The breakdown of cell membranes by electrical and mechanical stress.

We attempted to determine whether mechanical tension and electrical stress couple to cause membrane breakdown in cells. Using cell-attached patches from HEK293 cells, we estimated the mechanically produced tension from the applied pressure and geometry of the patch. Voltage pulses of increasing amplitude were applied until we observed a sudden increase in conductance and capacitance. For pulses of 50 micros duration, breakdown required >0.5 V and was dependent on the tension. For pulses of 50-100 ms duration, breakdown required 0.2-0.4 V and was independent of tension. Apparently two physically different processes can lead to membrane breakdown. We could explain the response to the short, high-voltage pulses if breakdown occurred in the lipid bilayer. The critical electromechanical energy per unit area for breakdown by short pulses was approximately 4 dyne/cm, in agreement with earlier results on bilayers. Our data suggest that, at least in a patch, the bilayer may hold a significant fraction (approximately 40%) of the mean tension. To be compatible with the large, nonlytic area changes of patches, the bilayer appears to be pulled toward the pipette tip, perhaps by hydrophobic forces wetting membrane proteins bound to the glass. Although breakdown voltages for long pulses were in agreement with earlier work on algae, the mechanism(s) for this breakdown remain unclear.     

Platinum complex toxicity in cultured renal epithelia.

BACKGROUND: Cisplatin is a potent antitumor drug but its clinical use is limited by nephrotoxic side effects. We have found recently, that nephrotoxicity of platinum complexes is related to basolateral organic cation transport. In this study, effects of cell culture conditions on platinum complex toxicity and organic cation transport were investigated by an in vitro system that utilizes the high TransEpithelial Electrical Resistance (TEER) of the C7-clone of the MDCK-(Madin-Darby-Canine-Kidney) cells. METHODS: TEER and caspase-3 activity of cells in microfilter membrane cups were measured after exposure of apical or basolateral membranes to 100 microM cis-, oxali-, or carboplatin. Caspase-3 activity after platinum complex exposure and uptake of the cation ASP+ (4-(4-(diethylamino)styryl)-N-methylpyridinium) of cells on filter membranes and impermeable supports (e.g. culture flasks) were compared. Atomic Force Microscopy (AFM) was used to depict morphometric differences between both culture conditions. RESULTS: In cells on filter membranes, cis-, oxali- and carboplatin induced loss of epithelial monolayer integrity by apoptosis via activation of caspase-3 to different extents. Basolateral application of platinum complexes enhanced toxicity dramatically and uptake of ASP+ from the basolateral side was higher than from the apical medium compartment. Intracellular accumulation of ASP+ was less distinct in cells grown on impermeable supports. Only cisplatin, the most lipophilic investigated complex, induced activation of caspase-3 in these cells. AFM disclosed more prominent cell-cell contacts in cells grown on filter membranes. CONCLUSION: We conclude that toxicity of hydrophilic substances can be underestimated in cells grown on solid supports, if basolateral transport mechanisms are involved. We suggest that unhindered access to basolateral transporters is responsible for higher levels of organic cation uptake and apoptosis in cells on filter membranes, even though more prominent cell-cell contacts indicate a better barrier function.     

Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices

Background

In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.

Methodology/Findings

This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.

Conclusions/Significance

The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.     

A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.

BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures. RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01). CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.     

Antinociceptive pattern of flavone and its mechanism as tested by formalin assay

Flavone, dextrose and long swim stress exhibited antinociception. Degree of antinociception was greater with long swim stress as compared to flavone or dextrose. Combination of these treatments resulted in potentiation of antinociception. Naloxone (opioid antagonist; 5 mg/kg i.p.) antagonised flavone or long stress induced antinociception showing opioid medicated mechanism, however, failed to reverse the potentiated antinociceptive component recorded in long stressed animals which received flavone and dextrose. Antinociceptive activity of flavone, dextrose and long swim stress which was documented by acetic acid assay has been confirmed in the present study. Role for opioid system in this action has been demonstrated. Therefore, formalin test can also be considered as an useful assay procedure for testing flavonoids. However, like acetic acid assay this assay procedure also has the limitation that it is unable to detect minor changes in the degree of antinociception produced by physiological interventions such as long swim and dextrose.     

