Changes in Free Radical Processes under the Influence of Low-Frequency Electromagnetic Field in Rats.

We studied the effect of a low-frequency (LF) electromagnetic field (EMF) on the state of the antioxidant system of Wistar rats in vivo. It was found that changes in activity of antioxidant enzymes and H2O2 content in the blood plasma of rats exposed to LF EMF depended on the frequency of EMF. We propose a mechanism of the protective effects of low doses of ROS the generation of which is stimulated by LF EMF.

Cytoprotective and Antioxidant Effects of Meconic Acid in Model Systems.

We studied the antioxidant and cytoprotective effects of meconic acid in the model systems. Meconic acid, similar to commercial drug Mexidol, reduced the intensity of chemiluminescence in the model system of yolk lipoproteins. Meconic acid also reduced the toxic effect of glutamate on neurons in the primary cerebellar culture, but had no effect on cell viability under normal conditions.

Neurotrophic Effect of Magnesium Comenate in Normal and under Conditions of Oxidative Stress in Culture of Chicken Spinal Ganglia.

The neurotrophic properties of magnesium comenate were studied under standard conditions and under conditions of oxidative stress. It was found that magnesium comenate has a stimulating effect on the neurotrophic processes of the spinal ganglia under normal conditions and under conditions of oxidative stress. Under standard conditions, magnesium comenate exhibits neurotrophic activity at a concentration of 0.0001 mM, under conditions of oxidative stress, magnesium comenate exhibits neurotrophic activity at concentration 0.1 mM.

[Antioxidant and anxiolytic effect of Bifidobacterium adolescentis and Lactobacillus acidophilus under conditions of normobaric hypoxia with hypercapnia].

Research in recent years has shown that there is a close connection between the brain and the intestine through neuronal, endocrine and immune pathways. The introduction of probiotics into the diet of animals and humans helps to reduce the level of anxiety and depression, as well as inflammatory processes during emotional stress. The aim of this work was to study the effect of intragastric administration of Bifidobacterium adolescentis and Lactobacillus acidophilus on oxidative processes in the brain tissues and the level of anxiety in rats under conditions of normoxia and acute hypoxia with hypercapnia. Material and methods. The experiment was performed on 64 male Wistar rats aged 2.5 months (body weight from 240 to 270 g). The animals were divided into 4 groups: group 1 - control; 2 - hypoxia; 3 - hypoxia + probiotics; 4 - probiotics. There were 16 animals in each group; half of them participated in the behavioral test, and the other half in the biochemical studies. Rats of groups 3 and 4 were orally administered lyophilized bacteria Bifidobacterium adolescentis MC-42, Lactobacillus acidophilus A-97, and Lactobacillus acidophilus A-630 for 30 days before hypoxia. The daily dose of probiotics was 1×109 CFU per animal, administered in a volume of 1 ml. Acute hypoxia with hypercapnia was simulated by placing rats in airtight vessels with a capacity of 1 L before the first agonal inhalation. A day later, in the brain tissues oxidative processes were assessed by the chemiluminescence method and by the level of malone dialdehyde (MDA). The activity of catalase in brain tissues was also determined. The level of anxiety of rats was investigated in the «elevated plus maze¼ test. Results. Compared to other groups, more intensive free radical oxidation took place in the brain tissues of hypoxified animals that did not receive B. adolescentis and L. acidophilus. There was a significant increase in chemiluminescence intensity and MDA level by 38 and 15%, respectively, compared with the control. In the brain tissues of these animals, catalase activity was reduced by 10% (p<0.01). Moreover, in the group of rats treated with B. adolescentis and L. acidophilus and subjected to acute hypoxia, the value of the light sum of chemiluminescence was 22% lower (p<0.01) than in the hypoxified group without taking probiotics, while the concentration of MDA and catalase activity remained at the level of physiological norms and did not differ from control. Hypoxified animals receiving biomass of lactobacteria and bifidobacteria had also a lower level of anxiety and a higher exploratory activity, expressed in an increase in the number of entries in the open and closed arms, a longer stay in the open arms and the center of the maze, and more frequent performance of orientation reactions and hanging. Conclusion. Pre-hypoxic administration of B. adolescentis and L. acidophilus reduces the development of oxidative stress in rat brain tissues and reduces anxiety indices in the "elevated plus maze" test, thereby exhibiting antioxidant and anxiolytic effects.

[Immunohistochemical assay of murine liver tissue using monoclonal antibodies to NO-synthase 2 and TNF-α under tetrachloromethane toxic injury conditions].

