Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay

1976 ◽  
Vol 54 (2) ◽  
pp. 110-119 ◽  
Author(s):  
J. Wlliam Lown ◽  
Asher Begleiter ◽  
Douglas Johnson ◽  
A. Richard Morgan
Keyword(s):  
Superoxide Dismutase ◽  
Free Radical ◽  
Mitomycin C ◽  
Free Radical Scavengers ◽  
Heat Denaturation ◽  
Cross Linking ◽  
Cytotoxic Action ◽  
Antitumor Antibiotic ◽  
The Cross ◽  
High Concentrations

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by St-endonuclease (EC 3.1.4.–) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoalkylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical [Formula: see text]. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechanisms.

scholarly journals Bound (“Glassy”) Rubber as a Free Radical Cross-linked Rubber Layer on a Carbon Black

Materials ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 1992 ◽  
Author(s):  
Alexey Kondyurin ◽  
Anastasia Eliseeva ◽  
Alexander Svistkov
Keyword(s):  
Free Radical ◽  
Carbon Black ◽  
Ion Beam ◽  
Cross Linking ◽  
Carbon Filler ◽  
Free Radical Reactions ◽  
Rubber Layer ◽  
Ion Beam Implantation ◽  
Cross Links ◽  
The Cross

A model of rubber with a cross-linked rubber layer on a carbon black filler has been proposed. The cross-links are the result of free radical reactions generated by carbon atoms with unpaired electrons at the edge of graphitic sheets in a carbon black filler. The experimental study of the cross-linking reactions in polyisoprene was done on a flat carbonized surface after ion beam implantation. The cross-linking process in the polyisoprene macromolecules between two particles was simulated. The model with a cross-linked rubber layer on a carbon filler as a “glassy layer” explains the mechanical properties of the rubber materials.

Studies related to antitumor antibiotics. Part XIV. Reactions of mitomycin B with DNA

1978 ◽  
Vol 56 (5) ◽  
pp. 296-304 ◽  
Author(s):  
J. William Lown ◽  
Gordon Weir
Keyword(s):  
Mitomycin C ◽  
Ph Dependence ◽  
Cross Linking ◽  
Antitumor Antibiotic ◽  
Antitumor Antibiotics ◽  
Previous Suggestion ◽  
Aziridine Ring ◽  
Strand Breakage ◽  
Radical Traps

The reactions of the antitumor antibiotic mitomycin B with DNA were examined using ethidium fluorescence assays. The following three aspects of mitomycin B action have been studied to compare its behavior with that of mitomycin C: (a) interstrand cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. The greater pKa value of 4.3 found for mitomycin B compared with that of mitomycin C, i.e., 3.2, together with the greater pH dependence of DNA alkylation and interstrand cross-linking and the faster and more extensive cross-linking by mitomycin B at low pH in the absence of reduction, support the suggestion that the aziridine moiety is involved in the initial alkylation of DNA. Mitomycin B, reduced in situ with NaBH4, nicks covalenty closed circular (CCC) PM2 DNA rapidly but less efficiently than mitomycin C in a reaction which is suppressed by (i) superoxide dismutase, (ii) catalase, and (iii) free radical traps showing the intermediacy of O−2∙, H2O2, and OH∙. DNA is cleaved by mitomycin B to which it is covalently attached as well as by the free antibiotic. The addition of intercalated ethidium bromide to DNA prior to treatment with reduced mitomycin B inhibits interstrand cross-linking but not strand scission. The reduced aziridine ring-opened mitomycin B (which lacks the 7-NH2 group of mitomycin C) alkylates DNA and thus provides evidence confirming a previous suggestion that the second covalent link to the DNA is formed at position 10 of the antibiotic.

