Abstract: Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that gamma-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e(-)(aq)) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, (*)OH, and reducing, e(-)(aq), radicals presents a case in which multiple ionization events along a gamma-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.
Abstract: The interaction of solvated electrons (e(-)(aq)) with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to (e(-)(aq))-induced damage is achieved by incorporation of the electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modelled as a localised e(-)(H2O6) anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble DNA-e(-)(aq) system, containing a pyrimidine-pyrimidine mismatched base pair, BUdR.T. The results show an overall dynamic pattern similar to that of the e(-)(aq) motion around normal DNA. However, the number of configuration states when e(-)(aq)) was particularly close to DNA is different. Moreover, in the (BUdR.T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR.A)-DNA. The structure and exchange of water at the sites of e(-)(aq) immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with e(-)(aq) approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the enhanced radiation damage of wobble DNA.
Abstract: Solvated electrons ((e-)(aq)) are produced during water radiolysis and can interact with biological substrates, including DNA. To augment DNA damage, radiosensitizers such as bromo-deoxyuridine (BUdR), often referred to as an "electron affinic radiosensitizer", are incorporated in place of isosteric thymidine. However, little is known about the primary interactions of (e-)(aq) with DNA. In the present study we addressed this problem by applying molecular modeling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modeled as a localized (e-)(H2O)6 anionic cluster. Our goals were to evaluate the suitability of the MD simulations for this application; to characterize the motion of (e-)(aq) around DNA (e.g., diffusion coefficients); to identify and describe configurational states of close (e-)(aq) localization to DNA; and to evaluate the structural dynamics of DNA in the presence of (e-)(aq). The results indicate that (e-)(aq) has distinct space-preferences for forming close contacts with DNA and is more likely to interact directly with nucleotides other than BUdR. Several classes of DNA - (e-)(aq) contact sites, all within the major groove, were distinguished depending on the structure of the intermediate water layer H-bonding pattern (or its absence, i.e., a direct H-bonding of (e-)(aq) with DNA bases). Large-scale structural perturbations were identified during and after the (e-)(aq) approached the DNA from the major groove side, coupled with deeper penetration of sodium counterions in the minor groove.
Abstract: Modification of 2'-deoxycytidine (dCyd) by hydroxyl radicals and direct ionization leads to the formation of various oxidation products, including dCyd 5,6-glycols, 5-hydroxy-2'-deoxycytidine, and ring fragmentation products. The mechanism of oxidation is complex and poorly understood. In the present work, we have prepared four cis- and trans-diastereomers of N1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine by bromination of dCyd followed by peroxidation of the resulting dCyd bromohydrins. The structure and stereochemistry of each product were determined by 1H NMR, 13C NMR, and 2D NOE analyses. The formation of imidazolidine products involves rearrangement of initial 5(6)-hydroxy-6(5)-hydroperoxides to C6-C2 endoperoxides, which subsequently decompose by a concerted pathway to imidazolidine products. A remarkable feature of the four diastereomers was their ability to interconvert via single and successive cycles of ring-chain tautomerism at N1-C5 and N3-C4, leading to epimerization of C5 and C4, respectively. The rate of isomerization was greater for cis-diastereomers compared to trans-diastereomers, and the rate sharply increased with pH (pH 9.0>7.0>5.5).
Abstract: The dynamic structure of 11-mer DNA duplexes of different sequences with or without homopyrimidine (T.T, or BrdU.T) mismatches was studied by molecular dynamics (MD) simulations on a time scale from 200 ps to 1 ns. The conformational analysis suggests that in mismatched duplexes the formation of classical T.T wobble H-bonding pairing is nearest-neighbor sequence-dependent and, in most cases, three-centered H-bonds and numerous alternative close cross-strand interatomic contacts exist. Thus, in duplex W1, where the central triplet is 5'd(CTA).d(TTG), two wobble conformations W upward arrow (alphabeta) and W downward arrow (betaalpha) are formed and exchange rapidly at 300 K. In contrast, when the central triplet is 5'd(TTT).d(ATA) (W2 duplex) wobble conformations are rarely observed at 300 K, and the T.T mispair most often adopts a "twisted" conformation with one largely persistent normal H-bond, plus a stable cross-strand contact involving a T flanking base. However, at elevated temperature (400 K) the same W2 duplex shows frequent exchange between the two classical wobble conformations (alphabeta<-->betaalpha), as is in the case when the central triplet is 5'd(TBrdUT).d(ATA) (W3 duplex at 300 K). It is suggested that in the W2 sequence, restrictions due to thymine-methyl/pi interactions prevent the formation of wobble pairing and thermal activation energy, and/or the chemical replacement of T by BrdU are required in order for the T(BrdU).T mismatch to adopt and exchange between wobble conformations. The specific short and/or long-lived (double/triple) cross-strand dynamic interactions in W1, W2 and W3 duplexes are throughout characterized. These frequent atomic encounters exemplify possible inter-strand charge transfer pathways in the studied DNA molecules.
