Cellular sites of H2O2-induced damage and their protection by nitroxides.

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.

Multifunctional antioxidant activity of HBED iron chelator.

The use of N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED) for iron chelation therapy is currently being tested. Besides its affinity for iron, bioavailability, and efficacy in relieving iron overload, it is important to assess its anti- and/or pro-oxidant activity. To address these questions, the antioxidant/pro-oxidant effects of HBED in a cell-free solution and on cultured Chinese hamster V79 cells were studied using UV-VIS spectrophotometry, oximetry, spin trapping, and electron paramagnetic resonance (EPR) spectrometry. The results indicate that HBED facilitates Fe(II) oxidation but blocks O2(.-)-induced reduction of Fe(III) and consequently pre-empts production of .OH or hypervalent iron through the Haber-Weiss reaction cycle. The efficacy of HBED as a 1-electron donor (H-donation) was demonstrated by reduction of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-derived nitrogen-centered radical cation (ABTS(.+)), accompanied by formation of a short-lived phenoxyl radical. HBED also provided cytoprotection against toxicity of H2O2 and t-BuOOH. Our results show that HBED can act both as a H-donating antioxidant and as an effective chelator lacking pro-oxidant capacity, thus substantiating its promising use in iron chelation therapy.

Radiation and the Apo2L/TRAIL apoptotic pathway preferentially inhibit the colonization of premalignant human breast cells overexpressing cyclin D1.

The role of cyclin D1 overexpression in human breast premalignancy was investigated using immortal, nontumorigenic MCF-10A cells. Previous work documented that cyclin D1 overexpression promoted in vitro anchorage-independent colonization. We now report that the colonization of MCF-10A cyclin D1 transfectants was preferentially inhibited by gamma-radiation and specific classes of apoptosis inducers [Apo-2 ligand (Apo-2L), but not tumor necrosis factor alpha]. Antibody inhibition studies and semiquantitative PCR indicated that radiation inhibition of colonization was partially mediated via the Apo2L/TRAIL pathway. The apoptotic removal of cyclin D1-overexpressing, colonization-competent premalignant breast cells by Apo2L/TRAIL or other biologicals may represent a novel approach to the prevention of breast cancer.

Regulation of transforming growth factor beta1 by nitric oxide.

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

The use of Zn-desferrioxamine for radioprotection in mice, tissue culture, and isolated DNA.

Redox-active metals mediate oxidative injury and might also potentiate radiation damage. The iron chelator desferrioxamine (DFO), which diminishes oxidative damage in many chemical and biological systems as well as in human subjects, has a controversial role in radiobiology and reportedly acts both as a radiosensitizer and a radioprotector. The present research focused on the radioprotective activity of its zinc complex. Zn-DFO was studied using three test systems differing by their complexities: isolated DNA from pUC 19 plasmid, cultured V79 Chinese hamster cells, and C3H mice. Zn-DFO (0.5-2 mM) protected isolated DNA against gamma-radiation better than each of its components alone; however, neither Zn-DFO nor DFO (50-100 microM) alone affected the radiation sensitivity of cultured cells. With total body irradiation, Zn-DFO, but not DFO alone at 100 micromol/kg body weight, administered to mice 30 min before irradiation provided significant radioprotection (P < 0.01). Zn-DFO had an LD(50/30) of 10.3 Gy, whereas DFO and vehicle alone had LD(50/30) of 8.03 Gy and 7.91 Gy, respectively. The effect of Zn-DFO on the hemodynamic parameters in mice did not differ from that of the vehicle (saline) alone. This excludes the explanation that the radioprotective activity of Zn-DFO results from its effect on oxygen levels. In addition to the possible direct effect of Zn, other potential modes of action underlying the radioprotective activity of Zn-DFO might involve a displacement of iron and its substitution by zinc, a greater proximity of the drug to DNA, and less likely an improved penetration of the drug into cells because of its structure. The failure of Zn-DFO to protect cells in tissue cultures indicates that it has some systemic role in the whole animal, possibly due to a prolonged half-life in the animal's circulation.

Redox generation of nitric oxide to radiosensitize hypoxic cells.

PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation. In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents. METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay. Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions. Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol. NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode. RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively. Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity. The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode. CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization. NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation.

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.

Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage.

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.

The cytotoxicity of nitroxyl: possible implications for the pathophysiological role of NO.

