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Biomedical subjects

M C Krishna

Publications and source records attributed to M C Krishna.

At least 37 records · Page 2Linked to original sources
PubMed1998

The role of nitric oxide chemistry in cancer treatment.

Over the last decade the role of nitric oxide (NO) in various disease states has become apparent. In cancer, NO plays a variety of roles which are at times contradictory. On one hand, NO is involved in different etiological mechanisms as well as promoting tumor growth. Yet, NO derived from leukocytes plays a seminal role in their tumoricidal activity. In cancer treatment, NO also has diverse effects. Whereas in vitro, NO can enhance the cytotoxic efficacy of some chemotherapeutic agents as well as radiation, NO donors can provide whole body protection against these same agents. This manuscript will discuss some mechanisms involved with NO and cancer treatment modalities and the potential application of these findings to cancer therapy.

Animals↗
PubMed1997

Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. Chemical aspects involved in the balance between oxidative and nitrosative stress.

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O-2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O-2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O-2 were nearly equivalent. The presence of superoxide dismutase reversed O-2-mediated inhibition, while catalase had no effect. Increasing the flux of O-2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O-2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.

Free Radicals↗
PubMed1997

In vivo imaging of a stable paramagnetic probe by pulsed-radiofrequency electron paramagnetic resonance spectroscopy.

Imaging of free radicals by electron paramagnetic resonance (EPR) spectroscopy using time domain acquisition as in nuclear magnetic resonance (NMR) has not been attempted because of the short spin-spin relaxation times, typically under 1 microsecond, of most biologically relevant paramagnetic species. Recent advances in radiofrequency (RF) electronics have enabled the generation of pulses of the order of 10-50 ns. Such short pulses provide adequate spectral coverage for EPR studies at 300 MHz resonant frequency. Acquisition of free induction decays (FID) of paramagnetic species possessing inhomogenously broadened narrow lines after pulsed excitation is feasible with an appropriate digitizer/averager. This report describes the use of time-domain RF EPR spectrometry and imaging for in vivo applications. FID responses were collected from a water-soluble, narrow line width spin probe within phantom samples in solution and also when infused intravenously in an anesthetized mouse. Using static magnetic field gradients and back-projection methods of image reconstruction, two-dimensional images of the spin-probe distribution were obtained in phantom samples as well as in a mouse. The resolution in the images was better than 0.7 mm and devoid of motional artifacts in the in vivo study. Results from this study suggest a potential use for pulsed RF EPR imaging (EPRI) for three-dimensional spatial and spectral-spatial imaging applications. In particular, pulsed EPRI may find use in vivo studies to minimize motional artifacts from cardiac and lung motion that cause significant problems in frequency-domain spectral acquisition, such as in continuous wave (cw) EPR techniques.

Animals↗
PubMed1997

Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin.

A major emphasis in cancer therapy research is finding mechanisms to enhance the effectiveness of clinically used chemotherapeutic agents. In this report, we show the effects of direct NO exposure or NO delivery agents such as NONOate NO donors, DEA/NO ((C2H5)2N[N(O)NO]-Na+) and PAPA/ NO (NH2(C3H6)(N[N(O)NO]C3H7)), or S-nitrosothiol NO donors (GSNO, S-nitrosoglutathione, and SNAP, S-nitroso-N-acetylpenicillamine) on the cytotoxicity of cisplatin with Chinese hamster V79 lung fibroblast cells. Cells pretreated with bolus NO or NO delivered from NONOate NO donors were markedly sensitized to subsequent cisplatin treatment, whereas S-nitrosothiol NO donors exerted little effect. The enhancement in cisplatin cytotoxicity from pretreatment with DEA/NO and PAPA/ NO persisted for approximately 180 and 240 min, respectively; thereafter cytotoxicity returned to a level consistent with cisplatin treatment alone. Pretreatment of cells with GSNO or SNAP did not enhance cisplatin cytotoxity. To discern why there were differential effects among the different NO donors, formation of NO over the time course of the experiment was assessed by the nitrosation of 2,3-diaminonaphthylene. Bolus NO, DEA/NO, and PAPA/NO produced more reactive nitrogen oxide species (RNOS) than did treatment with GSNO or SNAP. Previously reported electrochemical studies revealed that temporal NO concentrations measured from DEA/NO and PAPA/NO (1 mM) were greater than 5 microM. It appears that the flux of NO, as well as the amount of RNOS, is important in the NO-mediated enhancement of cisplatin cytotoxicity. Our results demonstrate the importance of NO delivery systems in the enhancement of cisplatin cytotoxicity and may provide insights into strategies for participation of NO donors and nitric oxide synthase with cisplatin therapy.