Abstract: Phthalocyanines (Pc), which are extensively studied as tumor localizing photosensitizers for photodynamic therapy, are transported by the blood circulatory system to target tissues. Binding interactions between human serum albumin and differently sulfonated aluminum phthalocyanines (AlPcSn; n = 1-4) were studied using optical and ESR spectroscopy. AlPcSn (n = 1-3) occupy one strong binding site and eight weaker sites. The high affinity binding site interactions differ with respect to the degree of sulfonation and isomeric composition of the Pc. Phthalocyanines without SO-3 groups on adjacent iso-indole rings exhibit a high affinity binding site constant of K approximately 3-4 x 10(7) M-1, while Pc with two or three adjacent SO-3 groups show binding for this high affinity site that is no longer independent, but cooperative (alpha = 2), with K approximately 2-6 x 10(6) M-1. Binding isotherms for AlPcS4 and its close analog, tempoyl spin-labeled SL-AlPcS3, do not approach saturation at high ligand concentrations. Competition analyses between AlPcSn and spin-labeled fatty acids (5- and 16-doxyl stearate isomers) reveal that all compounds participate in cooperative (allosteric) interactions with the high affinity binding site of 16-DS, while extruding 5-DS isomer from certain sites and increasing the binding affinity for the remaining. Protein conformational dynamics was studied by ESR spectroscopy using covalent (alkylation of Cys34 residue) and noncovalent spin labeling (employing SL-AlPcS3). Phthalocyanines perturb conformational dynamics parameters (tauc and S) depending on the degree of sulfonation and isomeric composition corresponding to the type of sites, i.e., independent or cooperative, occupied on the HSA molecule.
Abstract: Epipodophyllotoxin derivatives, such as etoposide (VP-16), constitute an important class of anticancer agents, the major cytotoxic effects of which are associated with trapping of the topoisomerase II/DNA cleavable complex and formation of protein-DNA cross-links and nicked DNA. VP-16, however, can be metabolized to several highly reactive products, including an ortho-quinone (VPQ). The inhibitory activity of VPQ against purified human topoisomerase II processing of supercoiled DNA was studied and compared with that of the parent compound, VP-16. Our results show that VPQ is a powerful inhibitor of topoisomerase II, which prevents DNA strand passage in the presence of ATP. As with VP-16, trapping of the cleavable complex is highly reversible upon removal of divalent ions, which indicating that VPQ alters the cleavage-reunion equilibrium of topoisomerase II and DNA mainly by noncovalent interactions, as does the parent compound. However, we observed several differences between the effects induced by VP-16 and VPQ, including a strong inhibition of the second DNA strand religation, which implies the involvement of additional (asymmetric) mode(s) of interactions of the VPQ, possibly by interference with ATP binding by the homodimeric enzyme, and/or involving covalent interactions. Reduced or oxidized glutathione prevented trapping of the topoisomerase/DNA cleavable complex by VPQ, but not by VP-16, probably by forming covalent adducts with the former.
Abstract: The effects of glutathione (GSH) depletion by buthionine sulfoximane (BSO) or by photosensitization-induced oxidative stress using metallo-phthalocyanines (MePcS4) on etoposide (VP-16) cytotoxicity against K562 human leukemic cells were investigated. Both treatments enhanced VP-16 toxicity in a markedly synergistic way, as revealed by combination index analysis procedure. Synergistic drug interactions were accompanied by a supra-additive induction of DNA strand breaks. The proposed role of intracellular GSH in preventing metabolic transformations of VP-16 and thus decreasing its toxicity was confirmed by electron spin resonance (ESR) monitoring of the accumulation of the VP-16 phenoxyl radical in cell cytoplasm subjected to GSH depletion. Taken together the results emphasize the beneficial effect of GSH-related oxidative stress in enhancement of etoposide toxicity and possibly in its anticancer applications.
