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Diverse Effects of Wideband Intense, Non-Ionizing Radiation on Cells and Tissues

EasternVirginia Medical School, Old Dominion University, and The Frank Reidy Research Center for Bioelectrics
Abstract - Ionizing radiation can be an environmental health risk as well as a cancer therapeutic, but similar roles for nonionizing radiation are controversial. We have examined effects of wideband, intense non-ionizing radiation applied to cells and tissues as nanosecond pulsed electric fields (nsPEFs). Compared to conventional electroporation pulses, nsPEFs have shorter pulse durations (210ns) and higher electric fields (-300kV/cm). In spite of the high electric field, measured thermal changes are negligible. For these short pulses we have observed a number of nsPEF-induced effects on biological cells and tissues, including apoptosis induction and tumor regression at high electric fields, as well as calcium mobilization, platelet aggregation and enhanced gene transcription at lower,
Thus, nsPEF effects are due to charging of the plasma membrane, as well as effects on intracellular structures and functions in cells and tissues that are defined by the Er scaling law. It is anticipated that when X significantly diminishes and charging mechanisms are not likely to predominate, the ET scaling law will not hold and a new paradigm of ultrashort pulsed electric field effects is expected.

S.J. Beebe, E.H. Hall and K.H. Schoenbach

I. INTRODUCTION
described

sublethal electric fields. When nsPEFs are below the plasma membrane (PM) charging time constant (-100ns), effects on intracellular membranes are appreciable. Thus, it is possible to observe effects on intracellular structures (membranes) and functions without observing effects on the PM. For Jurkat or HL-60 cells exposed to lOns or 60ns pulses, mobilization of calcium from

duration are increased, it is possible to observe effects on the PM. Thus, as the pulse duration increases there is a cell typespecific increase in effects at the PM, as well as on intracellular structures and functions. To determine nsPEF characteriastics that are responsible for these diverse cell responses, we designed experiments to determine if pulse duration, electric field, or energy density was the stimulus that recruited responses from cells to nsPEFs. The results indicate that nsPEF effects are not due to dose effects, but instead follow a scaling law (Et) defined by the product of electric field (E) and pulse duration (tau, ), which can be derived from circuit equations that describe membrane charging for pulses short compared to the membrane charging time constant. For both increases in ethidium homodimer fluorescence, which is used as a marker for plasma membrane electroporation and/or transport, and increases in phosphatidylserine externalization measured by Annexin-V-FITC fluorescence, which is a marker for plasma membrane lipid translocation, a threshold response to increases in both markers exhibited the same ET value. This law also holds true for intracellular effects that are measured on isolated cells and intact tissues. Thus, the intracellular activation of caspase proteases, which are the executioners for programmed cell death by apoptosis, was independent of the energy density when pulses were applied at 10, 60 and 300ns, but directly proportional to Er. Furthermore, caspase activation
and nsPEF-induced cell death in HCT116 colon carcinoma cells, the ET scaling law was extended to include pulse number

intracellular stores is observed at significantly lower electric fields than electric fields needed for PM electroporation or transport. However, as the electric field and/or the pulse

a unipolar voltage pulse is applied to the electrodes, the resulting current causes accumulation of electrical charges at the cell membrane and, consequently, a voltage across the membrane. If the membrane voltage exceeds a critical value, structural changes in the surface membrane occur with transmembrane pore formation, a process known as electroporation [1]. If the membrane voltage is not excessive and the duration of the pulse is limited, membrane poration
can be reversible and the cells survive. The time required to

placed in a conductive medium between two electrodes and

membrane

in cytoplasm

the

ace canb sicpef t of an b ctricefin s the n simplesttr cell is a conductor and the surrounding plasma iS an insulating dielectric layer. When cells are

charge the surface membrane is dependent upon parameters such as the cell diameter (D), resistivities of the cytoplasm (Pc) and suspension medium (Pa), and capacitance of the surface membrane per unit area (cm). For a spherical cell with a surface membrane that is an ideal dielectric (no of leakage currents), with a diameter of 10 pm, resistvities of
75 ns [2] [.c= (pc+pa/2)cmD/2]. The charging time constant is a measure of the time required for charges within the cell to redistribute in such a way as to screen out the imposed electric field. Therefore, it is during this charging time that the cell interior is exposed to the applied pulsed electric field intensity. A simple electrical model for living cells predicts t en the electricp l delafon is cells the subthat when the electric pulse duration is reduced into the submicrosecond range (time domain), there is an increasing probability for electric field interactions to occur at the level of cell substructures. Stated another way (frequency domain), the outer membrane becomes increasingly
cytoplasm and medium of 100 ohm cm, and a membrane capacitance of 1 uF/cm2, the charging time constant (cc) is

in intact fibrosarcoma tumors at 300ns was directly proportional to the electric field. Finally, for apoptosis markersThDeatntoDfnstrugteAiFrcOfcef (n) into the equation (E-un).

transparent for oscillating electric fields when the angular frequency of the oscillation exceeds a value given by the inverse of the charging time.
Scientific Research, the American Cancer Society, Old Dominion University, and Eastern Virginia Medical School supported this work.

