Changes In Membrane Integrity Research Articles

Propranolol cytotoxicity in rat and human lung in vitro.

Propranolol is a lipophilic nonselective beta blocker mainly eliminated via the liver. The specific architectural arrangement of the mammalian lung favors the filtration of so-called pneumophilic drugs out of the blood and retention within the tissue, as shown in particular for amphetamine, amiodarone, imipramine, chlorpromazine, propranolol and local anesthetics. In the current study we tested in vitro the susceptibility of freshly isolated rat type II pneumocytes (RTII), rat alveolar macrophages (RAM), human alveolar macrophages (HAM) and A549 human lung adenocarcinoma cell line (A549) to propranolol (0.001-1 mM). Cytotoxicity was evaluated by changes in membrane integrity (lactate dehydrogenase [LDH] assay) and mitochondrial metabolic activity (reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) after 3-h and 20-h incubation with propranolol. In all tested lung cells, propranolol caused concentration-dependent decreases of MTT reduction and LDH retention in the fraction of attached cells, which was associated with an increase of LDH activity in the medium and in the fraction of non-attached cells. After 3-h incubation, the reduction of MTT was significantly decreased compared with control at > or = 1 x 10(-4) M in HAM, at > or = 5 x 10(-4) M in A549 and significantly decreased of LDH retention in the fraction of attached cells in HAM and in A549 at > or = 5 x 10(-4) M. After 20-h incubation the reduction of MTT was significantly decreased compared with control at > or = 1 x 10(-6) M in RAM, at > or = 5 x 10(-5) M in RTII and HAM and > or = 5 x 10(-4) M in A549. Propranolol caused a significant decrease of LDH retention in the fraction of attached cells (> or = 5 x 10(-5) M, RAM and RTII; > or = 5 x 10(-4) M, HAM and A549). The cytotoxic effect of propranolol in HAM and A549 was more pronounced after prolongation of incubation time (from 3 h to 20 h). These results showed that rat and human lung cells were sensitive to propranolol concentration < or = 1 mM in vitro. We suppose that the damaging action of propranolol on the lungs might be mediated by physicochemical properties of moiety and its pulmonary metabolites.

Modulation of nonspecific binding in ultrafiltration protein binding studies.

The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units. UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 microM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37 degrees C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC. Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pre-treatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED. The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.

Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

Assessing pathogen removal efficiency of microfiltration by monitoring membrane integrity

This study was conducted to investigate the ability of various methods of monitoring membrane integrity to respond to changes in actual membrane integrity imposed by the compromised fibers within the microfiltration unit. In addition, the pilot-scale MF unit was challenged with high concentrations of coliform, Cryptosporidium, and spore, in order to assess the pathogen removal capability of microfiltration. A correlation between the integrity tests and microbial challenge data was also made. The integrity tests investigated in this study were pressure decay and diffusive air flow tests (direct integrity tests), and turbidity and particle counting (indirect integrity tests). Both pressure decay (PDT) and diffusive air flow (DAF) tests were sensitive enough to detect one damaged fiber out of 66,000. The extent of fouling did not affect the sensitivity of the PDT and DAF, showing that PDT and DAF tests are a simple, reliable means to monitor membrane integrity under field conditions. Indirect integrity monitoring using turbidity and particle counting, however, responded poorly to changes in membrane integrity. Microbial challenge study demonstrated that microfiltration was capable of removing various pathogens including Cryptosporidium, at the level required by drinking water regulations, under even adverse operating conditions. Finally, PDT and DAF tests showed a better correlation with actual microbial removal efficiency of microfiltration than turbidity and particle counting. The turbidity and particle counting grossly underestimated the removal of pathogen larger than MF membrane pore size due to poor sensitivity.

