrared spectroscopy (MAIR-IR) and SEM were used to analyze the samples pre and post PDT for validation. ResultsF. nucleatum was significantly reduced in a dose dependent manner by treatment with PDT. Changes in biofilm components and strength of bioadhesion were examined with MAIR-IR following jet impingement using calibrated water jets. SEM demonstrates significant morphological alterations in the bacteria, consistent with damage associated with exposure to reactive oxygen species. ConclusionThe results are indicative that aPDT is a method that can be used to eradicate micro-organisms associated with biofilm in peri-implantitis on relevant substrata. Data shows that the slime layer of the biofilm is removed and that further methods need to be employed to completely remove weakened or destroyed biofilm matrix components. Reactive oxygen species (ROS) mediated oxidative damage results in morphologic changes as a consequence of changes in cell membrane integrity.

Distinct patterns of expression of traumatic brain injury biomarkers after blast exposure: Role of compromised cell membrane integrity

Glial fibrillary acidic protein (GFAP), a protein enriched in astrocytes, and Tau, a protein abundant in neuronal microtubules, are being widely studied as biomarkers of brain injury, and persistent severity-dependent increases in brain and blood have been reported. Studies on the acute changes of these proteins after blast exposure are limited. Using a mouse model of closely-coupled repeated blast exposures, we have evaluated acute changes in the levels of GFAP and total Tau by Western blotting. Brain levels of GFAP and Tau proteins decreased significantly at 6h and increased considerably at 24h after repeated blast exposures. Plasma samples showed a similar initial decrease and later increase over this timeframe. This biphasic pattern points to possible absorption or sequestration of these proteins from plasma immediately after repeated blast exposures. Liver and spleen tissue showed significant increases in the levels of GFAP and Tau protein at 6 and 24h post-blast exposures whereas semi-quantitative RT-PCR analysis of liver showed no significant changes in the levels of GFAP or Tau mRNAs. These results suggest that blast exposure causes transient changes in cell membrane integrity in multiple organs leading to abnormal migration of proteins from the tissues to the plasma and vice versa. This transient changes in cell membrane permeability and subsequent bidirectional movement of molecules may contribute to the pathophysiology of TBI and polytrauma after blast exposure.

Trypanocidal activity of guaianolide obtained from Tanacetum parthenium (L.) Schultz-Bip. and its combinational effect with benznidazole

In the present study, we evaluated the in vitro antiprotozoal activity of a guaianolide (11,13-dehydrocompressanolide) isolated from Tanacetum parthenium against Trypanosoma cruzi and investigated the possible combinational effect of guaianolide and benznidazole. The isolated compound was shown to be effective against T. cruzi, with IC50 values of 18.1±0.8 and 66.6±1.3μM against the multiplicative epimastigote and amastigote forms, respectively. The best results were obtained against trypomastigotes, with an EC50 of 5.7±0.7μM. The guaianolide presented no toxicity in LLCMK2 cells (CC50 of 93.5μM) and was 16.4-fold more selective for trypomastigotes. The study of the combinational effect of benznidazole and guaianolide revealed the presence of a synergistic effect against the epimastigote form and marginal additive effect against the trypomastigote form. Striking morphological changes were observed in epimastigotes treated with guaianolide, such as thinning and stretching of the cell body and flagellum and changes in the format of the cell body with apparent leakage of the cytoplasmic content in trypomastigote forms. The ultrastructural analysis of epimastigotes revealed the presence of membranes that involved organelles and formation of myelin-like figures. Flow cytometry revealed a cell volume reduction and decrease in mitochondrial membrane potential. However, no major changes in cell membrane integrity were found in the epimastigote form treated with guaianolide.

Effect of Chronic Exposure to Aluminium on Isoform Expression and Activity of Rat (Na+/K+)ATPase