Abstract: Etoposide (VP-16) is a widely used anticancer drug whose toxicity involves poisoning of topoisomerase II. VP-16 undergoes enzymatic oxido-reductive transformations in cells, resulting in the formation of the ortho-quinone derivative (VPQ) as a major product. The actions of VP-16 and VPQ on purified human topoisomerase II have been compared. Both the parent drug and VPQ are very efficient at trapping the topoisomerase II-DNA cleavable complex, suggesting that methoxy groups on the E-ring are not a prerequisite for activity. Our data also imply that VPQ has more effect than VP-16 on the breakage-reunion equilibrium of topoisomerase II and DNA. The stronger inhibition of the religation of the second strand observed with VPQ suggests it interacts asymmetrically with the two homodimers of topoisomerase II bound to DNA.
Abstract: Etoposide (VP-16) is an anti-cancer drug commonly used against several types of tumours and leukaemia, either alone or in combination chemotherapy. Photodynamic therapy (PDT) is another, relatively new modality for treatment of various malignancies. The interactions between VP-16 and PDT, using aluminium tetrasulphophthalocyanine as photosensitiser, in K562 human leukaemic cells were investigated. Cell responses to individual and combined drug treatment under different experimental conditions revealed synergistic drug toxicity. The latter was evident from various events of cell response, including supra-additive accumulation of cells in G2/M cell cycle phase and endonucleolytic DNA fragmentation (apoptosis). The involvement of the cellular antioxidant system in the synergistic interactions of photosensitisation and VP-16 is proposed.
Abstract: In order to obtain information concerning the mechanism(s) of metallo-phthalocyanine (MePcS4) photosensitized damage of DNA constituents, the EPR-spin trapping method in conjunction with liquid chromatography was used to study thymidine (dThd) free radicals formed during photosensitization or exposure to gamma-radiation in solution. Under specified conditions two dThd free radical species, 5-hydroxy-5,6-dihydrothymidine-6-yl and 6-hydroxy-5,6-dihydrothymidine-5-yl, were formed both during exposure to ionizing radiation and photosensitization. These results imply that identical reactive intermediates (*OH radicals) are involved in the radiolytic and photosensitized oxidation of dThd. A light-dependent, Fenton-type mechanism is proposed to explain the generation of hydroxyl radicals during MePcS4 photosensitization.
Abstract: Catalase (CAT) in solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during photosensitization with tetrasulfonated metallophthalocyanines (MePcS4). The effect of added scavengers and D2O showed that both singlet oxygen and free radical species are involved in this process. Evidence was found that direct interactions of ground or excited-stated photosensitizer with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized protein damage included oxidation of cysteine residues as well as other amino acids, as demonstrated by the formation of carbon-centered free radicals and the loss of absorbance at lambda = 275 nm. In parallel, we detected degradation of the CAT heme groups, accompanied by release of Fe(II) ions in solution. These combined phenomena initiate cross-linkages between CAT subunits and subsequent degradation of the protein with formation of irreversible aggregates in solution. Phthalocyanine-mediated photoinactivation of cell-bound CAT results in loss of protection against accumulating H2O2, providing an additional pathway of phototoxicity.
Abstract: Photoirradiation of aqueous solutions of DNA in the presence of Al- or Zn-tetrasulphonated phthalocyanines (AIPcS4 and ZnPcS4) causes formation of strand breaks and liberation of nucleobases. The effect of added D2O, which enhances singlet oxygen (1O2) lifetime, radical scavengers including alcohols and the spin-trap DMPO, as well as superoxide dismutase, indicates that both singlet oxygen (1O2) and free radicals contribute to the production of strand breaks. However, in the case of base release, only free radicals, such as the hydroxyl radical (.OH), appear to be involved in the degradation process. Detection of the characteristic free-radical oxidation products of deoxyribose provides evidence that .OH are involved in the photosensitized DNA damage. EPR and spin trapping data suggest that superoxide (O2.-) is the most likely precursor of .OH and a Fenton-type mechanism is proposed for their formation.
Abstract: The photosensitizing properties of tetrasulphonated Al- and Zn-phthalocyanines (AlPcS4 and ZnPcS4) in lymphoma cells were studied as a function of the pre/post-illumination incubation time. Photocytotoxicity increased with incubation time, ranging from a transient cell-cycle arrest to cell killing. Under all experimental conditions, the phototoxicity of ZnPcS4 was markedly higher than that of AlPcS4. The primary photoprocesses initiated by metallo-phthalocyanines (MePcS4) in the cells were probed with DMPO/esr spin-trapping techniques. Under all incubation conditions the intracellularly bound MePcS4 sensitized formation of three different types of DMPO spin-adducts: DMPO/OH (hydroxyl radical), DMPO/R (organic carbon-centred radical(s)) and an unidentified simple nitroxyl, referred to as DMPO/ox. The yields of trapped radicals depended on the length of the incubation with the dyes prior to illumination and the formation of spin-adducts was shown to be intracellular. The ability of DMPO to protect cells from the photocytotoxic effects of Al- and ZnPcS4, combined with the generation of carbon-centred spin-adducts is direct evidence for the involvement of free-radical-mediated damage of cellular constituents.
