The Effect of pH on Peroxidase-Mediated Oxidation of and DNA Adduct Formation by Ellipticine

2006 ◽  
Vol 71 (8) ◽  
pp. 1169-1185 ◽  
Author(s):  
Jitka Poljaková ◽  
Martin Dračínský ◽  
Eva Frei ◽  
Jiří Hudeček ◽  
Marie Stiborová
Keyword(s):  
Horseradish Peroxidase ◽  
Dna Adducts ◽  
Cytochromes P450 ◽  
Antineoplastic Agent ◽  
Ph Optimum ◽  
Effect Of Ph ◽  
Order Of Magnitude ◽  
Enzymatic Activation

In order to understand the mechanism of enzymatic activation of an antineoplastic agent ellipticine, we investigated the effect of pH on the efficiency of three model peroxidases (bovine lactoperoxidase, human myeloperoxidase and horseradish peroxidase) in oxidation of ellipticine and in formation ellipticine-DNA adducts. The formation of the major ellipticine metabolite, ellipticine dimer, in which two ellipticine residues are connected through nitrogenN6in the pyrrole ring of one of the ellipticine moieties and carbon C9 of the other ellipticine, and formation of four ellipticine-DNA adducts were analyzed. All three peroxidases oxidize ellipticine to dimer and form ellipticine-DNA adducts, but lactoperoxidase and myeloperoxidase were less efficient in these processes than horseradish peroxidase. More than one order of magnitude higher rates of formation of dimer and amounts of the DNA adducts were found upon horseradish peroxidase than in reactions with lactoperoxidase or myeloperoxidase. An acid pH optimum was found for the formation of ellipticine dimer (pH 6.4), while the highest binding of ellipticine activated by peroxidases to DNA was detectable at pH 8.4. Likewise, the highest binding of 5-(hydroxymethyl)ellipticine, a metabolite of ellipticine generated by cytochrome P450, to DNA was found at pH 8.4. The results presented here are a contribution to the explanation of the reaction mechanism of formation of the major deoxyguanosine adduct in DNA generated from ellipticinein vivoandin vitroby its activation with cytochromes P450 and peroxidases.

scholarly journals Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2

2008 ◽  
Vol 1 (2) ◽  
pp. 160-168 ◽  
Author(s):  
Dagmar Aimová ◽  
Jitka Poljaková ◽  
Věra Kotrbová ◽  
Michaela Moserová ◽  
Eva Frei ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Dna Adducts ◽  
Cytochromes P450 ◽  
Antineoplastic Agent ◽  
Metabolic Activation ◽  
Experimental Models ◽  
Adduct Formation ◽  
Dna Adduct

Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by32P postlabelling analysis, levels of ellipticine-derived DNA adducts formedin vivoin the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolismin vivo. Inin vitroexperiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adductsin vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds).

Cytochrome P-450 and Peroxidase Oxidize Detoxication Products of Carcinogenic Aristolochic Acids (Aristolactams) to Reactive Metabolites Binding to DNA in vitro

1995 ◽  
Vol 60 (12) ◽  
pp. 2189-2199 ◽  
Author(s):  
Marie Stiborová ◽  
Eva Frei ◽  
Heinz H. Schmeiser ◽  
Manfred Wiessler
Keyword(s):  
Dna Adducts ◽  
Microsomal Cytochrome ◽  
Aristolochic Acids ◽  
Nuclease P1 ◽  
Order Of Magnitude ◽  
Prostaglandin H ◽  
Cytochrome P 450

We report the analysis of DNA adducts formed from aristolactams I and II, which are the final metabolites derived from carcinogenic aristolochic acids in vivo, after their oxidation by microsomal cytochrome P-450 and horseradish peroxidase in vitro. DNA adducts were detected and quantified using the nuclease P1-enhanced variation of the 32P-postlabeling assay. Quantitative analysis revelead that the extent of modification of DNA by aristolactams activated by peroxidase was more than one order of magnitude higher than for activation by microsomal cytochrome P-450. Peroxidase catalyzes the formation of active oxygen in the presence of NADH, H2O2 and aristolactams. Aristolactams are also oxidized by mammalian peroxidase prostaglandin H synthase. The possible role of aristolactams in carcinogenesis induced by aristolochic acid is discussed.

scholarly journals Ultra-strong bio-glue from genetically engineered polypeptides

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chao Ma ◽  
Jing Sun ◽  
Bo Li ◽  
Yang Feng ◽  
Yao Sun ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Covalent Bond ◽  
Genetically Engineered ◽  
Supramolecular Interactions ◽  
Molar Ratio ◽  
High Adhesion ◽  
Strong Adhesion ◽  
Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzene in vitro and in vivo