Animals↗
PubMed1997

Direct evidence for in vivo nitroxide free radical production from a new antiarrhythmic drug by EPR spectroscopy.

The new Class I anti-arrhythmic agent 2,2,5,5-tetramethyl-3-pyrroline-1-carboxamide derivative, is currently being evaluated in clinical trials in patients with a high risk for cardiac arrhythmias. In this study we show that this antiarrhythmic drug can be chemically converted to the nitroxide free radical analog. Further, using an in vivo Electron Paramagnetic Resonance (EPR) spectroscopy model by detecting free radicals in the distal portion of the tail of an anesthetized mouse, we demonstrate that the drug is oxidized to the corresponding nitroxide. In vitro studies using Chinese hamster V79 cells suggest that the oxidation products of the drug, namely, the hydroxylamine and the nitroxide protect against oxidative damage induced by hydrogen peroxide (H2O2). Taken together, our results suggest that, in addition to the antiarrhythmic effects of the parent drug, sufficient levels of nitroxides may accumulate from the parent drug in vivo to provide antioxidant defense to cardiac tissue that may be subject to ischemia and oxidation-driven injury.

Animals↗
PubMed1997

Nitric oxide enhancement of melphalan-induced cytotoxicity.

The effects of the diatomic radical, nitric oxide (NO), on melphalan-induced cytotoxicity in Chinese hamster V79 and human MCF-7 breast cancer cells were studied using clonogenic assays. NO delivered by the NO-releasing agent (C2H5)2N[N(O)NO]- Na+ (DEA/NO; 1 mM) resulted in enhancement of melphalan-mediated toxicity in Chinese hamster V79 lung fibroblasts and human breast cancer (MCF-7) cells by 3.6- and 4.3-fold, respectively, at the IC50 level. Nitrite/nitrate and diethylamine, the ultimate end products of DEA/NO decomposition, had little effect on melphalan cytotoxicity, which suggests that NO was responsible for the sensitization. Whereas maximal sensitization of melphalan cytotoxicity by DEA/NO was observed for simultaneous exposure of DEA/NO and melphalan, cells pretreated with DEA/NO were sensitized to melphalan for several hours after NO exposure. Reversing the order of treatment also resulted in a time-dependent enhancement in melphalan cytotoxicity. To explore possible mechanisms of NO enhancement of melphalan cytotoxicity, the effects of DEA/NO on three factors that might influence melphalan toxicity were examined, namely NO-mediated cell cycle perturbations, intracellular glutathione (GSH) levels and melphalan uptake. NO pretreatment resulted in a delayed entry into S phase and a G2/M block for both V79 and MCF-7 cells; however, cell cycle redistribution for V79 cells occurred after the cells returned to a level of cell survival, consistent with treatment with melphalan alone. After 15 min exposure of V79 cells to DEA/NO (1 mM), GSH levels were reduced to 40% of control values; however, GSH levels recovered fully after 1 h and were elevated 2 h after DEA/NO incubation. In contrast, DEA/NO (1 mM) incubation did not reduce GSH levels significantly in MCF-7 cells (approximately 10%). Melphalan uptake was increased by 33% after DEA/NO exposure in V79 cells. From these results enhancement of melphalan cytotoxicity mediated by NO appears to be complex and may involve several pathways, including possibly alteration of the repair of melphalan-induced lesions. Our observations may give insights for improving tumour kill with melphalan using either exogenous or possibly endogenous sources of NO.

Animals↗
PubMed1996

Stimulation by nitroxides of catalase-like activity of hemeproteins. Kinetics and mechanism.

The ability of stable nitroxide radicals to detoxify hypervalent heme proteins such as ferrylmyoglobin (MbFeIV) produced in the reaction of metmyoglobin (MbFeIII) and H2O2 was evaluated by monitoring O2 evolution, H2O2 depletion, and redox changes of the heme prosthetic group. The rate of H2O2 depletion and O2 evolution catalyzed by MbFeIII was enhanced by stable nitroxides such as 4-OH-2,2,6,6-tetramethyl-piperidinoxyl (TPL) in a catalytic fashion. The reduction of MbFeIV to MbFeIII was the rate-limiting step. Excess TPL over MbFeIII enhanced catalase-like activity more than 4-fold. During dismutation of H2O2, [TPL] and [MbFeIV] remained constant. NADH caused: (a) inhibition of H2O2 decay; (b) progressive reduction of TPL to its respective hydroxylamine TPL-H; and (c) arrest/inhibition of oxygen evolution or elicit consumption of O2. Following depletion of NADH the evolution of O2 resumed, and the initial concentration of TPL was restored. Kinetic analysis showed that two distinct forms of MbFeIV might be involved in the process. In summary, by shuttling between two oxidation states, namely nitroxide and oxoammonium cation, stable nitroxides enhance the catalase mimic activity of MbFeIII, thus facilitating H2O2 dismutation accompanied by O2 evolution and providing protection against hypervalent heme proteins.