Abstract: Phenoxyl radicals are intermediates in the oxidation of phenolic compounds to quinoid derivatives (quinones, quinone methides), which are known to act as ultimate mutagenic, carcinogenic, and cytotoxic agents by directly interacting with macromolecular targets or by generating toxic reactive oxygen species. One-electron reduction of phenoxyl radicals may reverse oxidative activation of phenolic compounds to quinoids, thus preventing their cytotoxic effects. In the present work, we studied interactions of ascorbate, thiols (glutathione, dihydrolipoic acid, and metallothioneins), and combinations thereof with the phenoxyl radical generated by tyrosinase-catalyzed oxidation of VP-16 [etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside)], a hindered phenol widely used as an antitumor drug. We found by liquid chromatography-ionspray mass spectrometry and electron spin resonance (ESR) that tyrosinase caused oxidation of VP-16 to its o-quinone and aromatized derivative via intermediate formation of the phenoxyl radical. Both ascorbate and thiols (GSH, dihydrolipoic acid, and metallothioneins) were able to directly reduce the VP-16 phenoxyl radical and prevent its oxidation. The characteristic ESR signal of the VP-16 phenoxyl radical was quenched by the reductants. The semidehydroascorbyl radical ESR signal was detected in the presence of ascorbate; thiols did not produce signals in the ESR spectra. In combinations, ascorbate plus GSH and ascorbate plus metallothionein acted independently and additively in reducing the VP-16 phenoxyl radical. Ascorbate was more reactive: the VP-16-dependent oxidation of GSH or metallothionein commenced only after complete oxidation of ascorbate. The semidehydroascorbyl radical ESR signal preceded the quenching of the VP-16 phenoxyl radical by GSH and metallothionein. In the presence of ascorbate plus dihydrolipoic acid, ascorbate was also more reactive toward the VP-16 phenoxyl radical than dihydrolipoic acid, but the ascorbate concentration was maintained at the expense of its regeneration from dehydroascorbate by dihydrolipoic acid. In ESR spectra, the semidehydroascorbyl radical ESR signal was continuously detected and then was abruptly substituted by the VP-16 phenoxyl radical signal. When VP-16 and tyrosinase were incubated in the presence of retina or hepatocyte homogenates, a two-phase lag period was observed by ESR for the appearance of the VP-16 radical signal: an ascorbate-dependent part (semidehydroascorbyl radical observable, sensitive to ascorbate oxidase) and thiol-dependent part (no radical signals in the spectra, sensitive to mersalyl acid). About 50% of the thiol-dependent part of the lag period could be accounted for by endogenous GSH (as revealed by treatment with GSH peroxidase+cumene hydroperoxide).(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: The Burkitt's lymphoma cell line Namalva was used as an in vitro model to study the accumulation and photocytotoxic properties of Photofrin, the photosensitizer currently in clinical trials. Photofrin uptake and intracellular protein binding were evaluated as a function of the incubation time using fluorescence spectroscopy. The drug concentration strongly affected the intracellular distribution pattern of Photofrin, which in turn was shown to correlate with the Namalva cell response to photodynamic action. At low drug load, a gradual cell response varying from transient cell cycle arrest (predominantly S-phase) to marked photocytotoxicity modulated by incubation time and light dose was observed. High drug load resulted in lethality without cell cycle selectivity. The data suggest possible Photofrin targeting of protein factors involved in cell proliferation.
Abstract: The 3-D quantitative structure-activity relationships/comparative molecular field analysis (QSAR/CoMFA) paradigm, which considers the primary importance of the molecular fields in biological recognition, is now widely used to analyze and predict receptor-binding properties of various ligands. CoMFA was applied to build 3-D QSAR models of substituted estradiol-receptor interactions, employing 3-D molecular databases of more than 40 molecules. Ligands included the 17 alpha-ethynyl- and isomeric 17 alpha (20E/Z)-(iodovinyl)estradiols and their 7 alpha-, 11 beta-, and 12 beta-methyl (-methoxy) and -ethyl (-ethoxy) derivatives as well as selected 2- and 4-halogenated analogs. The influence of different CoMFA descriptors was studied in order to achieve the highest possible cross-validated r2, as derived from partial least-squares calculations. Special emphasis was put on the analysis of the nature of H-bonding (donor/acceptor) interactions. The model with the best predictive performance (r2 = 0.895) was used to visualize steric and electrostatic features of the QSAR (standard deviation*coefficient contour maps) and to predict receptor-binding affinities (RBA) of substituted estradiols other than those included in the original database. Twenty-seven test molecules were selected, including five which had previously been reported by other investigators. For the latter, a very good correlation with literature RBA values was obtained, which together with the high cross-validated r2 provides evidence for the high predictive capacity of the model. Among the unknown structures, the model suggests several new substitutions to derive at reasonable affinity ligands for the estrogen receptor.
Abstract: The interaction of chloroaluminium-tetramethyl-tetrapyridinoporphyrazine (ClAlTMPyPa) with DNA was investigated by absorption/emission and circular-dichroic spectroscopy. Formation of a DNA-porphyrazine complex was evidenced by quenching of the ClAlTMPyPa fluorescence, a hypochromic red shift of the absorption band in the visible part of the spectrum and induction of a characteristic CD band with high positive ellipticity. Scatchard analysis of the spectral changes in low ionic strength buffer suggested an association constant of 9.7 +/- 0.6 x 10(7) M-1. Quenching of the fluorescence of a DNA-ethidium complex by added ClAlTMPyPa was shown to result from direct DNA-dye interaction rather than the displacement of ethidium from DNA. Our data suggest that porphyrazine binds to the outside of the DNA helix, involving interactions which are not limited to electrostatic forces only. The computer assisted modelling of ClAlTMPyPa-DNA interactions showed that the energy-minimized intermolecular orientation involves face-on binding of the ligand, preferably on the minor DNA groove.
Abstract: The effect of 2- and 4-fluoro substitution on the estrogen receptor-mediated tissue localization of radioiodinated 16 alpha-iodoestradiol (16 alpha-IE2) and its 11 beta-methoxy analogue (11 beta-OMe-16 alpha-IE2) was evaluated. Electrophilic substitution of estrone or 11 beta-methoxyestrone with N-fluoropyridinium salt gave the 2- and 4-fluoro derivatives which were subsequently converted to the 3,17 beta-enol diacetate and brominated to yield exclusively the 16 alpha-bromo analogues. Epimerization gave the corresponding 16 beta-bromoestrones which were reduced to the 17 beta-hydroxy derivatives. Halogen exchange with NaI or Na[125I]I provided the A-ring fluorinated 16 alpha-iodoestradiols. The 4-F analogue exhibited higher affinity for estrogen receptors than the corresponding 2-F analogue, and these differences were more pronounced at higher incubation temperatures. Biodistribution studies in immature female rats showed that 4-fluoro substitution had only a moderate effect on receptor-mediated tissue uptake of the parent molecules whereas 2-fluoro substitution resulted in strongly diminished tissue specificity. The lower target selectivity of the 2-F, compared to the 4-F, analogue correlates to some extent with their different receptor binding properties; however, the rate of catabolism may also be involved. Differences in blood clearance further accentuated the localization properties to yield particularly high uterus to blood ratios in the case of the 4-F-11 beta-OMe-16 alpha-IE2, suggesting the potential of the analog labeled with 123I as a radiopharmaceutical for receptor imaging in nuclear medicine. The isopotential maps of the fluorinated steroids, obtained via semiempirical computer modeling on the molecular structures, show striking differences between the 4-F and 2-F derivatives reflecting their varying biological properties.
Abstract: Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.
Abstract: Spin-trapping and electron spin resonance (ESR) spectroscopy were used to detect free radicals generated during light exposure of lymphoma cells sensitized in vitro by metallotetrasulfophthalocyanines (Al-PcS4 and Zn-PcS4). 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and alpha-phenyl-beta-tert-butylnitrone (PBN) were used as spin-trapping agents. Hydroxyl radical spin-adducts were detected under conditions of both extracellular and intracellular photosensitization. In addition, organic radicals of different origin and/or variable yields were trapped, depending on the photosensitization conditions and the spin-trap used. For comparison, analogous experiments were carried out with another tumor-localizing photosensitizer, Photofrin II.
Abstract: Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.
