Microsecond and nanosecond electric pulses in cancer treatments

New local treatments based on electromagnetic fields have been developed as non‐surgical and minimally invasive treatments of tumors. In particular, short electric pulses can induce important non‐thermal changes in cell physiology, especially the permeabilization of the cell membrane. The aim of this review is to summarize the present data on the electroporation‐based techniques: electrochemotherapy (ECT), nanosecond pulsed electric fields (nsPEFs), and irreversible electroporation (IRE). ECT is a safe, easy, and efficient technique for the treatment of solid tumors that uses cell‐permeabilizing electrical pulses to enhance the activity of a non‐permeant (bleomycin) or low permeant (cisplatin) anticancer drug with a very high intrinsic cytotoxicity. The most interesting feature of ECT is its unique ability to selectively kill tumor cells without harming normal surrounding tissue. ECT is already used widely in the clinics in Europe. nsPEFs could represent a drug free, purely electrical cancer therapy. They allow the inhibition of tumor growth, and interestingly, nsPEF can target intracellular organelles. However, many questions remain on the mechanism of action of these pulses. Finally, IRE is a new ablation procedure using pulses that provoke the permanent permeabilization of the cells resulting in their death. This technique does not result in any thermal effect, which is its main advantage in current physical ablation technologies. For both the nsPEF and the IRE, the preservation of the normal tissue, which is characteristic of ECT, has not yet been shown and their safety and efficacy still have to be investigated thoroughly in vivo and in the clinics. Bioelectromagnetics 33:106–123, 2012. © 2011 Wiley Periodicals, Inc.     

Paradoxical loss of excitation with high intensity pulses during electric field stimulation of single cardiac cells

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.     

Microdosimetric calculation of absorption fraction and the resulting dose conversion factor for radon progeny

It is an established fact that radon progeny can induce lung cancers. However, there is a well-known discrepancy between the epidemiologically derived dose conversion factor for radon progeny (4 mSv/WLM) and the dosimetrically derived value (15 mSv/WLM) (mSv is a unit of the dose while WLM is a unit of exposure to radon progeny). Up to now there is no satisfactory explanation to this. In the present study we propose that microdosimetry will help reduce the discrepancy significantly. The ICRP Human Respiratory Tract Model (HRTM) has been applied to calculate the effective dose conversion factor. All parameters have been kept at their best estimates. Modifications were made in the calculation of the absorbed fractions of alpha particles. In contrast to the ICRP approach where the energy has been considered to be deposited in the layer containing the sensitive cells, we used a microdosimetric approach in which the alpha particles deposit their energy only in the nuclei of sensitive cells. This modification alone has lowered the dose conversion factor by about one-third (from 15 mSv/WLM down to approximately 10 mSv/ WLM). Received: 19 February 2001 / Accepted: 10 July 2001     

Excitability changes of presumptive ectoderm following mesodermal induction

Dissociated ectodermal cells of the early newt gastrula which have been treated with CMF (Ca-Mg-free saline) for 5 hr differentiate into muscle cells when cultured in HFCS (heated fetal calf serum) for up to 9-12 days. Similarly dissociated cells placed into FCS (fetal calf serum) culture differentiate into epidermis. Differences in cell-cluster formation have been found between HFCS and FCS in early cell cultures (6 hr), and membrane excitability phenomena associated with the differentiation of these clusters into the muscle cells or epidermal cells have been investigated, respectively. The HFCS cultures consist of cell clusters which have few of microvilli at their surfaces and which form loose contacts by means of lamellipodia. FCS cultures consist of cell clusters which have numerous microvilli at their surfaces and which make tight contacts between cells by means of ridge-structure precursors. The different reaggregation pattern of dissociated ectoderm cells in HFCS reflects changes in the cell membrane surface induced by HFCS. The sequential genesis of action potentials in cells destined to form muscle cells in HFCS is very similar to those produced by somitic muscle cells in vivo and their ionic dependence for generating action potentials is related to epidermal action potentials in vitro (FCS).     