Semen treatment with progesterone and/or acetyl-L-carnitine does not improve sperm motility or membrane damage after cryopreservation-thawing

Objective: To assess the effects of progesterone and acetyl-L-carnitine used before semen cryopreservation-thawing on sperm motility parameters and plasma membrane integrity. Design: Prospective cohort study. Setting: Academic tertiary center. Patient(s): Subfertile men undergoing semen evaluation. Intervention(s): Before cryopreservation, spermatozoa were incubated with water-soluble progesterone (1 and 10 μM), acetyl-L-carnitine (2.5, 5, 10, and 20 mM), or both (progesterone, 1 μM; and acetyl-L-carnitine, 5 mM). Main Outcome Measure(s): Postthaw change of motility parameters (computer-assisted measurements) and vitality-membrane integrity (examined with eosin-Y staining and annexin V-Cy3 binding assay). Result(s): There were no statistically significant differences between control samples and samples treated with progesterone and/or acetyl-L-carnitine for cryosurvival rate, motility parameters, or membrane integrity. The percentages of postthaw cells identified as live showed significantly different results with use of the eosin-Y staining and annexin V binding assay. Conclusion(s): Neither progesterone nor acetyl-L-carnitine seemed to prevent cryodamage assessed by motility changes or membrane integrity in human spermatozoa of subfertile men. Annexin V binding, a reflection of membrane translocation of phosphatidylserine, provided more distinct information about postfreezing membrane integrity changes than eosin-Y staining.

Programmed cell death or desiccation tolerance: two possible routes for wheat endosperm cells

AbstractThe fate of cells in the endosperm of developing wheat kernels was investigated under normal conditions and upon premature slow drying on the cut ear. To follow the changes in membrane integrity and cellular ultrastructure, an electron spin resonance (ESR) spin probe technique and low temperature scanning electron microscopy (LTSEM) were used. ESR data indicated that during development, the relative amount of cells with intact membranes decreased and became almost zero at the stage of mass maturity, i.e. several days before the onset of maturation drying. This suggests that the death of starchy endosperm cells is a developmental phenomenon rather than one induced by water loss. Even at 8 days after anthesis (daa), early in the differentiation phase, a considerable proportion of endosperm cells had already lost plasma membrane integrity. Comparison of ESR data with LTSEM micrographs revealed that the loss of plasma membrane integrity occurred early in the starch accumulation process, from which it was concluded that the programmes of starch accumulation and developmental death are simultaneously switched on. When the differentiation into starchy endosperm was arrested by premature slow drying of kernels on the cut ear, meristematic cells (aleurone initials) acquired desiccation tolerance as assessed by the combination of the spin probe technique and LTSEM. In contrast, fast drying caused immediate death of these cells. Thus, meristematic endosperm cells have the competence to acquire desiccation tolerance. This occurs upon premature slow drying or during normal differentiation into aleurone cells, but is lost when the cells differentiate into starchy endosperm.

Paracetamol (acetaminophen) cytotoxicity in rat type II pneumocytes and alveolar macrophages In Vitro

Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro toxicity of APAP and its effect on the intracellular GSH level in rat type II pneumocytes (freshly isolated or 24-hr-old) and alveolar macrophages. Cytotoxicity was evaluated by changes in membrane integrity (lactate dehydrogenase, [LDH] assay) as well as by mitochondrial metabolic activity (reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT), following a 20-hr incubation with APAP (1–20 mM). APAP caused a concentration-related decrease in MTT reduction and LDH retention in the fraction of attached cells, which was associated with an increase in LDH activity in the medium and in the fraction of non-attached cells. The order of susceptibility was: freshly isolated type II pneumocytes > alveolar macrophages > 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (≤ 1 mM) APAP concentrations. In 24-hr-old type II pneumocytes, there were no changes in intracellular GSH concentration after APAP exposure. Potassium ethyl xanthate (a P450 inhibitor) and indomethacin (a PGHS inhibitor) significantly decreased APAP-induced GSH depletion in freshly isolated type II pneumocytes and alveolar macrophages, suggesting that P450 and/or PGHS are involved in APAP bioactivation in these cells.