Animals↗
PubMed1996

Do nitroxide antioxidants act as scavengers of O2-. or as SOD mimics?

Stable nitroxide radicals were reported to act as SOD mimics and catalyze the dismutation of O2-. through two different catalytic pathways including reductive and oxidative reaction mechanisms (Samuni, A., Krishna, C. M., Riesz, P., Finkelstein, E. & Russo, A. (1988) J. Biol Chem. 263, 17921-17924). Recent studies directly monitoring O2-. and employing kinetics analysis did not reveal SOD activity of nitroxides (Weiss, R. H., Flickinger, A. G., Rivers, W. J., Hardy, M. M., Aston, K. W., Ryan, U. S. & Riley, D. P. (1993) J. Biol. Chem. 268, 23049-23054). Such discrepancy may result in cases where distinction of stoichiometric scavengers from catalytic detoxifiers of O2-. is not readily feasible. Nitroxides are effective antioxidants that protect against oxidative injury in various pathological processes. The distinction of their SOD mimic activity from O2-. scavenging was established by examining the validity of direct and indirect methods employed to assay SOD-like catalytic activity. Kinetics analysis along with direct EPR monitoring were used to study the mechanism underlying nitroxide reactions with O2-.. The nitroxide EPR signal decayed in the presence of NADH but otherwise did not decrease with time, thus substantiating its catalytic role in O2-. dismutation. The catalytic rate constants for O2-., dismutation, determined for the nitroxides tested, were found to increase with [H+], indicating that .OOH rather than O2-. is oxidizing the nitroxide. The results demonstrate the limitations associated with direct kinetics analysis in evaluating SOD mimic activity, underscoring the need for independent assays for valid discrimination of SOD mimics from stoichiometric scavengers of O2-..

Antioxidants↗
PubMed1996

The effect of various nitric oxide-donor agents on hydrogen peroxide-mediated toxicity: a direct correlation between nitric oxide formation and protection.

The role that nitric oxide (NO) plays in various degenerative and disease states has remained a mystery since its discovery as a biological messenger, prompting the question, "NO, friend or foe?" Some reports have suggested that NO is cytotoxic, and yet others have shown that it possesses protective properties against reactive oxygen species (ROS). Many studies have used various NO donor complexes arriving at seemingly different conclusions. This report will address the effects of various NO donor compounds on ROS-mediated toxicity. Consistent with our previous study, the NO donor compound, DEA/NO ((C2H5)2N[N(O)NO]-Na+), afforded protection against hydrogen peroxide-mediated cytotoxicity in V79 Chinese hamster lung fibroblasts at concentrations as low as 10 microM DEA/NO. Furthermore, a survey of other NO donor complexes revealed that some either protected or potentiated hydrogen peroxide-mediated cytotoxicity. 3-Morpholinosynodiomine.HCl (SIN-1) and sodium nitroprusside (SNP) enhanced hydrogen peroxide-mediated cytotoxicity, while S-nitrosoglutathione (GSNO), and S-nitroso-N-acetylpenicillamine (SNAP) afforded protection. Electrochemical detection of NO in cell culture medium revealed that neither 1000 microM SIN-1 nor SNP yielded appreciable NO concentrations (<0.3 microM). In contrast, DEA/NO, SNAP, and GSNO yielded fluxes of NO >1.0 microM. Thus, a direct correlation between inhibition of hydrogen peroxide cytotoxicity and NO production was observed: agents that release NO during hydrogen peroxide treatment afford significant protection, whereas agents that do not release NO do not protect. Similar results were observed for NO donors studied when hypoxanthinesolidusxanthine oxidase was used as the source for ROS, although the S-nitrosothiol agents were much less protective. These results demonstrate that NO possesses properties which protect against ROS toxicity and demonstrate how the use of different NO donor compounds can lead to different conclusions about the role that NO can play in the cytotoxicity of ROS.

Animals↗
PubMed1996

Convenient colorimetric and fluorometric assays for S-nitrosothiols.

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use. Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.

Benzothiazoles↗
PubMed1996

Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin.