Modern aspects of studying phytohormones. Cytokinins

The review presents current data on mechanisms of cytokinin action in plants. By analogy with the first part (Ivanova et al., 1999), in which general principles of phytohormone action and cardinal trends of phytohormone investigations were examined, here the relevant information on mechanisms of action of auxins and gibberellins has been given, and taking cytokines as example an attempt has been done to summarize the literature data on the number of questions offered for analysing hormones of high animals (Gudwin, Merser, 1986). The review demonstrates that mechanisms of cytokine action at the cellular level are not known in many cases. One of the most significant factors in the action of phytohormones of this class on plants is their concentration, determined by their synthesis, transportation and further chemical conversions. This paper points to a poor knowledge of the relative role of these processes in regulation of cytokinin contents and their distribution among plant organs. Two possible ways of studying cytokinin action at the present day stage of investigations have been designated: 1) revealing the cytokinin expressed genes and establishing mechanisms of their action; 2) estimation of endogenous cytokinin alteration and the influence of this alteration on definite processes in the cell with the help of ipt-gene from t-DNA of Agrobacterium tumefaciens.     

Probabilistic approach to obtain hit-size effectiveness functions which relate microdosimetry and radiobiology

A probabilistic approach has been developed to relate microdosimetry, biological effects, and radiation quality. It is used to derive, and subsequently apply, microdosimetry-based cellular response functions for different biological end points of relevance for radiological protection. The approach makes use of measurable microdosimetry spectra and avoids assumptions concerning the course of mechanisms of radiation action. Instead, it postulates a response function that is, and behaves like, the cumulative probability that a subcellular target structure will respond to a specific target-averaged ionization density. Statistical distributions are applied and their parameters are evaluated to characterize the randomness involved in the localization of sensitive sites and in the reactivity of the whole sensitive structure. The resulting response functions can be used for prediction of the effects of low-level radiation. Such predictions for some selected effects of a stochastic nature (mutagenesis, chromosome abnormalities, etc.) are presented as relative biological effectiveness values based on low doses of radiations with a wide range of linear energy transfer and compared with various quality factor specifications. Cellular response relationships, termed hit-size effectiveness functions, can also be applied directly in radiation protection metrology by incorporating them into the software used to process the readings of microdosimetric spectrometers. The derivation of the functions, rather than their uses in radiation protection, is the principal subject of this report.     

Confocal microscopy for modeling electron microbeam irradiation of skin

For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the sample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differentiated tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal keratinocyte nuclei for the full-thickness EpiDerm™ skin model (MatTek, Ashland, VA). Application of these data to calculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.     

Rapid changes in flagellar rotation induced by external electric pulses.

The bacterial flagellar motor is the only molecular rotary machine found in living organisms, converting the protonmotive force, i.e., the membrane voltage and proton gradients across the cell membrane, into the mechanical force of rotation (torque). We have developed a method for holding a bacterial cell at the tip of a glass micropipette and applying electric pulses through the micropipette. This method has enabled us to observe the dynamical responses of flagellar rotation to electric pulses that change the membrane voltage transiently and repeatedly. We have observed that acceleration and deceleration of motor rotation are induced by application of these electric pulses. The change in the rotation rate occurred within 5 ms after pulse application.     

Microdosimetry model for boron neutron capture therapy: I. Determination of microscopic quantities of heavy particles on a cellular scale

Due to the limitations of existing microdosimetry models, a new model called MICOR has been developed to analyze the spatial distribution of microscopic energy deposition for boron neutron capture therapy (BNCT). As in most existing models, the reactions independent of the incident neutron energy such as the boron and the nitrogen capture reactions can be considered. While other models do not include reactions that are dependent on the neutron energy such as the proton recoil reaction, the present model is designed so that the energy deposition resulting from these reactions is included. The model MICOR has been extended to enable the determination of the biological effects of BNCT, which cannot be done with the existing models. The present paper describes the determination of several microscopic quantities such as the number of hits, the energy deposition in the cell nucleus, and the distribution of lineal and specific energy deposition. The companion paper (Radiat. Res. 155, 000-000 2001) deals with the conversion of these microscopic quantities into biological effects. The model is used to analyze the results of a radiobiological experiment performed at the HB11 facility in the HFR in Petten. This analysis shows the value of the model in determining the dose depositions on a cellular scale and the importance of the extension to the energy deposition of the proton recoil.     

