Background-Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8-and 9-μs EP.Methods-Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.Results-10-ns EP caused apoptotic or necrotic death within 2-20 hrs. Survival (S, %) followed the absorbed dose (D, J/g) as: S=αD (−K) , where coefficients K and α determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas α was cell type-and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.Conclusions-1.8-and 9-μs EP cause cell death efficiently and indiscriminately (LD 50 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD 50 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms.General significance-Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

BackgroundElectroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs.Methodology/Principal FindingsWe focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1–500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1–5 min delay enhanced the effects severalfold.Conclusions/SignificanceFor varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency.

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′,7′-dichlorodihydrofluoresein (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H2O2 and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3–100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. With settings optimal for sensitization, the second nsPEF treatment became 2.5 times (25 °C) or even 6 times (37 °C) more effective than the same nsPEF treatment delivered without conditioning. The minimum wait time required for sensitization development was 30 s, with still longer delays increasing the effect. We show that the delayed hypersensitivity was not mediated by either cell swelling or oxidative effect of the conditioning treatment; biological mechanisms underlying the delayed electrosensitization remain to be elucidated. Optimizing nsPEF delivery protocols to induce sensitization can reduce the dose and adverse side effects of diverse medical treatments which require multiple pulse applications.

Genes encoding proteins with antioxidant properties may influence susceptibility to endometrial hyperplasia (EH) and endometrial carcinoma (ECa). Patients with EH (n = 89), EH concurrent with ECa (n = 76), ECa (n = 186), and healthy controls (n = 1110) were genotyped for five polymorphic variants in the genes involved in metabolism of lipoproteins (APOE Cys112Arg and Arg158Cys), iron (HFE Cys282Tyr and His63Asp), and catecholamines (COMT Val158Met). Patients and controls were matched by ethnicity (all Caucasians), age, body mass index (BMI), and incidence of hypertension and diabetes. The frequency of the APOE E 2 allele (158Cys) was higher in patients with EH + ECa than in controls (P = 0.0012, P Bonferroni = 0.018, OR = 2.58, 95% CI 1.49–4.45). The APOE E 4 allele (112Arg) was more frequently found in patients with EH than in controls and HFE minor allele G (63Asp) had a protective effect in the ECa group, though these results appeared to be nonsignificant after correction for multiple comparisons. The results of the study indicate that E 2 allele might be associated with concurrent occurrence of EH and ECa.

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Резюме: Цели исследования. Ассоциации АРОЕ генотипа с уровнем железа в плазме и риском возникновения неопластических заболеваний эндометрия не изучались. Целью работы был анализ генотипических ассоциаций аполипопротеина Е (АРОЕ) с биохимическими показателями метаболизма железа и липидов у больных гиперплазией и раком эндометрия, а также у здоровых женщин. Материалы и методы. Биохимические показатели метаболизма железа и липидов в крови женщин с диагнозом гиперплазия эндометрия (53 человека), эндометриоидная аденокарцинома первой стадии (87 человек) и здоровые женщины (70 человек) определялись на автоматическом анализаторе «OLYMPUS AU-400». Генотипирование гена АРОЕ (Е2, rs7412 и Е4 rs429358) проводили с использованием методики полимеразной цепной реакции с последующей обработкой специфической рестриктазой Cfo1 (Promega) и определением длин фрагментов рестрикции. Основные результаты. Наблюдается статистически значимое снижение величин сывороточного железа, насыщения трансферрина, липопротеинов высокой плотности с одной стороны, и статистически значимое увеличение индекса массы тела, триглицеридов, липопротеинов очень низкой плотности, коэффициента атерогенности у пациенток с эндометриоидной аденокарциномой. Выявлены статистически значимые различия в распределении частот аллелей Е3, Е4, Е2 между группами больных и здоровых женщин (Р=0,048). Частота аллеля Е3 снижалась, а аллеля Е2 повышалась в ряду здоровые-ГЭ-РЭ. Показано, что у больных раком эндометрия и здоровых носительниц генотипа Е3/Е2 повышены показатели сывороточного железа по сравнению с другими генотипами АРОЕ (P= 0,0014 и 0,0363 соответственно). В группе больных гиперплазией носительницы минорных генотипов Е2/Е4, Е4/Е4 отличаются достоверно повышенным уровнем триглицеридов (0,0389). Выводы. Генотип Е3/Е2 АРОЕ ассоциирован с повышенным уровнем железа и риском рака эндометрия. Ассоциации генотипов APOE 2/2, 2/4, 4/4 и 3/2 с неопластическими заболеваниями эндометрия необходимо исследовать в дальнейшем. Полученные данные могут быть использованы для создания системы маркеров для определения высокого уровня предрасположенности к злокачественным заболеваниям женской репродуктивной системы.

(1) Background: There are no reliable and widely available markers of functional iron deficiency (FID) in cancer. The aim of the study was to evaluate the role of transferrin (Tf) as a marker of cancer of the ovary (CrO) and related FID. (2) Methods: The study groups consisted of 118 patients with CrO and 69 control females. Blood serum iron status was determined on a Beckman Coulter AU (USA) analyzer. Tf quantification was performed by immunoturbidimetry. The relative contents of apo- and holo-Tf (iron-free and iron-saturated Tf, respectively) were determined in eight patients and a control female by immunochromatographic analysis based on the use of monoclonal single-domain antibodies (nanobodies). (3) Results: Four groups of patients with different iron statuses were selected according to ferritin and transferrin saturation values: absolute iron deficiency (AID) (n = 42), FID (n = 70), iron overload (n = 4), normal iron status (n = 2). The groups differed significantly in Tf values (p < 0.0001). Lower values of Tf were associated with FID. Furthermore, FID is already found in the initial stages of CrO (26%). Immunosorbents based on nanobodies revealed the accumulation of apo-Tf and the decrease in holo-Tf in patients with CrO. (4) Conclusions: Tf may be a promising tool for diagnosing both CrO and associated FID.