The ability of aluminum to inhibit the (Na(+)/K(+))ATPase activity has been observed by several investigators. The (Na(+)/K(+))ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both catalytic (alpha) and regulatory (beta) subunits. For instance, three main alpha (alpha(1), alpha(2) and alpha(3)) and three beta (beta(1), beta(2) and beta(3)) subunit isoforms exist in vertebrate nervous tissue, whereas only alpha(1) and beta(1) have been identified in kidney. However, no studies have focused on determining the change in (Na(+)/K(+))ATPase isoforms caused by chronic exposure to aluminum and its relation with aluminum toxicity. In this study, adult male Wistar rats were submitted to chronic dietary AlCl(3) exposure (0.03 g/day of AlCl(3) for 4 months), and the activity and protein expression of (Na(+)/K(+))ATPase isozymes were studied in brain cortex synaptosomes and in kidney homogenates. The intracellular levels of adenine nucleotides, plasma membrane integrity, and aluminum accumulation were also studied in brain synaptosomes. Aluminum accumulation upon chronic dietary AlCl(3) administration significantly decreased the (Na(+)/K(+))ATPase activity measured in the presence of nonlimiting Mg-ATP concentrations, without compromising protein expression of alpha-subunit isoforms in brain and kidney. Aluminum-induced synaptosomal (Na(+)/K(+))ATPase inhibition was due to a reduction in the activity of isozymes containing alpha(1)-alpha(2) and alpha(3)-subunits. The onset of enzyme inhibition was accompanied by a decrease of the (Na(+)/K(+))ATPase sensitivity to submicromolar concentrations of ouabain, and it preceded major damage in plasma membrane integrity and energy supply, as revealed by the analysis of lactate dehydrogenase leakage and endogenous adenine nucleotides. The data suggest that, during chronic dietary exposure to AlCl(3), brain (Na(+)/K(+))ATPase activity drops, even if no significant alterations of catalytic subunit protein expression, cellular energy depletion, and changes in cell membrane integrity are observed. Implications regarding underlying mechanisms of aluminum neurotoxicity are discussed.

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI6.25, NRI12.5 and NRI25). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI6.25) - more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH) - and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI12.5 values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50µg ml−1, although this effect was not significant at lindane concentrations <200µg ml−1. 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI6.25, whereas in the case of lindane, this increase was only significant at doses of NRI25 and above.

Cyclodiene organochlorine insecticide-induced alterations in the sulfur-redox cycle in CHO-K1 cells

The effect of the cyclodiene organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHO-K1 cultures at fractions of their lethal doses, determined by the neutral red (NRI) incorporation assay (NRI 6.25, NRI 12.5 and NRI 25). Glutathione peroxidase, reductase and S-transferase, and total and oxidised glutathione were evaluated along the standard growth curve of the cultures. After a 24-h incubation with each insecticide, glutathione peroxidase incurred a large increase, while glutathione reductase and S-transferase activities were slightly higher than untreated controls. Unlike oxidised glutathione, the content of total glutathione declined significantly after exposure to cyclodiene insecticides. Changes in cell membrane integrity were assessed by the lactate dehydrogenase (LDH) release assay and lipid peroxidation for a wide range of pesticide concentrations. Membrane leakage and peroxide production were significantly enhanced at concentrations of aldrin and as low as 12.5 μg/ml, whereas dieldrin and endosulfan increased membrane fragility at much higher concentrations.

Analysis of Free Intracellular Calcium by Flow Cytometry: Multiparameter and Pharmacologic Applications

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca2+ in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca2+ in surface-marker-defined target cells as well as correlation of changes in Ca2+ with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca2+ by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca2+ in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca2+ with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca2+ changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.

The effect of substrate methylation and of detergent on the reaction of pig liver glutathione S-transferase with linoleate hydroperoxides

glucose and an increase in I4C incorporation into lactate of about 100%. Specific activities of lactate and pyruvate are also increased by more than 100%. If we consider the sum of radioactivity in (lactate plus pyruvate plus glucose) as an indication of the total d.p.m. which have passed through the stage of PEP, the d.p.m. in (lactate plus pyruvate) expressed as a percentage of the total d.p.m. in (lactate plus pyruvate plus glucose) can be taken as an indication of the extent of ‘futile cycling’ at this level of the gluconeogenic pathway. The value obtained for non-treated cells was lo%, which is in the range previously reported by Grunnet & Katz (1978). In the presence of DEM or phorone this values increases to 24% and to 21% respectively. Thus GSH depletion induced with any of both xenobiotics results in similar effects on propionate metabolism of isolated rat hepatocytes. We recently observed an inhibition of gluconeogenesis induced by GSH depletion (Saez et al., 1985a,b). Here we show that this inhibition can be explained by an increase in the flux through pyruvate kinase. Membrane integrity and other indicators of metabolic competence, such as ATP levels, were not affected by GSH depletion. Thus GSH depletion by treatment with substrates of GSH S-transferase may have other metabolic effects. Indeed, Krack et al. ( 1 980) showed an increase in glycogen degradation of rat hepatocytes induced by 0.9 mM-DEM, without changes in cell membrane integrity. I t is worthwhile emphasizing that inhibition of gluconeogenesis and the increase in PEP recycling occurs only when GSH concentrations fall below a critical level ( I .5 pmol/g) in agreement with the glutathione threshold theory of Jollow (1 980).

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.