The Importance of Subcellular Structures to the Modeling of Biological Cells in the Context of Computational Bioelectromagnetics Simulations

Numerical investigation of the interaction of electromagnetic fields with eukaryotic cells requires specifically adapted computer models. Virtual microdosimetry, used to investigate exposure, requires volumetric cell models, which are numerically challenging. For this reason, a method is presented here to determine the current and volumetric loss densities occurring in single cells and their distinct compartments in a spatially accurate manner as a first step toward multicellular models within the microstructure of tissue layers. To achieve this, 3D models of the electromagnetic exposure of generic eukaryotic cells of different shape (i.e. spherical and ellipsoidal) and internal complexity (i.e. different organelles) are performed in a virtual, finite element method-based capacitor experiment in the frequency range from 10 Hz to 100 GHz. In this context, the spectral response of the current and loss distribution within the cell compartments is investigated and any effects that occur are attributed either to the dispersive material properties of these compartments or to the geometric characteristics of the cell model investigated in each case. In these investigations, the cell is represented as an anisotropic body with an internal distributed membrane system of low conductivity that mimics the endoplasmic reticulum in a simplified manner. This will be used to determine which details of the cell interior need to be modeled, how the electric field and the current density will be distributed in this region, and where the electromagnetic energy is absorbed in the microstructure regarding electromagnetic microdosimetry. Results show that for 5 G frequencies, membranes make a significant contribution to the absorption losses. © 2023 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.     

Effects of temperature on a moth auditory receptor

Measurements of the thoracic temperature and recordings of the spike activity of the most sensitive auditory receptor (A1 cell) were made in Empyreuma pugione (Arctiidae, Ctenuchinae). The temperature range tested (19–36 °C) is relevant for the behavior and ecology of this species. Experiments were performed during the hours of maximal flying activity in the wild: sunrise and sunset. The thoracic temperature during rest reflects that of the surrounding air; there is an increase of 3–4 °C immediately after ceasing free flying in the laboratory. The spike activity of the tympanic organ was recorded with a stainless-steelhook electrode placed beneath the tympanic nerve in the mesothorax. The A1 cell activity was studied without acoustic stimulation (spontaneous) and in response to 35-kHz acoustic pulses of 20, 40, or 100 ms duration. At all of these durations A1 cell response to saturating stimulus was analysed, while with 40-ms pulses different stimulus intensities were used (20–90 dB SPL in 10-dB steps). The number of action potentials per pulse, mean spike rate, maximal instantaneous discharge, and latency period depend strongly on air temperature, while the variation coefficients of the interspike intervals during the responses were not temperature dependent and vary non-monotonically with stimulus intensity. During responses to a saturating stimulus, the stimulus duration does not affect the activation energy, calculated from an Arrhenius plot, of different physiological features. Adaptation, studied in the responses to 100-ms pulses, is also temperature dependent. This phenomenon has two components, each of which shows different activation energies, suggesting a different membrane origin. High stimulus intensity (90 dB SPL) significantly affects the activation energy of the action potentials and mean spike rate, while the activation energy, of the maximal instantaneous discharge and latency period do not show this strong dependency. The spontaneous A1 cell spike rate varies with temperature, as does the value of the mode of the relative frequency distribution of the interspike interval. The activation energy of the spike rates measured at A1 cell responses to saturating stimuli is in good agreement with that described in amphibian innerear hair cells. It is suggested that this moth auditory receptor cell also has mechanosensitive protein channels.Abbreviations AP/p action potentials per pulse - AP/s action potentials per second - CI confidence interval - E _a activation energy - ISI interspike interval - SD standard deviation - VC variation coefficient     

Evidence for functional sodium and calcium ion channels in the membrane of cultured cardiomyocytes of the adult rat

Characteristics are reported for electrical activity of adult rat cardiomyocytes in long-term primary culture. Cells in vitro for 12 to 28 days have mean membrane potential of -53 mV, are electrically excitable, and some are spontaneously contractile. The action potential of these cells has a slow rate of depolarization and is abolished by methoxyverapamil (D-600) but not by tetrodotoxin (TTX). When cells are hyperpolarized by passage of an inward current, spontaneous action potentials cease and action potentials evoked by depolarizing pulses are then TTX sensitive. Fetal bovine serum is a constituent of the culture medium. Its temporary removal causes spontaneous contractility to cease but the cells remain electrically excitable.     