The Mitochondrion in Cell Death Control: Certainties and Incognita

Apoptosis research has recently experienced a change from a paradigm in which the nucleus determined the apoptotic process to a paradigm in which caspases and, more recently, mitochondria constitute the center of death control. Mitochondria undergo major changes in membrane integrity before classical signs of cell death become manifest. These changes concern both the inner and the outer mitochondrial membranes, leading to the dissipation of the inner transmembrane potential (ΔΨm) and/or the release of intermembrane proteins through the outer membrane. An ever-increasing number of endogenous, viral, or xenogeneic effectors directly act on mitochondria to trigger permeabilization. At least in some cases, this is achieved by a direct action on the permeability transition pore complex (PTPC), a multiprotein ensemble containing proteins from both mitochondrial membranes, which interact with pro- and antiapoptotic members of the Bcl-2 family. At present, it is elusive whether opening of the PTPC is the only physiological mechanism leading to mitochondrial membrane permeabilization. Proteins released from mitochondria during apoptosis include caspases (mainly caspases 2, 3, and 9), caspase activators (cytochrome c, hsp 10), as well as a caspase-independent death effector, AIF (apoptosis inducing factor). The functional hierarchy among these proteins and their actual impact on the decision between death and life is elusive.

Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection.

Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 micrometer according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.

Mitochondria as regulators of apoptosis: doubt no more

Scientific revolution [ 1] implies a transformation of the world view in which a dominant paradigm is substituted by a new one, one which furnishes an ameliorated comprehension of facts, as well as an advantage for the design of informative experiments. Apoptosis research has recently experienced a change from a paradigm in which the nucleus determined the apoptotic process to a paradigm in which mitochondria constitute the center of death control. Several pieces of evidence imply mitochondria in the process of apoptosis. Kinetic data indicate that mitochondria undergo major changes in membrane integrity before classical signs of apoptosis become manifest. These changes concern both the inner and the outer mitochondrial membranes, leading to a disruption of the inner transmembrane potential (ΔΨ m) and the release of intermembrane proteins through the outer membrane. Cell-free systems of apoptosis demonstrate that mitochondrial products are rate limiting for the activation of caspases and endonucleases in cell extracts. Functional studies indicate that drug-enforced opening or closing of the mitochondrial megachannel (also called permeability transition pore) can induce or prevent apoptosis. The anti-apoptotic oncoprotein Bcl-2 acts on mitochondria to stabilize membrane integrity and to prevent opening of the megachannel. These observations are compatible with a three-step model of apoptosis: a premitochondrial phase during which signal transduction cascades or damage pathways are activated; a mitochondrial phase, during which mitochondrial membrane function is lost; and a post-mitochondrial phase, during which proteins released from mitochondria cause the activation of catabolic proteases and nucleases. The implication of mitochondria in apoptosis has important consequences for the understanding of the normal physiology of apoptosis, its deregulation in cancer and degenerative diseases, and the development of novel cytotoxic and cytoprotective drugs.

Effects of the pharmaceutical cosolvent hydroxypropyl-beta-cyclodextrin on porcine corneal endothelium.

The influence of the pharmaceutical cosolvent hydroxypropyl-beta-cyclodextrin (HPBCD) on porcine corneal endothelium was investigated. The purpose was to find out if this substance causes severe damage to the cornea. METHODS; One hundred and ninety-five pig corneas were preserved in Eagle's minimal essential medium with dextran and HPBCD. They were stained with trypan blue, examined using a light microscope and then reincubated. Changes in cell density and cell morphology as a function of the duration of preservation and the HPBCD concentration were evaluated. We developed a morphological classification combining the morphological aspects observed using the light microscope and the scanning electron microscope. The vitality of the endothelium was analyzed by cell separation and monolayer cultivation. The cell density stayed stable without significant alterations in 0.1% HPBCD, 1% HPBDC and control solutions. In 10% HPBDC, however, the endothelium showed significant loss of cells. The morphological classification revealed high-grade endothelial damage in 10% HPBDC and low-grade damage in 1% HPBCD. The changes observed in 0.1% HPBCD and control medium were comparable. The degree of alteration conformed to the results of monolayer cultivation: endothelial cells of damaged corneoscleral buttons were limited in their ability to proliferate. Severe endothelial destruction in 10% HPBCD and changes in membrane integrity at lower concentrations limit the use of HPBCD in ophthalmic solutions.