Diabetic human platelets release a substance that inhibits platelet-mediated vasodilation

1997 ◽  
Vol 273 (1) ◽  
pp. H371-H379 ◽  
Author(s):  
H. J. Oskarsson ◽  
T. G. Hofmeyer
Keyword(s):  
Superoxide Dismutase ◽  
Free Radical ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Radical Scavengers ◽  
Radical Scavengers ◽  
Short Acting ◽  
Vasodilatory Response ◽  
Dilatory Response ◽  
A2 Receptors

This study was performed to investigate the mechanism for impaired vasodilation in response to activated diabetic human platelets. As observed previously, diabetic platelets failed to cause vasorelaxation, whereas normal platelets produced normal vasodilation. However, when activated and perfused through quiescent, NG-nitro-L-arginine-pretreated arteries, diabetic and normal platelets caused similar degrees of vasoconstriction. Inhibition of serotonergic and thromboxane A2 receptors in preconstricted normal arteries also failed to improve vasodilatory responses to diabetic platelets. The amount of ADP released into the supernatant from activated diabetic and normal platelets was similar. Concomitant perfusion of activated diabetic platelets impaired vasodilation produced by abluminally applied acetylcholine but perfusion of normal platelets did not. Whereas activated diabetic platelets failed to produce vasodilation, supernatant from the same platelets caused normal vasorelaxation. Dimethylthiourea and Tiron, intracellular free radical scavengers, normalized the vasodilatory response to diabetic platelets, whereas superoxide dismutase, catalase, and mannitol did not. We conclude that the impaired vasorelaxation in response to activated diabetic platelets is caused by an unidentified, short-acting, platelet-derived substance(s) that interferes with the normal dilatory response.

Free-radical scavengers, thiol-containing reagents and endothelium-dependent relaxation in isolated rat and human resistance arteries

1993 ◽  
Vol 84 (3) ◽  
pp. 287-295 ◽  
Author(s):  
W. Sunman ◽  
A.D. Hughes ◽  
P.S. Sever
Keyword(s):  
Superoxide Dismutase ◽  
Free Radical ◽  
Methyl Ester ◽  
Enzyme Inhibitor ◽  
Subcutaneous Fat ◽  
Free Radical Scavengers ◽  
Radical Scavenger ◽  
Resistance Arteries ◽  
Rat Mesentery ◽  
Endothelium Dependent Relaxation

1. Small arteries were isolated from either rat mesentery or human subcutaneous fat, and mounted in a myograph for the measurement of isometric force. 2. Superoxide dismutase, either in the presence or absence of catalase, relaxed noradrenaline-induced tone. This effect was abolished by removal of the endothelium or incubation with an inhibitor of NO synthase, N-ω-nitro-L-arginine methyl ester. Catalase alone had a negligible effect on noradrenaline-induced tone. 3. Captopril, an angiotensin-converting enzyme inhibitor and putative free-radical scavenger, did not relax pre-contracted isolated vessels. N-Acetylcysteine caused an endothelium-independent relaxation of rat vessels. Similar effects were observed in human vessels. 4. Acetylcholine induced a concentration-dependent relaxation of isolated resistance arteries, which was inhibited by removal of the endothelium or N-ω-nitro-L-arginine methyl ester, but unaffected by indomethacin. Preincubation with captopril, N-acetylcysteine or catalase alone did not alter the acetylcholine concentration-response relationship, but superoxide dismutase in combination with catalase enhanced responses to acetylcholine, causing a six-fold increase in potency. 5. Superoxide dismutase causes endothelium-dependent relaxation of resistance arteries and potentiates responses to acetylcholine. This action is probably due to the ability of the enzyme to scavenge superoxide anions which inhibit endothelium-dependent relaxation. 6. N-Acetylcysteine causes an endothelium-independent relaxation of resistance arteries which is probably unrelated to the putative ability of this compound to scavenge superoxide radicals and may reflect a direct action on vascular smooth muscle.