Electrophysiology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Two cell types can be distinguished with intracellular recording from the pars intercerebralis of the American cockroach (Periplaneta americana). The first type, which corresponds morphologically to the medial neurosecretory cell, always had spontaneously occurring, overshooting action potentials. These action potentials are probably endogenously produced. Tetrodotoxin experiments revealed that sodium is the dominant ion of the action potential. The action potentials are followed by a relatively long after-hyperpolarization. The input resistance of these cells ranged from 120 to 390 M omega. A mathematical model, based on cellular morphology and response to current pulses, revealed a membrane time constant of about 100 msec and an axonal:somatic conductance ratio of approximately 13. Area-specific membrane resistance was estimated at 33 k omega cm2. These cells also often had reversible and spontaneous inhibitory postsynaptic potentials. The second cell type, which is non-neurosecretory, never produced spontaneous action potentials and rarely had synaptic potentials. Action potentials could be evoked by current injection into the cell body or by extracellular stimulation of their axons in the posteroventral portion of the the protocerebrum. These action potentials also depend on sodium ions. Their input resistance ranged from 16 to 35 M omega. They had a membrane time constant of approximately 15 msec and an axonal:somatic conductance ratio of about 9. Their area specific membrane resistance was estimated at 14 k omega cm2.     

Nanosecond Electric Pulses Affect a Plant-Specific Kinesin at the Plasma Membrane

Electric pulses with high field strength and durations in the nanosecond range (nsPEFs) are of considerable interest for biotechnological and medical applications. However, their actual cellular site of action is still under debate—due to their extremely short rise times, nsPEFs are thought to act mainly in the cell interior rather than at the plasma membrane. On the other hand, nsPEFs can induce membrane permeability. We have revisited this issue using plant cells as a model. By mapping the cellular responses to nsPEFs of different field strength and duration in the tobacco BY-2 cell line, we could define a treatment that does not impinge on short-term viability, such that the physiological responses to the treatment can be followed. We observe, for these conditions, a mild disintegration of the cytoskeleton, impaired membrane localization of the PIN1 auxin-efflux transporter and a delayed premitotic nuclear positioning followed by a transient mitotic arrest. To address the target site of nsPEFs, we made use of the plant-specific KCH kinesin, which can assume two different states with different localization (either near the nucleus or at the cell membrane) driving different cellular functions. We show that nsPEFs reduce cell expansion in nontransformed cells but promote expansion in a line overexpressing KCH. Since cell elongation and cell widening are linked to the KCH localized at the cell membrane, the inverted response in the KCH overexpressor provides evidence for a direct action of nsPEFs, also at the cell membrane.     

Microdosimetry model for boron neutron capture therapy: II. Theoretical estimation of the effectiveness function and surviving fractions

A model has been developed to obtain a better understanding of the effects of boron neutron capture therapy (BNCT) on a cellular scale. This model, the microdosimetry model MICOR, has been developed to include all reactions important for BNCT. To make the model more powerful in the translation from energy deposition to biological effect, it has been designed to be capable of calculating the effectiveness function. Based on this function, the model can calculate surviving fractions, RBE values and boron concentration distributions. MICOR has been used to analyze an extensive set of biological experiments performed at the HB11 beam in Petten. For V79 Chinese hamster cells, the effectiveness function is determined and used to generate surviving fractions. These fractions are compared with measured surviving fractions, which results in a good agreement between the measured and calculated surviving fractions (within the uncertainties of the measurements).     