1-4244-0019-8/06/$25.OO 2006 IEEE

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Although this is well-known [3, 4], only recently have high frequencies (short durations) been used for achieving intracellular effects, e.g., the electroporation of intracellular membranes. Conventional electroporation (EP) using pulsed electric fields 0.1-10 millisecond (ms) in duration and volts to low kilovolts/centimeter in amplitude have been used for decades for the transient electroporation of cells to allow the entry of xenomolecules through aqueous channels or pores [1, 5]. Although the physical nature of the pores is not well characterized, the experimental conditions that allow intracellular delivery of membrane impermeant molecules with good cell survival are well known. Conditions for optimal electroporation depend on the waveform, the composition of the medium in which the cells are suspended, and the cell type [1, 6]. These pulses have minimal effects on intracellular membranes [7]. This method has been used to introduce genes into cells for gene therapy. An approach called electrochemotherapy or electroporation therapy (EPT), is a method to deliver poorly permeable chemotherapeutic agents, such as bleomycin in tumor cells in vivo that can be appropriately oriented between two electrodes [8-10]. Both electroporation and EPT are dependent on electric effects on the plasma membrane of the cells or tissues. We have been able to generate much shorter electrical pulses in the nanosecond range (10 to 300 ns) and electric field intensities as high as 350 kV/cm [11-19]. We refer to these pulses as nanosecond pulsed electric fields or nsPEFs. Over the past several years, we have analyzed the effects of nsPEF on living cells and tissues in vitro, ex vivo, and in vivo. NsPEF conditions defined here, are distinctly different than electroporation pulses, not only in their temporal and electrical characteristics, but also in their effects on intact cells and tissues. Electroporation pulses and nsPEF, respectively, exhibit different electric field strengths (1-5 kV/ cm3 vs. 10-350 kV/ cm3; different pulse durations (0.1-20 milliseconds vs. 1-300 nanoseconds); different energy densities (joules/ cm3 vs. millijoules/ cm3), and power (500W vs. 180MW). Thus, nsPEF can be five to six orders of magnitude shorter with electric fields and power several orders of magnitude higher and energy densities considerably lower than EP pulses. In addition to the unique short duration and rapid rise time, nsPEF are exceptional because they are very low energy and extremely high power. Thus, they are measurably non-thermal in vitro [20] and in vivo [21]. However, the most important differences are in biological responses that are due to inclusion of intracellular effects that are mainly absent in EP. When the electric pulse duration is reduced into the sub-microsecond range and the pulse rise-time is fast, there is an increasing likelihood for electric fields to modify cell substructures. NsPEFs have been shown to penetrate living using vital stains (propidium iodide, ethidium homodimer, or calcein), while modifying intracellular structures including granules in human peripheral eosinophils [11], endoplasmic reticulum [17, 22, 23], and DNA [24, 25]. Effects on the ER
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cells without measurable effects on the plasma membrane