Subcellular action of Neocarzinostatin. Intracellular incorporation, DNA breakdown and cytotoxicity

The subcellular site of action of a proteinaceous antitumor antibiotic neocarzinostatin (NCS) was studied using normal human lymphocytes, Epstein-Barr virus transformed lymphoblastoid cells, osmotically burst lymphoblastoid cells, and colicin E1 plasmid DNA. The rate of DNA strand break in these different types of DNA was found to be in the following order: Colicin DNA > burst cell DNA > lymphoblastoid cell DNA > normal lymphocyte DNA. Furthermore, fluorescence microscopy revealed that lymphoblastoid cells incorporated more fluorescein isothiocyanate labeled NCS than normal cells. High uptake of NCS in lymphoblastoid cells coincided with a high killing rate; low uptake of NCS in lymphocytes resulted in very little cell killing. Uptake velocity using fluorescein diacetate (FDA) also showed that the lymphoblastoid cells exhibited a higher uptake of FDA coinciding with a higher killing rate. The cell killing activity of NCS appears to be closely associated with the rate of intracellular uptake of NCS and subsequent direct degradation of DNA by the drug. This notion is reinforced by the reported finding that the dose required for DNA strand scission is only about 1/100 of that for the inhibition of cap formation. Thus DNA strand scission, rather than the cell membrane, appears to be the primary target of NCS. Enhanced incorporation of many substances is commonly observed upon transformation of cells by viruses, and our present results may provide an important clue toward the explanation of the selective toxicity toward tumor cells of NCS.     

Fluorometric assay of tubulin-colchicine complex.

A fluorometric assay method for the tubulin-colchicine complex has been developed. This assay method is based on the fact that the binding of colchicine to tubulin leads to a 300-fold enhancement of fluorescence intensity of colchicine at 430 nm. Since the excitation wavelength can be set at 350 nm, far from an absorption band of tubulin, the assay does not necessitate the separation of the tubulin-colchicine complex from free colchicine. Fluorescence intensity is linear up to 2 mg/ml of the tubulin-colchicine complex.     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

Stomatocytosis of latex particles (0.26 micron) by rat erythrocytes by the electrical breakdown technique

The uptake of macromolecules by erythrocytes can be achieved with the electrical breakdown technique [2, 4]. In this technique the erythrocyte membranes are subjected to a high external electrical field pulse for a short period. Local, reversible breakdowns of the cell membrane occur above a critical field strength which lead to a time-dependent increase in the permeability of the membrane. By this means, human erythrocyte membranes can be made permeable to DNA, pharmaceutical compounds, and latex particles following an electrical field pulse [1, 3, 5]. Larger particles should also be taken up by erythrocytes using this method. Vienken et al. [5] demonstrated the entrapment of latex particles with a diameter of 0.091 micron in human erythrocyte ghosts, although this was shown with only a single electron micrograph which does not prove that the ghost membrane was intact. In our experiments in order to entrap latex particles with a diameter of 0.26 micron rat erythrocytes were subjected to an electrical field pulse of 12 kV/cm with a decay time of 60 microseconds. Experiments using the electron microscope show that after such an electrical field pulse the uptake of latex particles by rat erythrocytes follows the stomatocytotic pathway. We show further that using electron microscopic techniques, a single section cannot demonstrate the completed uptake of a latex particle by the erythrocyte.     

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. II. Electric field-induced electrical breakdown of the photosynthetic membrane and its recovery

Preilluminated suspensions of swollen thylakoid vesicles (‘blebs’) were exposed to uni- and bipolar pairs of identical electric field pulses of variable duration, intensity and spacing. The resulting field-stimulated luminescence (electrophotoluminescence) was used as an intrinsic, voltage-sensitive optical probe to monitor electrical phenomena at the membrane level. The application of a pair of voltage pulses of opposite polarity made it possible to produce electric changes in the membrane by the first pulse and to analyse these effects by a second pulse of opposite polarity. It was found that the relative amplitudes of the two electrophoto-luminescence signals depended on the intensity of the applied electric field and on the time interval (t*) between the two pulses. When t* varied from 0.4 to 12 ms, the second stimulated luminescence signal was at first much smaller than the first one and then increased exponentially until the two signals were equal for t* ≥ 3 ms. We analysed these differences between the two field-stimulated luminescence signals as a measure of the electrical breakdown of the membrane, induced during the first pulse. In this way a distinction between irreversible and reversible breakdown could be made with an estimation of the recovery kinetics of the reversible breakdown, which was found to be complete within 3 ms. Irreversible breakdown of the membrane was found to increase with lengthening the exposure time from 0.1 to 1.3 ms especially when applying high electric field of at least 2000 V/cm.     

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 μs. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1 V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$. Between charging times of 300 ns and 5 μs at 25°C and between 100 ns and 2 μs at 40°C, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$ showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25°C) and from 1 to 0.4 V (40°C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns.The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25°C) and 100 ns (40°C) other processes (such as the Born energy) become possible.     