Auxin, cytokinin and the control of shoot branching

BACKGROUND: It has been known for many decades that auxin inhibits the activation of axillary buds, and hence shoot branching, while cytokinin has the opposite effect. However, the modes of action of these two hormones in branching control is still a matter of debate, and their mechanisms of interaction are equally unresolved. SCOPE: Here we review the evidence for various hypotheses that have been put forward to explain how auxin and cytokinin influence axillary bud activity. In particular we discuss the roles of auxin and cytokinin in regulating each other's synthesis, the cell cycle, meristem function and auxin transport, each of which could affect branching. These different mechanisms have implications for the main site of hormone action, ranging from systemic action throughout the plant, to local action at the node or in the bud meristem or leaves. The alternative models have specific predictions, and our increasing understanding of the molecular basis for hormone transport and signalling, cell cycle control and meristem biology is providing new tools to enable these predictions to be tested.     

Time disparity sensitive behavior and its neural substrates of a pulse-type gymnotiform electric fish, Brachyhypopomus gauderio

Roles of the time coding electrosensory system in the novelty responses of a pulse-type gymnotiform electric fish, Brachyhypopomus, were examined behaviorally, physiologically, and anatomically. Brachyhypopomus responded with the novelty responses to small changes (100 μs) in time difference between electrosensory stimulus pulses applied to different parts of the body, as long as these pulses were given within a time period of ~500 μs. Physiological recording revealed neurons in the hindbrain and midbrain that fire action potentials time-locked to stimulus pulses with short latency (500–900 μs). These time-locked neurons, along with other types of neurons, were labeled with intracellular and extracellular marker injection techniques. Light and electron microscopy of the labeled materials revealed neural connectivity within the time coding system. Two types of time-locked neurons, the pear-shaped cells and the large cells converge onto the small cells in a hypertrophied structure, the mesencephalic magnocellular nucleus. The small cells receive a calyx synapse from a large cell at their somata and an input from a pear-shaped cell at the tip of their dendrites via synaptic islands. The small cells project to the torus semicircularis. We hypothesized that the time-locked neural signals conveyed by the pear-shaped cells and the large cells are decoded by the small cells for detection of time shifts occurring across body areas.     

Recoil proton, alpha particle, and heavy ion impacts on microdosimetry and RBE of fast neutrons: analysis of kerma spectra calculated by Monte Carlo simulation

Fast neutrons (FN) have a higher radio-biological effectiveness (RBE) compared with photons, however the mechanism of this increase remains a controversial issue. RBE variations are seen among various FN facilities and at the same facility when different tissue depths or thicknesses of hardening filters are used. These variations lead to uncertainties in dose reporting as well as in the comparisons of clinical results. Besides radiobiology and microdosimetry, another powerful method for the characterization of FN beams is the calculation of total proton and heavy ion kerma spectra. FLUKA and MCNP Monte Carlo code were used to simulate these kerma spectra following a set of microdosimetry measurements performed at the National Accelerator Centre. The calculated spectra confirmed major classical statements: RBE increase is linked to both slow energy protons and alpha particles yielded by (n,alpha) reactions on carbon and oxygen nuclei. The slow energy protons are produced by neutrons having an energy between 10 keV and 10 MeV, while the alpha particles are produced by neutrons having an energy between 10 keV and 15 MeV. Looking at the heavy ion kerma from <15 MeV and the proton kerma from neutrons <10 MeV, it is possible to anticipate y* and RBE trends.     

The microdosimetry of the (10)B reaction in boron neutron capture therapy: a new generalized theory

The microdosimetry of (10)B thermal neutron capture reactions should be considered as an essential step to be followed before studying the radiobiological aspects of boron neutron capture therapy. The boron dose itself is insufficient as the only quantity used to describe the biological effectiveness of the (10)B reaction for two important reasons: the specific microdistribution that the (10)B carrier compound exhibits at the cellular level and the primarily stochastic nature of the energy deposition process, which influences the biological response to the particulate radiation. In this work, these two aspects are analyzed in detail and an innovative rigorous analytical framework is developed in the microdosimetry domain. This formalism provides the necessary microdosimetric tools for more precisely describing the (10)B dose distribution deposited in sensitive microscopic structures and offers improved approaches for analyzing the biological dose--effect relationship of (10)B reactions.     

Seprase: an overview of an important matrix serine protease

Seprase or Fibroblast Activation Protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which includes dipeptidyl peptidase IV (DPPIV/CD26) and related type II transmembrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localisation of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis.