Nutritional composition, mineral content, antioxidant activity and quantitative estimation of water soluble vitamins and phenolics by RP-HPLC in some lesser used wild edible plants.

The present study aimed to investigate the nutritional properties and antioxidant activities of six underutilized wild edible plants viz. Ipomoea aquatica, Achyranthes aspera, Aasystasia ganjetica, Enhydra fluctuans, Oldenlandia corymbosa and Amaranthus viridis that are commonly consumed as food in the India The antioxidant properties of the plants were evaluated by using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, ABTS radical scavenging ability, reducing power capacity, metal chelating activity, lipid peroxidation assay, estimation of total phenolic content and flavonoids content in different solvent extraction system like benzene, chloroform, methanol and 70% aqueous (aq.) ethanol. The quantitation of phenolic acids and flavonoids and water soluble vitamins in these plants were carried out by HPLC using Acclaim C 18 column (5 µm particle size, 250 × 4.6 mm), Dionex Ultimate 3000 liquid chromatograph and detection was carried out in photo diode array (PDA) detector. The results of investigation showed that these plants are rich sources of protein, carbohydrate minerals and vitamins, especially the B group of vitamins that can contribute immensely to nutrition, food security, and health and therapeutic benefits. The different levels of antioxidant activities were found in the solvent systems used. The HPLC analysis also showed the presence of phenolic acids and flavonoids in various amounts in these plants which could be utilized as natural antioxidant.

OK-432: an effective sclerosing agent for the treatment of lymphangiomas of head and neck.

OBJECTIVE: The basic objective of this study was to find out the efficacy of OK-432 for the conservative treatment of lymphangiomas of head and neck regions at the Department of ENT-Head & Neck Surgery of Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal. METHODS: It was a hospital based prospective, cross-sectional, case series, conducted among the patients suffering from different lymphangiomas of head and neck regions and treated with intra-lesional injection of OK-432, from March, 2005 to September 2006. Altogether eleven patients were enrolled in this modality of treatment out of which one patient was excluded from the study, which was treated surgically due to very ugly scar at the site of lesion resulting from previous surgery for the same. The data were analyzed by using simple mathematical tools like percentage and frequency. RESULTS: Out of ten patients treated with intralesional injection of OK-432, absolute response, i.e. total resolution of swelling was found in nine patients (90%) after the first dose. Remaining one patient also responded well on the treatment but some residual swelling was in situ for which second dose was given. After the second dose it was also totally resolved. In this way we achieved 100% response after the second dose of OK-432. CONCLUSION: The results of this study so far indicate that OK-432 is an effective and safe tool for the treatment of lymphangioma of head and neck region. As the procedure can be done in an out patient department, without hospitalization and has got minimal side effects, it can be proposed as the first line treatment of lymphangiomas of head and neck.

Synergistic activity of suramin with tumor necrosis factor alpha and doxorubicin on human prostate cancer cell lines.

We evaluated the action of suramin, doxorubicin, and tumor necrosis factor alpha (TNF-alpha) on the testosterone-responsive human prostate cell line LNCaP and on the testosterone-independent human prostate cell line PC-3. The synergistic action of these agents in combination was tested by the Chou and Talalay method (quantitative analysis of dose-effect relationships) to determine whether in vitro doses were active at levels safely achieved in vivo. The action of suramin was potentiated threefold by doxorubicin for the PC-3 line and seven-fold by doxorubicin for the LNCaP line. Both the suramin-TNF-alpha and the doxorubicin-TNF-alpha combinations showed synergistic action against the LNCaP line. Synergistic activity was noted at drug concentrations routinely achieved clinically. This study demonstrates that suramin, doxorubicin, and TNF-alpha are active agents against prostate cancer cell lines and that their activity can be enhanced when they are used in combination.

Synergistic antitumor activity of etoposide and human interleukin-1 alpha against human melanoma cells.