Predicting log removal performance of membrane systems using in-situ integrity testing

Two of the most sensitive methods currently available for monitoring membrane integrity are the pressure decay test (PDT) and diffusive air flow (DAF) tests. Both these tests are capable of monitoring small changes in membrane integrity, even in very large membrane arrays. The DAF test, for example, is capable of detecting a single broken fibre in a membrane array containing over one million fibres. This enables operators to control membrane integrity at levels well above 4 log removal of Giardia or Cryptosporidium, without relying on filtered water quality monitors such as turbidity or particle counters. The tests form part of an overall membrane control strategy known as the Memsure™ process, involving process integrity monitoring, and identification and isolation of faulty modules for later repair.

Superoxide Disrnutase Does Protect the Cdtured Rat Cardiac Myocytes Against Hypoxia/Reoxygenation Injury

The effect of superoxide dismutase (SOD) on membrane integrity and fluidity of the cultured neonatal rat cardiac myocytes in vitro was investigated under the condition of hypoxia and hypoxia/reoxygenation. Lactate dehydrogenase (LDH) concentration was used as the biochemical indicator for the loss of cell membrane integrity. Fluorescence polarization (FP), average microviscosity (N) and anisotropy (Ast), which are inversely proportional to the fluidity of cell membrane, were assayed. Cells were respectively exposed to hypoxia or hypoxia/reoxygenation for different periods of time in the absence or presence of SOD at various concentrations. Hypoxia alone or hypoxia/ reoxygenation brought injury to the cultured myocytes. This was demonstrated by changes in LDH and membrane fluidity. In the former LDH concentration gradually increased in a time-dependent manner and the values of FP, N and Asf were significantly increased. The changes in membrane integrity and fluidity induced by hypoxia or hypoxia/reoxygenation could be prevented by adding SOD to the culture medium. The results provide a direct evidence that SOD (740 u.ml-1, the effective dose) was effective in protecting cultured myocytes against the injury as well as an indirect evidence of free radical generation. Based on the results obtained from this study and the establishment of concept of optimally effective dose by Bernier and Omar et al, it was suggested that some previous reports, in which no evidence was found both in protective effect of SOD and in free radical generation by using only one dose in hypoxia/reoxygenation model, should be interpreted with caution.

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 ± 1.6 to 34.5 ± 1.9% (P < 0.05) and membrane integrity from 27.0 ± 2.1 to 34.3 ± 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 ± 1.6 to 30.0 ± 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol.

A systematic approach was taken to assess and optimize a protocol for intracellular vitrification by introducing high concentrations of the cryoprotectant agent (CPA) ethylene glycol (EG) into unfertilized murine oocytes. The effects of EG on membrane integrity, microfilament organization, and developmental potential were evaluated. During exposure to 0.5-2 M EG, oocytes showed maximum shrinkage to 55.5% of the isotonic volume within the first minute and reexpanded to their initial volume within 15 min. Transferral of oocytes to higher concentrations of EG (4-8 M EG) for 1-5 min after 15 min of equilibration at 2 M EG was tolerated well. Microfilament organization appeared normal after this equilibration period. During prolonged exposure (> 5 min) to high concentrations of EG (> 4 M), membrane blebs were noticed on the surface of the cells, and microfilament distribution was disturbed. After treatment with 6 M EG and vitrification with 6 M EG + f2p40.5 M sucrose, there were no significant differences in development to the two-cell and blastocyst stages between CPA-treated, vitrified, and control oocytes. These results indicate that EG is an effective CPA for mouse oocyte vitrification protocols without any observed compromise in morphology and developmental functions.

Apoptosis as a mechanism of cell death induced by different chemotherapeutic drugs in human leukemic T-lymphocytes

The involvement of apoptosis in the mechanism of cell death induced by six clinically relevant anticancer drugs [methotrexate (MTX), doxorubicin (ADR), daunorubicin (DNR), vincristine (VCR), 6-mercaptopurine (6MP), and prednisolone (PRD)] in human leukemic T-lymphocytes (CCRF-CEM and Jurkat) was investigated by analysing changes in cell size and morphology, changes in membrane integrity, alterations in [Ca 2+] i and induction of DNA fragmentation. MTX, ADR, and DNR showed pronounced dose- and time-dependent cytotoxic effects on both cell lines, whereas cell viability was not considerably reduced by 6MP or PRD. On the other hand, the cytotoxic activity of VCR was much higher on Jurkat cells than on CEM cells. With the exception of 6MP and PRD, all the other compounds induced extensive chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation of apoptotic bodies and fragmentation of DNA in both cell lines. Occurrence of DNA fragmentation always preceded loss of membrane integrity. These observations are consistent with cell death being mediated by apoptosis. Significant increases in [Ca 2+] i were only observed in CEM cells preincubated with MTX or DNR (10 μM). In contrast, MTX as well as VCR induced a reduction in the basal intracellular Ca 2+concentration in Jurkat T-cells. Although the ability to induce changes in [Ca 2+] i correlated with higher cytotoxic potency of the anticancer drugs, a causal relationship between increased [Ca 2+] iand induction of apoptosis could not be clearly established. These results, therefore, suggest no determinant role for Ca 2+ in triggering the process of endonucleolytic cleavage of genomic DNA in these leukemic T-lymphocytes.