The mechanism of electrical breakdown in the membranes ofValonia utricularis

Summary The dielectric breakdown in the membranes of cells ofValonia utricularis was investigated using intracellular electrodes and 500-sec current pulses. Electrical breakdown, which occurs when the membrane potential reaches a well-defined critical value, is not associated with global damage to the cell or its membranes (the membrane reseals in <5 sec). It was thus possible to investigate the effect of temperature on dielectric breakdown in single cells. It was found that the critical potential for breakdown was strongly dependent on temperature, decreasing from 1000 mV at 4°C to 640 mV at 30°C. The decrease in the breakdown potential with increasing temperature and the very short rise-time of the breakdown current (1 sec) suggests that the Wien field dissociation does not play a major role in the breakdown process. It is shown that the nonlinearI–V characteristics observed at different temperatures can be accurately accounted for with no adjustable parameters, by considerations of the mechanical compression of the membrane due to stresses induced by the electric field. Electrical breakdown on this scheme results from an electromechanical instability in the membrane. On this basis the present results indicate that the elastic modulus of the region of the membrane where breakdown occurs, decreases by a factor of 2 with increasing temperature from 4 to 30°C. On the assumption of a thickness of 4.0 nm and a dielectric constant of 5, the elastic modulus is estimated to have a value of 5×10⁶ Nm^–2 at 20°C.     

Validation of an automated determination of pulmonary resistance by electrical subtraction.

An analog computer to determine dynamic pulmonary compliance (C) and pulmonary resistance (R) on a breath-by-breath basis was tested in guinea pigs and dogs. C was determined by dividing volume by transpulmonary pressure at instants of zero flow. R was determined by the method of electrical subtraction at predetermined flows. In both species the computer outputs and the results of direct analysis were in close agreement. In guinea pigs, the device reliably followed the rapid three- to fourfold changes in C and R resulting from histamine infusion. In unanesthetized dogs, the dispersion and mean values of C and R were similar by the two methods.     

The efficiency and linearity of the radiochromium release assay for cell-mediated cytotoxicity.

The release of radiochromium from EL-4 cells lysed by anti EL-4 peritoneal lymphocytes is a first order decay process with respect to time. The rate of ⁵¹Cr release from damaged cells was independent of the effector: target ratio but was slower from pelleted cells than cells in suspension. We conclude that under normal assay conditions the fraction of chromium released is nearly proportional to the number of cells damaged but find that the proportionality constant is influenced by assay conditions. There is a lag time of 23 ± 5 min before ⁵¹Cr is rapidly released from the cells.     

Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.     

The FRAME alternatives laboratory database. 1. In vitro basal cytotoxicity determined by the Kenacid blue total protein assay

A database of over 280 chemicals has been compiled by using a mouse 3T3-L1 fibroblast-like cell line in exponential growth, exposed to chemicals for 72 hours in a 96-well tissue culture plate format, and determining cell number via the Kenacid blue (KB) assay for total protein. Ranking the chemicals according to their basal cytotoxicity, expressed as the concentration (mM) that inhibits increase in total cellular protein over 72 hours by 50% (the ID50 value) shows a wide range of ID50 values, from 0.00003 mM to 10,096 mM. This information includes the results for MEIC chemicals 1-50, and we have now added basal cytotoxicity data for 23 of the next 25 MEIC chemicals. When the neutral red uptake (NRU) assay was performed with the same cell cultures, before the KB assay, very similar indications of basal cytotoxicity were obtained. Comparisons between the results with 3T3-L1 cells and with a human fibroblast-like cell line, BCL-D1 showed a significant difference in order of magnitude of the ID50 value for only 5 of 52 chemicals. However, there was a difference in ID50 value of more than one order of magnitude for 8 of 24 chemicals tested with an undifferentiated teratocarcinoma cell line, F9.     

Modulation of HL-A antigens by anti-HLA antiserum: effects on the cytotoxicity assay and mixed leukocyte reaction.

Modulation of human leukocyte antigen (HLA) was attempted by treating leukocytes with specific anti-HLA antiserum or by their passage through columns coated with anti-HLA or a double layer of HLA-anti-HLA. The modulated cells were resistant to the cytotoxic effects of the anti-HLA, and they were poor stimulators and good responders to allogeneic cells in the unidirectional mixed leukocyte reaction. Modulated cells regained their HLA 16 hr after modulation if kept in cell suspension alone. The proliferative responses of modulated cells to mitogens were as good as non-modulated cells, indicating that modulation was probably not caused by depletion of lymphoid cells. Supernatants of modulated cells that were incubated overnight or preformed HLA-anti-HLA complexes were capable of suppressing and enhancing the MLR of specific cells depending on the dose used. The similarities of modulation of HLA to other lymphocyte receptors and the limitation of application of the modulation phenomenon to transplantation of allogeneic cells are discussed.