To investigate the possibility of increased activity of cytotoxic anticancer drugs combined with cytokines, we treated human melanoma cells with combinations of etoposide (VP-16) and human recombinant interleukin-1 alpha (rIL-1 alpha). We evaluated the combined cytotoxic effects of VP-16 and rIL-1 alpha using A375-C6 cells, which are sensitive to rIL-1 alpha, and A375-C5 cells, a clonal variant line resistant to rIL-1 alpha. We used the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromid e) assay and the inhibition of [3H]thymidine incorporation into DNA. We analyzed data using the median effects principle of Chou and Talalay (Chou's analysis). The calculated combination index values, at a dose ratio of VP-16 to rIL-1 alpha of 12:1 in simultaneous exposure, indicated synergistic cytotoxicity toward both A375-C6 cells and A375-C5 cells. We observed more pronounced synergism with VP-16 and rIL-1 alpha toward the A375-C5 IL-1 alpha-resistant melanoma cells. These results suggest that rIL-1 alpha combined with cytotoxic antitumor drugs may provide increased benefit in the treatment of malignant melanoma.

Peroxidase-catalyzed metabolism of etoposide (VP-16-213) and covalent binding of reactive intermediates to cellular macromolecules.

The horseradish peroxidase- and prostaglandin synthetase-catalyzed oxidative metabolism of the highly active anticancer drug, etoposide (VP-16-213), has been studied in vitro. This oxidation of VP-16 resulted in the formation of VP-16 quinone, an aromatic VP-16 derivative and the corresponding aromatic VP-16 quinone. This oxidative metabolism of VP-16 also resulted in the formation of reactive species that covalently bound to exogenously added DNA and heat-inactivated microsomal proteins. The peroxidase-catalyzed binding was time dependent and required the presence of cofactors (hydrogen peroxide or arachidonic acid). The prostaglandin synthetase/arachidonic acid-catalyzed metabolism and binding of VP-16 were inhibited by indomethacin, an inhibitor of the cyclooxygenase, and were shown to involve the peroxidative arm of prostaglandin synthetase. Our studies show that the protein covalent binding species were formed as a result of O-demethylation of the drug as shown by the loss of specifically labeled (O-14CH3) radioactivity from O-methoxy group and by incubating proteins with VP-16 quinones. In contrast, the covalent binding intermediates for DNA appeared to be different and VP-16-derived quinone methides are suggested as DNA binding species. Co-oxidation of VP-16 and the related drug, VM-26, during prostaglandin biosynthesis may be an important pathway for the metabolism of these agents and may play a role in their cytotoxic properties.

A role for the interleukin 1 receptor in the synergistic antitumor effects of human interleukin 1 alpha and etoposide against human melanoma cells.

To investigate the possibility that anticancer drugs combined with cytokines may show increased activity, human tumor cells were treated with combinations of human recombinant interleukin 1 alpha (rIL-1 alpha) and etoposide (VP-16). The cytotoxicity of these combinations was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay using rIL-1 alpha-sensitive A375-C6 melanoma cells and A375-C5 cells, a clonal variant line that is resistant to IL-1 alpha. Data were analyzed for synergism by the median effect principle of T-C. Chou and P. Talalay (J. Biol. Chem., 252: 6438-6442, 1977). At a dose ratio of VP-16 to rIL-1 alpha of 12 nM:1 unit/ml in either simultaneous or sequential exposure (VP-16 first), the calculated combination index values indicated synergistic cytotoxicity toward both A375-C6 cells and A375-C5 cells. IL-1 alpha treatment 24 h prior to VP-16 exposure had no advantage over simultaneous treatment. Surface IL-1 alpha receptors on both A375-C6 and A375-C5 cells were measured using 125I-radiolabeled rIL-1 alpha binding; A375-C6 cells had 701 +/- 128 (SD) receptor molecules/cell and A375-C5 cells only had 58 +/- 33 receptor molecules/cell. The dissociation constants for IL-1 alpha were similar in both cell types (19 +/- 6 pM for A375-C6 and 17 +/- 2 pM for A375-C5). The specific binding of rIL-1 alpha to the surface IL-1 alpha receptors of both sensitive and resistant cells was significantly increased in a dose-dependent fashion by the prior treatment with VP-16 (1.75-fold on A375-C6 cells and 3.5-fold on A375-C5 cells). VP-16 also enhanced the internalization of receptor-bound rIL-1 alpha, suggesting that a possible mechanism of the synergistic cytotoxicity of rIL-1 alpha and VP-16 might be related to the modulation of rIL-1 alpha receptors by VP-16, resulting in increased internalization of rIL-1 alpha.