1980 ◽  
Vol 31 (1) ◽  
pp. 1-17 ◽  
Author(s):  
F.A. Beland ◽  
D.L. Tullis ◽  
F.F. Kadlubar ◽  
K.M. Straub ◽  
F.E. Evans
Keyword(s):  
Dna Adducts

Formation of DNA adducts by the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo [4,8-f]quinoxaline (4,8-DiMeIQx) in vitro and in vivo. Identification of a N-(2′-deoxyguanosin-8-yl)-4,8-DiMeIQx adduct

1994 ◽  
Vol 15 (11) ◽  
pp. 2553-2558 ◽  
Author(s):  
Henrik Frandsen ◽  
Spiros Grivas ◽  
Robert J. Turesky ◽  
Rolf Andersson ◽  
Lars O. Dragsted ◽  
...  
Keyword(s):  
Dna Adducts ◽  
Food Mutagen

scholarly journals Targeting Cancer Cell Tight Junctions Enhances PLGA-Based Photothermal Sensitizers’ Performance In Vitro and In Vivo

Pharmaceutics ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 43
Author(s):  
Victoria O. Shipunova ◽  
Vera L. Kovalenko ◽  
Polina A. Kotelnikova ◽  
Anna S. Sogomonyan ◽  
Olga N. Shilova ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
3D Culture ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Multicellular Spheroids ◽  
Mesenchymal Transition ◽  
Non Invasive ◽  
Order Of Magnitude

The development of non-invasive photothermal therapy (PTT) methods utilizing nanoparticles as sensitizers is one of the most promising directions in modern oncology. Nanoparticles loaded with photothermal dyes are capable of delivering a sufficient amount of a therapeutic substance and releasing it with the desired kinetics in vivo. However, the effectiveness of oncotherapy methods, including PTT, is often limited due to poor penetration of sensitizers into the tumor, especially into solid tumors of epithelial origin characterized by tight cellular junctions. In this work, we synthesized 200 nm nanoparticles from the biocompatible copolymer of lactic and glycolic acid, PLGA, loaded with magnesium phthalocyanine, PLGA/Pht-Mg. The PLGA/Pht-Mg particles under the irradiation with NIR light (808 nm), heat the surrounding solution by 40 °C. The effectiveness of using such particles for cancer cells elimination was demonstrated in 2D culture in vitro and in our original 3D model with multicellular spheroids possessing tight cell contacts. It was shown that the mean inhibitory concentration of such nanoparticles upon light irradiation for 15 min worsens by more than an order of magnitude: IC50 increases from 3 µg/mL for 2D culture vs. 117 µg/mL for 3D culture. However, when using the JO-4 intercellular junction opener protein, which causes a short epithelial–mesenchymal transition and transiently opens intercellular junctions in epithelial cells, the efficiency of nanoparticles in 3D culture was comparable or even outperforming that for 2D (IC50 = 1.9 µg/mL with JO-4). Synergy in the co-administration of PTT nanosensitizers and JO-4 protein was found to retain in vivo using orthotopic tumors of BALB/c mice: we demonstrated that the efficiency in the delivery of such nanoparticles to the tumor is 2.5 times increased when PLGA/Pht-Mg nanoparticles are administered together with JO-4. Thus the targeting the tumor cell junctions can significantly increase the performance of PTT nanosensitizers.

scholarly journals Repair of cis-platinum-DNA adducts by ABC excinuclease in vivo and in vitro.

1985 ◽  
Vol 163 (3) ◽  
pp. 817-823 ◽  
Author(s):  
I Husain ◽  
S G Chaney ◽  
A Sancar
Keyword(s):  
Dna Adducts

scholarly journals TNFα Potently Activates Osteoclasts, through a Direct Action Independent of and Strongly Synergistic with RANKL

Endocrinology ◽  
2002 ◽  
Vol 143 (3) ◽  
pp. 1108-1118 ◽  
Author(s):  
Karen Fuller ◽  
Chiho Murphy ◽  
Barrie Kirstein ◽  
Simon W. Fox ◽  
Timothy J. Chambers
Keyword(s):  
Direct Action ◽  
Ring Formation ◽  
Actin Ring ◽  
Independent Manner ◽  
Low Concentrations ◽  
Order Of Magnitude ◽  
Extreme Sensitivity ◽  
Osteoclastic Differentiation

Abstract TNFα is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFα promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFα promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFα was equipotent with RANKL for this action. Activation by TNFα was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFα promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFα strongly synergized with RANKL, such that miniscule concentrations of TNFα were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFα suggests that the most sensitive osteolytic response of bone to TNFα is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.

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