Cerebral vascular responsiveness after experimental traumatic brain injury: the beneficial effects of delayed hypothermia combined with superoxide dismutase administration

2008 ◽  
Vol 109 (3) ◽  
pp. 502-509 ◽  
Author(s):  
Anna I. Baranova ◽  
Enoch P. Wei ◽  
Yuji Ueda ◽  
Milton M. Sholley ◽  
Hermes A. Kontos ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Superoxide Dismutase ◽  
Free Radical ◽  
Brain Injury ◽  
Vascular Function ◽  
Free Radical Scavengers ◽  
Therapeutic Window ◽  
Radical Scavenger ◽  
Vascular Protection ◽  
Cerebral Vascular

Object Traumatic brain injury (TBI) induces cerebral vascular dysfunction reflected in altered responses to vasodilators such as acetylcholine and hypercapnia. It has been demonstrated that the use of either posttraumatic hypothermia or free radical scavengers offered vascular protection when those treatments were delivered early after the injury, losing efficacy when the initiation of either treatment was delayed. Because immediate posttraumatic treatment is not realistic in the clinical setting, the authors undertook this study to investigate whether the combination of delayed hypothermia and the delayed administration of the free radical scavenger superoxide dismutase (SOD) could result in improved vascular protection. Methods Male Sprague–Dawley rats were anesthetized and subjected to either an impact-acceleration or sham injury. Animals were treated either with hypothermia (32°C) initiated 60 minutes after TBI, delayed SOD (60 U/ml) applied 90 minutes after TBI, or a combination of delayed hypothermia (32°C) and delayed SOD (60 U/ml) applied 15 minutes prior to the cessation of hypothermia. In this investigation, the diameter of cerebral pial arterioles was measured at rest and then challenged with vasodilator acetylcholine and hypercapnia. Four vessels were assessed per animal prior to injury and then again up to 6 hours after injury. Results Delayed SOD treatment did not enhance vascular function, while delayed hypothermia treatment only partially preserved pial vascular function. However, the combination of delayed hypothermia and delayed SOD significantly preserved vascular function after the injury. Conclusions The results of these studies demonstrate that delayed hypothermia partially preserves vascular function after TBI, while expanding the therapeutic window over which agents such as SOD can now provide enhanced protection.

scholarly journals Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes

1980 ◽  
Vol 187 (1) ◽  
pp. 227-237 ◽  
Author(s):  
P R McIntosh ◽  
R B Freedman
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Molar Ratio ◽  
Cross Linking ◽  
Thiol Compounds ◽  
Microsomal Membranes ◽  
The Cross ◽  
High Concentrations ◽  
Cytochrome P 450

1. In liver microsomal membranes from adult rabbits treated with beta-naphthoflavone, reaction with Cu2+ salts plus 1,10-phenanthroline leads to the cross-linking of the two specifically beta-naphthoflavone-inducible cytochrome P-450 species, form 4 and form 6, to form homo- and hetero-dimer species. 2. The cross-linking is not reversed by treatment with 2-mercaptoethanol, so that it can be observed conveniently and specifically on conventional reducing sodium dodecyl sulphate/polyacrylamide gels. 3. The reaction occurs rapidly, and significant cross-linking is observed after 30s at all temperatures from −10 to 40 degrees C. 4. The cross-linking can be brought about by Cu2+ alone at concentrations greater than 0.5 mM, but not by 1,10-phenanthroline alone; at low Cu2+ concentrations, 1,10-phenanthroline enhances the cross-linking reaction, but high concentrations of 1,10-phenanthroline are inhibitory; the optimal molar ratio of Cu2+ to 1,10-phenanthroline is 4:1.5. The effect of Cu2+ is not mimicked by Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+ or Ag+; Cu+ is probably also ineffective. 6. The cross-linking reaction is inhibited by the prior addition of high concentrations of EDTA or thiol compounds, by sodium dodecyl sulphate at greater than or equal to 0.1% and by sodium deoxycholate and non-ionic detergents at greater than or equal to 1%; the reaction cannot be reversed by incubation with EDTA or with thiol compounds after reaction with cupric phenanthroline; the cross-linking reaction is not inhibited by prior treatment of microsomal membranes with N-ethylmaleimide. 7. The chemical nature of the cross-linking reaction is unknown, but it is most unlikely that it involves the formation of intermolecular disulphide bonds. 8. The great specificity of the reaction makes it a promising tool for the study of molecular interactions between cytochrome P-450 species in intact microsomal membranes.

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