Experimental data showing a significant role of enzyme NO-synthase type 2 and cytokine TNF-α enzymes in pathogenesis of CCl4-induced liver injury have been obtained by immunohistochemical assay. It is established that the hepatotoxic agent leads to an increase in NO-synthase 2 and TNF-α levels. The use of both studied phytoadaptogens (eleutherococcus extract and ginseng tincture) at a doses of 48.5 mg/kg and 47 mg/kg, respectively, and the reference drug karsil (100 mg/kg daily for 5 weeks) prevented to a significant degree the accumulation of enzyme NO-synthase 2 and the cytokine TNF-α, which is apparently one of the mechanisms of hepatoprotective action.

The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics.

The extracellular pH is lower in tumor than in normal tissue, whereas their intracellular pH is similar. In this study, we show that the tumor-specific pH gradient may be exploited for the treatment of cancer by weak acid chemotherapeutics. i.v.-injected glucose substantially decreased the electrode estimated extracellular pH in a xenografted human tumor while its intracellular pH, evaluated by (31)P magnetic resonance spectroscopy, remained virtually unchanged. The resulting increase in the average cell pH gradient caused a parallel increase in tumor growth delay by the weak acid chlorambucil (CHL). Regardless of glucose administration, the effect of CHL was significantly greater in tumors preirradiated with a large dose of ionizing radiation. This suggests that CHL was especially pronounced in radioresistant hypoxic cells possessing a larger transmembrane pH gradient. These results indicate that the naturally occurring cell pH gradient difference between tumor and normal tissue is a major and exploitable determinant of the uptake of weak acids in the complex tumor microenvironment. The use of such drugs may be especially effective in combination with radiation.

Vascular endothelial growth factor receptor-2-blocking antibody potentiates radiation-induced long-term control of human tumor xenografts.

Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.

Topotecan selectively enhances the radioresponse of human small-cell lung carcinoma and glioblastoma multiforme xenografts in nude mice.

PURPOSE: To evaluate the therapeutic efficacy of different combinations of the DNA topoisomerase I-targeting drug, topotecan (TPT), with radiation for treatment of two human tumor xenografts. METHODS AND MATERIALS: The small cell lung carcinoma 54A and glioblastoma multiforme U87 were transplanted into nude mice. Equal i.p. injections of TPT and/or equal fractions of tumor irradiation were administered daily, for 5 consecutive days. When combined, TPT was injected at different constant time intervals prior to or after each radiation fraction. The tumor growth delay and changes in skin radiation reaction by TPT were evaluated. Tumor oxygenation was measured using the Eppendorf pO(2) histography. RESULTS: The tumor growth delay induced by such chemoradiotherapy was independent of interval and sequencing of the agents for either tumor model. The efficacy of TPT alone or in combination with radiation was always dose-dependent, although of different magnitude in the two xenografts. In 54A xenografts, TPT alone induced longer growth delay, but its combined effect with radiation was not more than additive. In contrast, U87 responded less to TPT alone, however the drug and radiation interacted synergisticly in this tumor model. Using both a radiobiological approach (tumor irradiation under normoxia vs. clamp hypoxia conditions) and the polarographic electrode measurements, it was shown that TPT did not modify tumor oxygenation and, thus, unlikely modulated oxygen-related tumor radiosensitivity. In contrast to tumors, TPT virtually unchanged skin radiation reaction. CONCLUSIONS: Our data suggest that TPT, when combined with radiation treatment of tumors, provides a therapeutic gain without substantial local and systemic adverse effects.

Hydralazine at thermoradiotherapy: tumor size and blood flow effects.

PURPOSE: This study was aimed to assess the dependence on tumor size and blood flow of the efficacy of a vasoactive drug hydralazine with thermoradiotherapy. METHODS AND MATERIALS: Experiments were performed on mice bearing SCC-VII tumors with volumes of about 85 and 340 mm3 (7-8 or 11-12 days after transplantation, respectively). Local hyperthermia (water bath, 43 degrees C, 0.5 h) was started 3 h after irradiation of tumors. Hydralazine (2.5 mg/kg, IP) was given 0.5 h before heating. Tumor blood flow was evaluated by laser Doppler flowmetry before, during and up to 2 days after the treatments. RESULTS: It was shown that hydralazine and hyperthermia, even in combination with each other, had very weak anti-tumor effect, especially for 85 mm tumors. The agents also insignificantly enhanced the efficacy of radiotherapy excluding the case of polyradiomodification for 340 mm3 tumors when a dose modifying factor of about 2.0 was achieved. Thermometry showed only a small improvement by HDZ in heating patterns of tumors of both sizes. Meanwhile, the therapeutic efficacy of hydralazine and heat was correlated with the changes in tumor blood flow, first of all with the delayed effects. The radiomodifiers induced only minor and transient suppression of perfusion in the smaller tumors, and more markedly and for longer time decreased blood flow in the larger tumors. In the latter case, the inhibiting effect of the drug plus hyperthermia remained for at least 48 h after the treatment. CONCLUSION: (a) The combined use of hydralazine and heat seems to be advisable only at radiotherapy of rather large advanced tumors; (b) the efficacy of such radiomodification is correlated with prolonged inhibition of tumor blood flow by these agents; and (c) hydralazine and hyperthermia are likely to kill selectively both acutely and chronically hypoxic radioresistant cancer cells.