result in intracellular Ca2+ release followed by capacitative influx through store-operated channels in the plasma membrane [17, 23]. This mimics Ca2+-mediated signaling response through ligands such as purinergic receptors [17, 23], and thrombin in human platelets [26]. Effects on DNA are complex. Applications of low electric fields and/or short pulse durations in non-ionic, isotonic buffer, enhance gene expression following transfection by EP [16, 17]. At higher electric fields and/or longer pulse durations, DNA damage is likely. Furthermore, effects on the plasma membrane potential have been defined with a 5ns resolution during a 60ns pulse in Jurkat cells [27]. While membrane signaling is usually initiated by ligands acting on receptors, it is possible that nsPEFs initiate a "ligandless" signaling from the plasma membrane, as well as from intracellular membranes. Responses from one or more of these nsPEF-recruited targets lead to apoptosis signaling by mitochondrialmediated mechanisms and cell death in cultured cells [15] and tumor tissues [14, 16, 17]. In general, these effects appear to be tunable. At relatively low electric fields and pulse durations, it is possible to enhance cell functions through pathways that activate and aggregate human platelets [26], induce lipolysis in isolated rat adipocytes [28], enhance expression of transfected genes [16, 17], or delay cell division transiently with continued survival of a subpopulation of cells [29]. At relatively high electric fields, it is possible to terminate cell function through pathways that delete cells in culture and tissues by programmed cell death, including activation of apoptosis cascades [14-17]. Thus, applications of nsPEF provide a new approach to physically target plasma and intracellular structures and modulate cell behavior and fate. Furthermore, application of nsPEFs to tumors provides a potential cancer therapy. Effects of nsPEFs to inhibit fibrosarcoma tumor growth and reduce tumor size [14, 16-17], and most recently, to cause shrinkage and even complete elimination of melanoma tumors [21], have encouraged us to continue to investigate nsPEFs as a potential therapeutic and/or as a basic science tool to reveal cell behaviors to external stimuli. The diversity of cell responses to nsPEFs encourages us to determine mechanisms and sites of action within biological cells. It is clear that specificity for lethal and non-lethal nsPEF actions depend on the pulse duration, electric field and pulse number. In the results described here, we have focused on nsPEF conditions that lead to effects on the plasma membrane, as well as on apoptosis markers and survival in cells and tumor tissues to better understand the relationship between pulse duration, electric field and pulse number. The results indicate a scaling rule that helps understand these relationships and shed light on a generalized mechanism of action that can explain biological responses to nsPEFs. II. RESULTS Relationship between pulse duration and electric field on plasma membrane integrity using flow cytometry and ethidium homodimer-] as a marker in HL-60 cells: In order to define the relationship between pulse number and electric field we first analyzed plasma membrane effects defined by ethidium

homodimer (EtHd) uptake into HL-60 cell in cell suspensions in cuvettes at three different pulse durations and various electric fields. EtHd is used as a marker for plasma membrane integrity and does not enter cells until the plasma membrane is breached. At 300ns pulses were varied between 12 and 60kV/cm; at 60ns pulses were varied between 18 and 60kV/cm, and at lOns pulses were varied between 26 and 300 kV/cm. Cells were analyzed by flow cytometry 10-15 minutes post-pulse. For single pulses, ethidium homodimer fluorescence became significantly greater than non-pulse background fluorescence at 18, 26, and 300 kV/cm for 300, 60, and lOns pulses, respectively. Thus, higher electric fields were required as the pulse duration was decreased. It appears that nsPEF effects on the plasma membrane as determined by EtHd can be estimated by the product of the electric field [E in kV/cm) and pulse duration [I or tau in ns). Effects of nsPEFs on plasma membrane structures in Jurkat cells: In order to determine if this scaling relationship was specific to HL-60 cells, we tested a range of electric field intensities, at two different pulse durations, and analyzed Jurkat cells for EtHd fluorescence and Annexin-VFITC-fluorescence using flow cytometry. Annexin-V-FITCfluorescence is used as a marker for phosphatidylserine (PS) externalization. PS is a lipid that is on the inner leaflet of the plasma membrane lipid bilayer of normal non-apoptotic cells. Jurkat cells were exposed to single pulses of lOns (26300 kV/cm), or 60ns (16-60 kV/cm) at different electric fields. Cells were exposed to nsPEFs in the presence of EtHd and after pulsing, were incubated with Annexin-V-FITC (10 minutes) before analysis by flow cytometry. For 1 Ons pulses, the threshold for fluorescence of both membrane markers was about 150 kV/cm with an Etau value of 1500. At 60 ns the threshold for both membrane markers was about 26 kV/cm with an Etau value of 1560. Thus, the scaling rule held true for two different cell types and for two different effects that were determined at the plasma membrane. NsPEF effects on caspase activation in HL-60 cells in vitro are independent of energy density: To determine if the scaling law included a factor for energy density, we compared different pulse durations at electric fields that equalized energy density and analyzed a cell response that is not directly related to a plasma membrane effect. We investigated caspase activation in HL-60 cells. Single pulses were delivered to HL-60 cells in suspension for 10, 60, or 300ns with electric fields of 150, 60 and 26 kV/cm, respectively, which were adjusted to equalize the energy density ( 1.7 joules/cm3) for all conditions. For each nsPEF condition, the Etau value is indicated as the product of electric field (E) and pulse duration (I or tau). At a constant energy density the magnitude of the effect on caspase activation was directly proportional to the product Etau. Thus, nsPEF-induced caspase activation is independent of the energy density and relates more specifically to the pulse duration and the electric field. Thus, greater caspase activation was observed as the Etau increased. This suggests that, like effects on the plasma membrane, caspase activation is related to charging events at membranes, either at the plasma membrane and/or at intracellular membranes. 560