In addition to the broad repertoire of regulatory functions nitric oxide (NO) serves in mammalian physiology, the L-arginine:NO pathway is also involved in numerous pathophysiological mechanisms. While NO itself may actually protect cells from the toxicity of reactive oxygen radicals in some cases, it has been suggested that reactive nitrogen oxide species formed from nitric oxide synthase (NOS) can be cytotoxic. In addition to NO, the one electron reduction product NO- has been proposed to be formed from NOS. We investigated the potential cytotoxic role of nitroxyl (NO-), using the nitroxyl donor Angelis's salt, (AS; sodium trioxodinitrate, Na2N2O3) as the source of NO-. As was found to be cytotoxic to Chinese hamster V79 lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-(CN6)3-), which converts NO- to NO, afforded dramatic protection against AS-mediated cytotoxicity. Treatment of V79 cells with L-buthionine sulfoximine to reduce intracellular glutathione markedly enhanced AS cytotoxicity, which suggests that GSH is critical for cellular protection against the toxicity of NO-. Further experiments showed that low molecular weight transition metal complexes associated with the formation of reactive oxygen species are not involved in AS-mediated cytotoxicity since metal chelators had no effect. However, under aerobic conditions, AS was more toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA double strand breaks in whole cells, and this effect was completely prevented by coincubation of cells with ferricyanide or Tempol. The data in this study suggest that nitroxyl may contribute to the cytotoxicity associated with an enhanced expression of the L-arginine:NO pathway under different biological conditions.

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.

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.

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.

Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli.

We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB. Both phenotypes require the same genes. Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells. The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested. We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense. Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding.

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.

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.

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.

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.

Protection from radiation-induced chromosomal aberrations by the nitroxide Tempol.

BACKGROUND: The nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, free radical that exhibits protection from ionizing radiation damage and from oxidative stress mediated through exposure of cells to superoxide or hydrogen peroxide. Radiation protection has been observed in both in vivo and in vitro models. To understand the mechanism of Tempol-mediated radioprotection better, the production of radiation-induced chromosome aberrations was evaluated. This study analyzed Tempol-mediated radioprotection of human peripheral blood lymphocytes (PBLs). METHODS: Peripheral blood lymphocytes were exposed to control (0mM), 10 mM (Tp10), and 50 mM (Tp50) concentrations of Tempol for 20 minutes before irradiation with 0, 150, 300, and 450 cGy. One quarter ml whole blood was cultured in F12 medium and phytohemagglutinin at 37 degrees C for 49, 54, 59, and 64 hours. Colcemide was added to each sample for the last 5 hours before harvest. Cells were harvested, treated with hypotonic solution, and fixed before dropping on cold clean slides. Mitotic indices and frequency of dicentric, ring, and triradial chromosomal aberrations were determined at 1000x magnification for each treatment group at each collection point. RESULTS: Treatment of cells with Tempol alone did not induce the chromosomal aberration frequency above that for unirradiated controls. Radiation dose response curves for total chromosome aberration production revealed radioprotection for Tempol treatment for both 10 and 50 mM exposures. Tempol protection factors (assessed at 0.2 aberrations/cell level) for Tp 10 and Tp 50 were 2.2 and 2.8, respectively. CONCLUSIONS: Tempol protects against radiation-induced chromosome aberrations in human PBLs. This finding is consistent with and lends support to previous studies in which Tempol was reported to enhance cell survival and reduce radiation-induced DNA double strand breaks.

Free radical modes of cytotoxicity of adriamycin and streptonigrin.

Free radical modes of cytotoxicity of streptonigrin (STN) and Adriamycin (ADR) in Chinese hamster V79 cells under aerobic conditions were evaluated using 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TP), a low molecular weight stable nitroxide free radical with antioxidant properties and desferrioxamine (DF), a transition metal chelator. In addition, exogenous superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), were tested for cytoprotective effects. EPR studies showed that TP reacts with the semiquinones of both ADR and STN and also with O2- radicals generated during aerobic redox cycling of the respective semiquinone radicals. Pulsed field gel electrophoresis studies confirmed that DNA double-strand breaks (dsb) induced by STN in V79 cells were inhibited completely by TP, whereas ADR-induced DNA dsb were not affected by TP. Clonogenic cell survival studies showed that STN-induced cytotoxicity could be inhibited completely by DF or TP. Both agents were ineffective in inhibiting ADR-induced cytotoxicity. SOD and CAT were ineffective in protecting against both STN and ADR cytotoxicity. Our results are consistent with a mechanism requiring the semiquinone radical intermediate of STN for cytotoxicity and minimal free radical involvement in ADR-induced V79 cell cytotoxicity.