Relative biological effectiveness of proton beams in clinical therapy.

PURPOSE: In clinical proton beam radiation therapy, an RBE of 1.1 relative to megavoltage X-rays is currently being employed at most treatment centers. This RBE pertains to radiation in the spread out Bragg-peak (SOBP) for all tissue systems, all dose levels per fraction and all proton beam energies. As the number of centers and treatment sites for which proton beam therapy continues to increase and additional experimental data is accrued, a re-assessment of the justification for a generic RBE is warranted. In this paper we address: (1) the constancy of the RBE along the central axis from the plateau entrance to the distal SOBP (upstream of the distal edge); (2) RBE as a function of dose (or cell survival level); and (3) the target cell or tissue (alpha/beta) dependency of the RBE. This analysis pertains to modulated proton beams of initial energies of approximately 70-200 MeV and SOBPs of approximately 2-10 cm, respectively. RESULTS AND CONCLUSIONS: With exceptions, the available experimental data indicate that the RBE of SOBP protons increases with decreasing dose or dose per fraction and increasing depth in the SOBP, with the magnitude of both effects likely being dependent on the alpha/beta ratios of the target cells or tissues. The use of a generic RBE of 1. for all tissues, especially those exhibiting low alpha/beta values such as CNS, may be too low, especially at dose levels of < or = 2 Gy/fraction. Systematic determination of the RBE values dependent upon the three interdependent variables identified in this manuscript (beam depth, dose size and target tissue) will provide an enhanced data base for detailed treatment planning and institutional trial comparisons, thereby maximizing the therapeutic benefit of proton beams.

Thermoradiotherapy with hydralazine: the effect of preirradiation of the tumor bed on blood flow and growth delay of Ehrlich carcinoma.

Ehrlich carcinoma transplanted into preirradiated calf muscle of mice was used as a model for tumor recurrence after unsuccessful radiotherapy. Due to the tumor bed effect (TBE), these grafts grew more slowly than control tumors implanted in the unirradiated tissue. When these tumors achieved the same volume (0.3-0.4 cm3), in 10-11 days for tumors implanted in irradiated tissue and 7-8 days for control tumors, they were treated with radiation, the tumor blood flow inhibitor hydralazine, and hyperthermia, alone or in different combinations. In the case of the trimodality treatment, single irradiation of tumors at a dose of 12.5 Gy was followed 2.5-3 h later by administration of hydralazine (2.5 mg/kg) and local hyperthermia (water bath, 43 degrees C for 30 min). The growth delay induced in the different tumor types by irradiation, hydralazine and hyperthermia, alone or in different combinations, was related to the blood flow measured in the tumors by the 133Xe clearance technique 24-48 h after treatment. It was shown that the reduction of blood flow after treatment with hyperthermia or hydralazine was approximately equal in both types of tumors. However, the combined inhibiting effect of these agents differed in the tumors: It was synergistic in control tumors and close to additive in tumors implanted in irradiated tissue. In terms of the specific tumor growth delay, the latter tumors were slightly more sensitive to hyperthermia, but were more resistant to radiation and thermoradiotherapy compared to control tumors. Hydralazine potentiated the tumoricidal effects of heat alone and heat combined with radiation. The enhancement was more substantial in control tumors compared to tumors implanted in irradiated tissue. A general correlation between the hydralazine-induced enhancement of the effects of heat on tumor blood flow and growth delay was observed. In tumors implanted in irradiated tissue, the inhibition of perfusion after treatment with hydralazine plus hyperthermia was smaller, and presumably a less marked treatment response to these agents (with or without radiation) was therefore achieved as a result in these tumors compared to the control tumors.