NsPEF-induced cell death in HCT116 colon carcinoma cells follows the scaling rule that includes pulse number. Thus far data indicate that the scaling rule holds for cells that grow in suspension. To determine if adherent cells respond according to the scaling rule, we determined if cell survival could be predicted by the scaling rule in HCT 116 colon carcinoma cells. Cells were exposed to various numbers of pulses at 60ns (10-50 pulses) or 300ns pulses (5-30 pulses) with electric fields of 60 kV/cm. Cells were analyzed for cell number using a Coulter counter 24 hours post pulse. For the 60ns pulse about 5000 of the cells survived after 50 pulses. The product of the Etau including the pulse number was 180,000. For 300ns pulses 500o survival occurred with 5 or 10 pulses. The 10-pulse condition has a value of 180,000. Thus, the scaling rule holds for cell death in adherent cells and is extended to include pulse number. Furthermore, cell death was pulse number-dependent at both pulse durations. Caspase activation in fibrosarcoma tumors exposed to nsPEFs ex vivofollows the scaling rule. To determine ifthe scaling rule held for tumor tissues, we grew fibrosarcoma tumors in mice, removed them and treated them in cuvettes with 5 pulses 300ns and various electric fields between 18 and 60 kV/cm. After pulsing, tumor tissues were incubated in cell culture media in a cell culture incubator for 3 hours, homogenized, and then analyzed for caspase enzyme activity using a peptide substrate that fluoresces when cleaved (DEVD). At 18, 26, 40 and 60 kV/cm, E values were (xlOOO) 5.4, 7.8, 12, and 18 respectively. Caspase activity was directly proportional to the electric field and increased as the Etau value increased. Furthermore, the caspase increase was independent of energy density as it was for caspase activation in HL-60 cells. III. DISCUSSION The data presented above indicate that nsPEF-induced effects follow a scaling rule in cells and solid tumors that include the product of the pulse duration and electric field. This is described in more detail elsewhere [17] and has been considered in simulated models [30, 31]. This rules applies to cells in culture, as well as tumor tissue, and defines events that are related to membrane charging. Both conventional plasma membrane electroporation- and nsPEF-mediated effects are determined by membrane charging. However, cell responses to the two paradigms are defined by different rules. First, nsPEFs are independent of energy density and are thus non-thermal. More importantly, differences between the two PEF conditions are further defined by inclusion of plasma membrane and intracellular membrane charging determined for nsPEFs, but only plasma membrane charging for conventional electroporation. Thus, effects on membrane integrity defined by ethidium homodimer uptake and phosphatidylserine externalization are not surprising. Interestingly, however, are effects that are not immediately associated with membrane charging, such as caspase activation. Caspase activation in cultured cells and tumors then appears to be due to membrane charging. Furthermore, cell survival in HCT1 16 cells followed this rule indicating, that cell death by apoptosis is also determined by membrane charging.

The scaling rule was shown to include pulse number for demise of HCT1 16 cells. This is curious because
this would be expected to include
a

"dose" factor that would

include an energy density term, which was shown to be unimportant for nsPEFs. Thus, the inclusion of pulse number into the scaling rules requires further investigation.

[16] SJ Beebe, J White, PF Blackmore, Y Deng, K Somers, and KH Schoenbach. "Diverse effects of nanosecond pulsed electric fields on cells and tissues," DNA Cell Biol, vol. 22(12), pp 785-96 Dec 2003. [17] SJ Beebe, PF Blackmore, J White, RP Joshi, and KH Schoenbach,

decrease the pulse duration and increase the electric fields. Thus, for a Ins pulse, extremely high electric fields will be required to induce apoptosis. It remains to be determined if
conditins canbe r eaced to to cause ause cel deat byapotosis. can be reached cell death by apoptosis. conditions However, other cell responses, such as calcium mobilization, do not require such high electric fields [22, 23]. Thus, it is likely that signal transduction events can be triggered by

As work continues with nsPEFs, we strive to

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determined for pulse durations that are sufficiently short to diminish or exclude membrane charging. Nevertheless, the nsPEF scaling rule for pulse durations used in this study will facilitate translation in facilitate translation of of responses determined determined for for cells cells in culture to tissues in vivo.
responses

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