Electron paramagnetic resonance (EPR) imaging utilizing stable nitroxyl radicals is a promising technique for measuring free radical distribution, metabolism, and tissue oxygenation in organs and tissues [Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D. J., Giannella, E., & Zweier, J. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3388-3392]. However, the technique has been limited by the rapid reduction of nitroxide in vivo to its hydroxylamine derivative, a diamagnetic, EPR-inactive species. In this report a novel, polynitroxylated derivative of human serum albumin is shown to be capable of reoxidizing the hydroxylamine back to nitroxide in vivo. Polynitroxyl-albumin (PNA) is shown to be effective in maintaining the signal intensity of the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL or TPL) in the ischemic isolated rat heart, allowing the acquisition of high-resolution three-dimensional (3D) EPR images of the heart throughout a prolonged 2.5 h period of global cardiac ischemia. In serial transverse sections of the 3D image, TPL intensity maps of the heart showed cardiac structure with submillimeter resolution. TPL intensities in coronary arteries and myocardium showed that nitroxide concentration decreases with increasing distance from large blood vessels. These results demonstrate that EPR imaging in vivo is possible using nitroxides in conjunction with PNA. In addition to its utility in the emerging technology of EPR imaging, the greatly prolonged half-life of TPL observed in the presence of PNA may facilitate the therapeutic application of nitroxides in a variety of disease processes.

Animals↗
PubMed1996

Radiation sensitisation by nitric oxide releasing agents.

Previous studies have shown that nitric oxide (NO) sensitises hypoxic cells to ionising radiation. In the present study, four different nitric oxide (NO) donor agents were evaluated for both NO release and hypoxic radiosensitisation. The S-nitrosothiol NO donor agents, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), were shown to release sustained NO concentrations (microM) and significantly radiosensitise hypoxic cells. The extent of hypoxic radiosensitisation by both of these agents at 1.0 mM concentration was similar to that obtained with molecular oxygen. In contrast, neither 3-morpholinosydnonimine (SIN-1) nor sodium nitroprusside (SNP) released detectable NO concentrations and neither agent enhanced the hypoxic radiation response to the extent of that observed for GSNO or SNAP. NO-mediated hypoxic cell radiosensitisation by NO donor drugs may offer a new approach for clinical consideration, particularly if such agents can be selectively delivered to hypoxic cells.

Animals↗
PubMed1995

Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli.

Previously, we reported that nitric oxide (NO) provides significant protection to mammalian cells from the cytotoxic effects of hydrogen peroxide (H2O2). Murine neutrophils and activated macrophages, however, produce NO, H2O2, and other reactive oxygen species to kill microorganisms, which suggests a paradox. In this study, we treated bacteria (Escherichia coli) with NO and H2O2 for 30 min and found that exposure to NO resulted in minimal toxicity, but greatly potentiated (up to 1,000-fold) H2O2-mediated killing, as evaluated by a clonogenic assay. The combination of NO/H2O2 induced DNA double strand breaks in the bacterial genome, as shown by field-inverted gel electrophoresis, and this increased DNA damage may correlate with cell killing. NO was also shown to alter cellular respiration and decrease the concentration of the antioxidant glutathione to a residual level of 15-20% in bacterial cells. The iron chelator desferrioxamine did not stop the action of NO on respiration and glutathione decrease, yet it prevented the NO/H2O2 synergistic cytotoxicity, implicating metal ions as critical participants in the NO/H2O2 cytocidal mechanism. Our results suggest a possible mechanism of modulation of H2O2-mediated toxicity, and we propose a new key role in the antimicrobial macrophagic response for NO.

Catalase↗
PubMed1995

Nitric oxide protects against alkyl peroxide-mediated cytotoxicity: further insights into the role nitric oxide plays in oxidative stress.

Endogenously formed nitric oxide (NO) possesses diverse properties such as regulating physiological functions, exerting specific toxic effects, and protecting against various toxic substances. Recent studies suggest that in the presence of reactive oxygen species, NO can serve as an antioxidant. We show here that NO delivered from the NO donor compound, PAPA/NO (NH2(C3H6)(N[N(O)NO](C3H7)), protects Chinese hamster V79 lung fibroblasts from the cytotoxicity of t-butyl hydroperoxide and cumene hydroperoxide. In contrast, the other end products of PAPA/NO degradation in aqueous solution, NH2(C3H6)NH(C3H7) and nitrite, did not protect. The NONOate DEA/NO releases NO six times faster than PAPA/NO, yet did not afford protection, which implies that NO must be present throughout the alkyl hydroperoxide exposure. Measurements of NO concentrations released from PAPA/NO suggest that micromolar levels protect against cytotoxicity induced by alkyl hydroperoxides. These findings demonstrate that the flux of NO sustained over the duration of the peroxide exposure determines protection and not the total of NO delivered. These results suggest that concentrations of NO produced in the microenvironment of endothelial cells are high enough to protect cells from Fenton-type-mediated toxicity and support the premise that NO may exert a salutary effect in certain diseases associated with membrane damage.

Alkylating Agents↗