[Postradiation impairment of tumor blood supply using polyradiomodification for increasing the effectiveness of radiotherapy]. / Postradiatsionnoe narushenie krovosnabzheniia opukholei pri poliradiomodifikatsii dlia povysheniia éffektivnosti luchevoi terapii.

A therapeutic effect of the postradiation use of methods disturbing the blood supply of malignant neoplasms: short-term hyperglycemia (STH), mexamine (M) and a tourniquet (T)--was assessed in experiments on mice with solid Ehrlich carcinoma. It was shown that an independent use of STH, M and T without radiation exposure did not possess a significant antitumor effect whereas their use shortly after irradiation enhanced the effect of the latter on tumors without increasing skin radiation injury in the zone of tumor growth. The utmost antitumor effect was achieved in combination of irradiation with immediate STH followed either by M or T: in these cases a tumor radiation effect was enhanced in different series of experiments 1.5-2-or over 3-fold. A conclusion has been made as to the importance of the blood supply inhibition in malignant tumors in the postradiation period.

Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient.

Theory suggests that the transmembrane pH gradient may be a major determinant of the distribution of lipophilic weak electrolytes across the cell membrane. The present study evaluates the extent to which this factor contributes to pH-dependent changes in the cytotoxicity of two such chemotherapeutic drugs: chlorambucil and mitoxantrone. Experiments were performed with two cell types of the same origin but exhibiting different pH gradients at the same extracellular pH (pHe): CHO cells cultured under normal physiological conditions (pH 7.4) and acid-adapted cells obtained by culturing under low pH conditions (6.8). Over the pHe range examined (6.0-7.6), the difference between intracellular pH (pHi) and pHe increased with decreasing pHe. Acid-adapted cells were more resistant to acute changes in pHi than normal cells, resulting in substantially larger gradients in these cells. Drug cell survival curves were performed at pHe values of 6.4, 6.8 and 7.4. The cytotoxicity of chlorambucil, a weak acid, increased with decreasing pHe, and low pH-adapted cells were more sensitive than normal cells at the same pHe. In contrast, for the weak base, mitoxantrone, cytotoxicity increased with pHe and was more pronounced in normal cells. As predicted by the theory, the cytotoxicity of both drugs changed exponentially as a function of the pH gradient, regardless of cell type. For mitoxantrone, the rate of such change in cytotoxicity with the gradient was approximately two times greater than for chlorambucil. This difference is probably due to the presence of two equally ionizable crucial groups on mitoxantrone vs one group on chlorambucil. It is concluded that the cellular pH gradient plays a major role in the pH-dependent modulation of cytotoxicity in these weak electrolytes. The data obtained also suggest that a pronounced differential cytotoxicity may be expected in vivo in tumour vs normal tissue. In comparison with normal cells at a pHe of 7.4 (a model of cells in normal tissues), acid-adapted cells at a pHe of 6.8 (a model of cells distal from supplying blood vessels in tumours) were more sensitive to chlorambucil, with a dose-modifying factor of approximately 6, and were more resistant to mitoxantrone by a factor of 14.

Antitumour effect of irradiation followed by hyperglycemia and hyperthermia: the dependence on tumour size and blood flow.

The interrelationship of three tumour parameters-volume, blood flow and growth delay-was evaluated after irradiation alone or combined with hyperglycemia (HG) and hyperthermia (HT). The experiments were performed on Ehrlich carcinoma tumours 5, 7 or 9 days after intramuscular implantation when they reached a mean volume of approximately 0.17, 0.46 or 0.90 cm3, respectively. Tumours were irradiated at a dose of 12.5 Gy. In groups of trimodality therapy, induction of HG (5i.p. injections of glucose during a 2-h period, total dose of glucose 10.4 g/kg) was started immediately after irradiation, and HT (water bath, 43 degrees C, 30 min) was given 3 h later. Regardless of the method of therapy, blood flow in the central part of each tumour was measured 4 and 7 h after irradiation by 133Xe clearance technique. It was shown that if tumours were treated by irradiation alone, both tumour blood flow and growth delay declined with increasing tumour volume. There was no correlation between individual values of tumour blood flow intensity and growth delay within each size group, and only all the data pooled together showed a significant direct relationship between these two parameters. In contrast, for radiotherapy with HG and HT, the antitumour effect increased with tumour volume. Blood flow was strongly inhibited by radio-modifiers; the larger tumours, the lower levels of perfusion were observed. There was a pronounced trend of increased tumour growth delay with decreased blood flow for tumours of the same volume, and these parameters were highly correlated if all individual data were analysed together regardless of tumour size. It was concluded that postirradiation blood flow inhibition plays a significant role in the tumouricidal effect of irradiation with HG and HT. The monitoring of tumour perfusion may be useful for the prediction of the effectiveness of such postirradiation modification, which is especially attractive in radiotherapy of large advanced tumours.