Effects of saponins and glycoalkaloids on the permeability and viability of mammalian intestinal cells and on the integrity of tissue preparations in vitro

The effects of potato and tomato glycoalkaloids and a saponin mixture from Gypsophila were investigated in cytotoxicity studies (neutral red uptake, mitochondrial MTT reduction and release of lactate dehydrogenase), using cultured cell lines of rat and human intestinal mucosal epithelium. Experiments to assess the effects of these compounds on the integrity of the intestinal epithelium were also carried out using preparations of isolated rat jejunum in vitro. By investigating the effect of these compounds on cultured cells and on intestinal tissue preparations, changes in membrane integrity, as evidenced by lactate dehydrogenase leakage in cell culture, could be confirmed in a system more relevant to the whole gut. Of the compounds tested, α-tomatine was consistently the most potent in all tests, and indications of a synergistic effect on membrane depolarization were observed between α-chaconine and α-solanine at total glycoalkaloid concentrations of less than 1 mM (< 0.86 mg/ml), with an optimum when the former comprised 25% of the mixture. An increase in the apparent permeability of the brush border was observed at sublethal concentrations of the compounds, and this may have important implications with respect to enhanced uptake of macromolecules, such as allergens, whose passage through the epithelium is normally somewhat restricted.

Metabolism and Cytotoxicity of Aflatoxin B1 in Cultured Rat Hepatocytes and Nonparenchymal Cells: Implications for Tumorigenesis

Liver cells, isolated from male Sprague-Dawley rats and established in primary culture, were treated with a dose of 180 pmol aflatoxin B1 (AFB1) for 2 hr. Reticuloendothelial nonparenchymal cells (NPC) metabolized a total of 28% of the AFB1. In contrast, parenchymal epithelial cells or hepatocytes (HC) were highly efficient in conjugating AFB1 to water-soluble products (50-70% of the dose) but also bound a large proportion (27%) of the toxin to cellular macromolecules. in comparison to NPC, HC bound 90-fold more AFB1 per cell. Cytotoxicity in primary cultures was evaluated by changes in membrane integrity (lactate dehydrogenase release), inhibition of glutathione-S-transferase activity and cell monolayer morphology over a range of AFB1 doses. No response was detected in NPC, whereas HC exhibited dose-related cytotoxic responses to AFB1. In rat liver both cell types form AFB1-DNA adducts in a dose-dependent fashion and yet only epithelial cell carcinomas have been observed in whole animal studies. The possibility that cell-specific AFB1 cytotoxicity produces a promotional stimulus for hepatocellular carcinoma is discussed.

Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells.

The goal of this investigation was to determine if human natural killer (NK) cells were susceptible to the cytolytic effects of the Actinobacillus actinomycetemcomitans leukotoxin (LTX). Following treatment with LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was evaluated. LTX inhibited the IL-2-induced expression of both CD69 and the IL-2 receptor. Furthermore, the up-regulation of CD56 was also impaired. To determine whether the observed functional deficits were the result of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with cell death were observed within 60 min. Direct analysis of cell viability by measuring propidium iodide exclusion, however, indicated little change in the viability of LTX-treated NK cells. Electron microscopic analysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condensation of nucleoplasm. However, no change in membrane integrity was initially noted. Finally, LTX caused a rapid and sustained elevation in the intracellular levels of Ca2+. These morphological and biochemical changes are consistent with the notion of programmed cell death.