Adriamycin-induced free radical formation in the perfused rat heart: implications for cardiotoxicity.

Adriamycin is an anthracycline drug with a wide spectrum of clinical antineoplastic activity. However, the usefulness of the drug is limited by its dose-dependent cardiotoxicity. Adriamycin-stimulated free radical formation has been suggested as one of the mechanisms for its cardiotoxic effects. In order to evaluate this underlying mechanism, we have perfused rat hearts with Adriamycin, using a modified Langendorf technique, and the free radicals formed were analyzed by electron spin resonance spectroscopy using spin-trapping techniques. Our studies show that Adriamycin stimulated the formation of .OH in the heart, and the maximum .OH was formed with 1 microM of the drug. The addition of superoxide dismutase (600 units/ml) inhibited the hydroxyl radical formation by 2- to 3-fold, while catalase (550 units/ml) abolished it completely, showing the intermediacy of superoxide and H2O2. Furthermore, ICRF-187, an iron chelator and a cytotoxic drug, was also an effective inhibitor of .OH formation in the rat heart. The heart rate was not significantly modified by all the above experiments. This study demonstrates that Adriamycin stimulates the formation of .OH in the isolated rat heart and suggests that this mechanism may be significant in Adriamycin-induced cardiotoxicity.

DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action.

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.

Adriamycin activation and oxygen free radical formation in human breast tumor cells: protective role of glutathione peroxidase in adriamycin resistance.

Previous studies with Adriamycin-sensitive and -resistant (ADRR) MCF-7 human breast tumor cell lines indicated that Adriamycin formed significantly less hydroxyl radical (.OH) as the result of enhanced detoxification of reactive oxygen intermediates in the ADRR cell line. In order to further define the sites of drug activation and the role of detoxification mechanisms in free radical levels, subcellular fractions were isolated from these two cell lines and free radical formation in the presence of Adriamycin was examined by using electron spin resonance spectroscopy. Studies reported here show that considerable NADPH-cytochrome P-450 reductase and NADH dehydrogenase activities were present in microsomes and mitochondria, respectively, and in nuclei obtained from these cells, and the relative activity of NADH dehydrogenase was 2-fold higher in the mitochondrial fraction of ADRR cells compared to the mitochondrial fraction from the parental wild type cells. In the presence of Adriamycin and a reducing cofactor (NADPH or NADH), Adriamycin semiquinone free radical, superoxide anion, and .OH were detected in all these fractions. Although only a small difference in the relative amount of oxy radical formation was detected in tumor microsomes, both mitochondria and nuclei of ADRR cells showed an overall 2-fold decreased formation of oxy radicals. The formation of the free radicals was significantly inhibited by superoxide dismutase, catalase, and dimethyl sulfoxide, indicating that free .OH generation was both superoxide and hydrogen peroxide dependent. The addition of purified glutathione peroxidase likewise inhibited .OH formation in a dose-dependent fashion. Similarly, when the lysate from ADRR cells, which contains 12- to 14-fold more glutathione peroxidase than Adriamycin-sensitive cells, was added to reaction mixtures containing Adriamycin-sensitive cells and Adriamycin, the .OH formation was diminished. Decreased free radical formation in nuclei and mitochondria, as a result of detoxification of hydrogen peroxide by glutathione peroxidase, may be significant in the protection of ADRR cells from Adriamycin-induced cell killing.

Potentiation of doxorubicin cytotoxicity by buthionine sulfoximine in multidrug-resistant human breast tumor cells.