[Quantitative assessment of morphologic changes in tumors during combined use of irradiation, induced hyperglycemia and local hyperthermia]. / Kolichestvennaia otsenka morfologicheskikh izmenenii v opukholiakh pri sochetannom primenenii oblucheniia, iskusstvennoi giperglikemii i lokal'noi gipertermii.

The paper is concerned with morphometric evaluation of changes in Ehrlich mouse solid carcinoma after a separate, dual and combined use of x-ray irradiation, induced hyperglycemia (IH) and local hyperthermia (HT), using two-staged quantitative analysis of tumor tissue injuries. A portion of viable tissue was determined at the first stage, and the density of location of undestroyed tumor cells was evaluated at the second stage. The general result was characterized by the product of these indices. Disorders in the tumor tissue structure were noted in 24 h, the main damage being done by IH and HT in the center of a tumor. In 48 h the total effect was on the increase only in groups with irradiation as a result of cell death in the peripheral zone. In the double use of the agents their combined effect was less than the additive one, and the combined use of all three methods resulted in obvious synergism, and the number of viable tumor cells (by morphological criteria) was decreased by more than two orders.

[Effect of blood flow inhibition in tumors on the effectiveness of hyperglycemia and hyperthermia when used following radiation]. / Vliianie ingibirovaniia krovotoka v opukholiakh na éffektivnost' giperglikemii i gipertermii pri ikh postradiatsionnom primenenii.

The role of tumor blood supply inhibition associated with hyperthermia (HT) and short-term hyperglycemia (SH) combined with HT in enhancement of antitumor action of x-ray radiation was assessed in experiments on solid Ehrlich carcinoma in mice. It was shown that blood flow inhibition assessed by 133Xe clearance, and tumor growth delay were more pronounced after irradiation at a dose of 10 Gy with subsequent SH and HT at 43 degrees C for 30 min as compared to a dose of 15 Gy combined with HT at 44 degrees C for 30 min. Both schemes showed correlation between a degree of blood supply damage in some tumors and the efficacy of their therapy. The authors suggest a significant role of blood flow inhibition in tumors in postradiation HT and especially HT in a background of SH for increasing antitumor effect of irradiation. The assessment of tumor blood supply inhibition is possible to predict the efficacy of SH and HT in radiation therapy.

[Hypoxic proton radiotherapy of solid Ehrlich tumors with different dose magnitudes]. / Protonnaia gipoksiradioterapiia solidnykh opukholei Erlikha pri raznoi moshchnosti dozy.

The paper is devoted to an experimental evaluation of the efficiency of hypoxiradiotherapy with irradiation of tumors by protons at different dose rates. In experiments performed at ITEP synchrotron it has been shown that the use of the hypoxic gas mixture containing 6% O2 for breathing appreciably diminishes the degree of skin reactions with only weak tumor protection. It is concluded that the use of hypoxic hypoxia during proton irradiation afford the same advantages as during gamma- or x-ray therapy. The skin protection can be also achieved by use of ultra-high dose rate and in this case it is associated with radiochemical depletion of oxygen. This effect may be used for the improvement of radiotherapy of malignant tumors.

The pH partition theory predicts the accumulation and toxicity of doxorubicin in normal and low-pH-adapted cells.

The accumulation and toxicity of the weak base doxorubicin has been investigated as a function of extracellular pH, intracellular pH and the cellular pH gradient in cells previously cultured under normal (pH 7.4) and low-pH (6.8) conditions. Low-pH-adapted cells exhibit transmembrane pH gradients which substantially differ from normal cells at the same extracellular pH. No relationship was obtained between intracellular pH and the uptake or toxicity of doxorubicin in the two cell types. In contrast, doxorubicin accumulation and toxicity increased with increasing extracellular pH in both normal and low-pH-adapted cells. However, at the same extracellular pH, drug cytotoxicity was more pronounced in normal than in low-pH-adapted cells. The difference in doxorubicin accumulation and cytotoxicity at the same extracellular pH was found to be dependent on the difference in the transmembrane pH gradient of the two cell types. As the cellular pH gradient differs between tumour and normal tissue, this observation suggests a basis for enhancing cellular drug uptake in either tissue type.