Resistance to antineoplastic drugs is a major problem in the clinical management of cancer. Previous studies have demonstrated that the cytotoxicity of certain anticancer drugs is increased by lowering the glutathione (GSH) levels with buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. In this study we report that the resistance to doxorubicin, an anthracycline antibiotic and the most active agent in the treatment of breast cancer, can be partially reversed by exposing MCF-7 doxorubicin-resistant breast tumor cells (MCF-7/ADRR) to minimally cytotoxic doses of BSO. We found that the BSO treatment (50 microM, 48 h) of MCF-7/ADRR cells resulted in 80 to 90% depletion in total GSH concentrations. The toxicity of doxorubicin, as determined by growth inhibition and clonogenic assays, was significantly potentiated in the BSO-treated GSH-depleted cells relative to control breast tumor cells, and a dose-modifying factor of 5 to 7 was observed. Since the cytotoxicity of doxorubicin has been associated with its ability to undergo enzymatic activation and to form hydroxyl (OH) radicals in this cell line, we also quantitated the OH formation in the BSO-treated and untreated MCF-7/ADRR cells using electron spin resonance spintrapping techniques. These results show that doxorubicin stimulated at least 2-fold more OH formation in the tumor cells after GSH levels were decreased by 90%. These results indicate that GSH plays an important role in modulating doxorubicin-induced OH formation via the scavenging of hydrogen peroxide by glutathione peroxidase and thus partially protects MCF-7/ADRR cells from the cytotoxic effect of doxorubicin.

Effects of glutathione and ethylxanthate on mitomycin C activation by isolated rat hepatic or EMT6 mouse mammary tumor nuclei.

Mitomycin C (MC) activation to a reactive species was studied in nuclei isolated from rat liver and EMT6 tumor cells. Both preparations were similar in the rate of 4-(p-nitrobenzyl)pyridine (NBP) alkylation by MC and the levels of NADPH-cytochrome P-450 reductase. MC activation by both hepatic and EMT6 cell nuclei was inhibited by the presence of O2 and by heat inactivation. NADPH was preferred over NADH as the source of reducing equivalents by both types of isolated nuclei. MC activation to alkylating metabolites was not affected when EDTA or diethylenetriaminepentaacetic acid, two Fe2+ chelating agents, was present in the incubation system with either preparation of isolated nuclei. Glutathione (1 and 5 mM) and N-acetylcysteine (1 and 10 mM) both inhibited MC alkylation of NBP in nuclear preparations from rat liver and EMT6 tumor cells by 50-60%. Ethylxanthate (1 mM) effectively inhibited the MC alkylation of NBP by hepatic nuclei but was unable to inhibit MC alkylation of NBP by tumor cell nuclei. At 100 mM, ethylxanthate produced a slight stimulation in the rate of MC alkylation of NBP. These data are consistent with the hypothesis that MC activation in EMT6 tumor cells proceeds via a one electron reduction pathway which is inhibitable by glutathione but not inhibitable by ethylxanthate. Hepatic nuclei are apparently able to activate MC by either a one- or two-electron pathway.

Adriamycin-enhanced membrane lipid peroxidation in isolated rat nuclei.

Isolated rat liver nuclei enzymatically activated Adriamycin to the electron spin resonance-detectable semiquinone free radical in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). This process resulted in the enhancement of oxyradical-mediated peroxidation of the nuclear envelope membrane unsaturated phospholipids, measured as malonaldehyde equivalents by the thiobarbituric acid method. Peroxidation required the inclusion of NADPH and catalytically active protein, presumably NADPH:cytochrome P-450 reductase, and was enhanced more than 5-fold by Adriamycin. It was observed that Adriamycin-stimulated nuclear membrane peroxidation was diminished by superoxide dismutase, reduced glutathione, the hydroxyl radical scavenger, 1,3-dimethylurea, and by the metal ion chelator, diethylenetriaminepentaacetic acid, indicating that multiple species of reactive oxygen and trace amounts of metal ions (iron) were required in the peroxidation reaction. The generation of superoxide and hydroxyl radicals was confirmed by 5,5-dimethyl-1-pyrroline-N-oxide spin trap electron spin resonance spectroscopy. Calf thymus DNA added to incubations containing nuclei and NADPH caused a pronounced concentration-dependent inhibition of Adriamycin-stimulated lipid peroxidation. It was found that nuclei incubated with Adriamycin (300 microM) accumulated 128 nmol of the drug per mg of nuclear protein within 1 h, apparently because the Adriamycin was internalized and bound to the DNA and nuclear protein. These results suggest that some of the toxic effects of Adriamycin observed in the nucleus could result, directly or indirectly, through the peroxidation of the unsaturated lipids of the nuclear membrane.

Enzymatic activation and binding of adriamycin to nuclear DNA.

Rat liver microsomal activation of the anthracycline antitumor drug, Adriamycin, in the presence of reduced pyridine nucleotide under anaerobic conditions produces reactive species that bind covalently to cellular macromolecules including DNA. Since the nuclear membrane contains enzymes capable of activating Adriamycin, we have examined activation of Adriamycin by isolated nuclei. The anaerobic incubation of Adriamycin with rat hepatic nuclei resulted in the formation of the Adriamycin semiquinone free radical. Moreover, this activation resulted in the covalent binding of Adriamycin to nuclear DNA. The binding of Adriamycin to DNA was reduced pyridine nucleotide and time dependent and was significantly decreased in the presence of reduced glutathione or ethylxanthate . Dicumerol , an inhibitor of DT-diaphorase, in contrast, had no effect on this binding. When the incubation was carried out in the presence of oxygen, no semiquinone radical was detected; however, superoxide and hydroxyl radicals were readily detected by a spin-trapping technique. Furthermore, little binding of Adriamycin to nuclear DNA was observed under aerobic conditions. These observations suggest that the nuclear activation and covalent binding of Adriamycin to DNA may be important in the biochemical actions of this drug.

DNA interstrand cross-link and free radical formation in a human multidrug-resistant cell line from mitomycin C and its analogues.

A subline of the human breast tumor cell line (MCF-7), selected for resistance to Adriamycin and having the multidrug resistance phenotype, also developed significant cross-resistance to mitomycin C and its two analogues, BMY 25282 and BMY 25067. Because mitomycin C and the analogues contain both quinone and aziridine moieties, the mechanism of tumor cell kill is thought to involve alkylation and cross-linking of DNA molecules, hence they are not expected to show cross-resistance to cells selected for resistance to a DNA intercalator. Studies to understand this novel observation show that the resistant MCF-7 cells form significantly less hydroxyl radical and DNA cross-linking in the presence of mitomycin C and BMY 25282 than the sensitive cells. Although BMY 25067 formed less free radicals in the resistant cells, similar to the other two drugs, the formation of DNA cross-links was identical in both cell lines, indicating a somewhat different mechanism of tumor cell kill by this analogue. DNA cross-link formation increased slightly with time in the sensitive cells while there was a small decrease in the resistant cells. This difference in the formation of toxic intermediates appeared to result from enhanced detoxification of reactive species (hydrogen peroxide and alkylating intermediates) as a result of significantly higher glutathione peroxidase (14-fold) and glutathione S-transferase (44-fold) activities in the resistant cell line. These events, i.e., free radical formation and DNA alkylation, showed a good correlation with the cytotoxicity in drug-sensitive cells, indicating that both mechanisms contribute to cell killing of human breast tumor cells.

Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance.

Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro. However, the relevance of these to tumor cell resistance in vivo remains unclear. We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy. One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522. The sensitivities of six other sarcoma lines were similar to that of AG1522. STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment. Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations. Accumulation of [3H]daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil. Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher. This was due mostly to an increase in selenium-dependent peroxidase activity. After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry. Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine. These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.

Hearing results after myringoplasty.

BACKGROUND: Myringoplasty is one of the various surgical techniques for the management of chronic supurative otitis media of tubotympanic type (CSOM-TT). The presence of a perforation of tympanic membrane with intermittent discharge and hearing loss of conductive nature are the indications of myringoplasty. It is a beneficial procedure done for closing tympanic membrane perforation and improving hearing. OBJECTIVE: The aim of this study was to assess hearing improvement after myringoplasty within ten weeks following surgery. MATERIAL AND METHODS: The study population consisted of 50 patients who were suffering from CSOM-TT. Preoperative and postoperative examinations of the patients were conducted clinically as well as audiologically. Pre and postoperative air-bone (A-B) gap were calculated by taking the averages of bone conduction and air conduction at the frequencies of 500, 1000 and 2000 Hz. Myringoplasty was performed with underlay technique under local anaesthesia by either permeatal or endaural approach. Temporal muscle fascia was used as grafting material for reconstruction of the tympanic membrane. RESULTS: Preoperatively, air-bone gap of 30 db or more was observed in 39 (76%) patients whereas post operatively A-B gap of 30 db or more was observed in only one patient. Using hearings gain exceeding 15 dB as the criterion, thirty-nine (78%) patients had their hearing gain exceeding 15 dB. Using postoperative A-B gap within 20 dB as the criterion, 42 (84%) patients had their A-B gap within 20 dB. CONCLUSION: Myringoplasty is a beneficial procedure for hearing improvement. Using the proportion of patients with a postoperative A-B gap of 30 dB as the criterion, in this study, 98% of patients achieved their A-B gap closer within 30 dB. Using hearing gain exceeding 15 dB as the criterion, 78% patient had their hearing gain exceeding 15 dB.

Activation of hydrazine derivatives to free radicals in the perfused rat liver: a spin-trapping study.

Spin-trapping techniques and electron spin resonance spectroscopy have been used to study bioactivations of hydrazine and its derivatives by isolated perfused rat livers. Using phenylbutylnitrone (PBN) as the stable spin trap, it was found that the liver perfusion of hydrazine, acetylhydrazine and isoniazid resulted in the formation of the same carbon-centered radical which was shown to be the acetyl radical. The identity of the acetyl radical was confirmed after organic extraction of the liver perfusates, by comparing its coupling constants with those of the in vitro metal ion- or horseradish peroxidase-catalyzed oxidation products of the hydrazines in the same solvents. The liver perfusion of iproniazid formed the isopropyl radical which was previously observed to result from peroxidase-catalyzed oxidation.

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors.

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.

Free-radical formation by mitomycin C and its novel analogs in cardiac microsomes and the perfused rat heart.

Using a spin-trapping technique, we have examined free-radical formation by mitomycin C and its analogs, BMY 25282 and BMY 25067, in rat cardiac microsomes and isolated perfused rat hearts. All three drugs stimulated 2--4-fold OH radical formation in cardiac microsomes which was inhibited by SOD and catalase. Superoxide anion radical was also detected in the presence of diethylenetetraaminopentaacetic acid. Addition of DMSO yielded methyl radicals, thus indicating the production of free OH under these conditions. Similar stimulation of OH formation (2--3-fold) in the perfusates from rat hearts was detected with all three drugs. Perfusion with catalase (550 U/ml) completely suppressed the OH signal both in the presence and absence of the drugs, thus suggesting the intermediacy of hydrogen peroxide. However, BMY 25067-induced OH formation was more sensitive to inhibition by superoxide dismutase (SOD) and the iron chelator ICRF-187. Perfusion with DMSO produced methyl radicals at the expense of OH in the presence of all three drugs. SOD and catalase inhibited DMPO-OH signals, indicating that most of the OH formation was extracellular in this setting. While mitomycin C and BMY 25067 (up to 10 microM) did not affect the heart rate, perfusion with 10 microM BMY 25282 caused acute arrhythmia and cardiac standstill within 20 min. An initial surge in OH formation (2-fold) accompanied this cardiotoxic effect. Both the arrhythmia and the free radical signal were partially blocked by SOD, catalase and ICRF-187, indicating that iron-dependent oxygen radical formation from BMY-25282 (and possibly other compounds) is involved, in part, in inducing toxic manifestations in the rat heart and possibly in clinic.

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones.

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.