Abstract: Complex formation between free-base tetrasulphophthalocyanine (TSPC) and tryptophan (Trp) in solutions of high dielectric constant was studied using absorption/emission spectroscopy. ESR experiments demonstrated dye/substrate electron or hydrogen atom transfer reactions during illumination, competing with superoxide radical generation. To discriminate between the well-known Trp photodynamic (1O2 mediated) oxidation and other reactions, Fe(III)-EDTA was added to initiate the Haber-Weiss reaction. The TSPC/Trp complex showed an enhanced ability to reduce Fe(III)-EDTA. Spin-trapping experiments and the SOD and catalase-independent formation of 2-deoxyribose/thiobarbituric acid products imply involvement of a photoinduced Haber-Weiss reaction with generation of Trp-peroxyl and Trp-alkoxy radicals as intermediates, followed by formation of Trp carbon-centred radical(s).
Abstract: Using solutions of different polarity and various reducing (oxidizing) substrates, the type I (free radical) and type II (singlet oxygen) photosensitizing efficiencies of haematoporphyrin (HP), haematoporphyrin derivative (HPD, Photofrin II) and free-base tetrasulphophthalocyanine (TSPC-H2) were investigated. The quantum yields of 1O2-mediated oxidation of 2,2,6,6-tetramethyl-4-oxopiperidine (TEMP) followed the order QHP greater than QHPD greater than QTSPC-H2 in all the reaction media investigated. Monomeric TSPC-H2 effectively generates O2-. as shown by spin-trapping measurements. It is probable that both HPD and TSPC-H2 can oxidize L-tryptophan (Trp) via a mixed type I and type II mechanism, depending on the polarity of the medium. Both HP and TSPC-H2 in the monomeric form can be readily photoreduced by mild electron donors (ethylenediaminetetraacetic acid (EDTA), cysteine, sodium ascorbate, etc.). However, the reaction efficiencies differ because of the higher net negative electrical charge of TSPC-H2.
Abstract: Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).
Abstract: Hematoporphyrin (Hp) solutions were subjected to a wide range high intensity (0.2-10.0 GW/cm2) near-UV laser pulse radiation (lambda exc = 355 nm, pulse duration 30 ps). The formation of stable Hp photoproducts was followed by UV-VIS absorption spectroscopy and liquid-gel column chromatography. As judged by the influence of free radial scavengers, a significant part of the products is assigned to arise from the reaction of Hp with OH.(and H.) radicals. Using nitroxide radicals (TEMPO and TEMPONE) and the spin trap DMPO the generation of primary transient photoproducts, hydrated electrons (eaq-), OH. and H. radicals, was studied varying the pulse intensity at a constant absorbed light energy. The results showed that bi-photonic processes are responsible for the observed product generation (different for eaq- photoejection and OH.(H.) formation). A tentative diagram of the Hp excitation routes involved in the present high intensity laser flash photolysis is suggested. According to it, OH. and H. radicals are supposed to be generated in a resonance energy transfer reaction from highly excited Hp** to water molecules (H2O sensitization).
Abstract: Hemoglobin A (HbA) and hemoglobin F (HbF) dynamic structures have been studied using spin-label ESR spectra analysis technique, which permits quantitative separation of slow macromolecular rotation (described by rotational correlation time, tau c) and fast anisotropic nitroxide radical motion (described by the 'order parameter', S). The hardly restricted motion of the maleimide spin-label reflects the overall macromolecular rotation and small dynamic structure differences between HbA and HbF were observed (tau c is equal to 26 and 27 ns, respectively). On the other hand, the dynamic equilibrium of the iodoacetamide spin-label demonstrates significant differences between beta- and gamma-chain C-terminus flexibility. Thus, there are different states of alpha,beta and alpha,gamma intersubunit contacts which may be expected to determine the different O2 affinity of HbA and HbF. The antibiotic, chloramphenicol, strongly affects the O2 affinity and the Hill constant of HbF, and also provides detectable changes of gamma-subunit C-terminus flexibility (tau c changes from 20 ns to 27 ns after chloramphenicol treatment of HbF), while the HbA tetramer structure remains almost unaffected. The HbF domain structure rearrangements are accompanied by a decrease of the steric restriction of the spin-label motion (S changes from 0.75 to 0.72).
Abstract: The influence of the polarity of the medium on the efficiency of Type I and Type II photosensitization as carried out by hematoporphyrin and its derivative Photofrin II was studied by EPR techniques. Our results suggest that porphyrin aggregates do not participate as such in the photosensitized processes. Thus, the solvent-induced breakdown of the aggregated components of Photofrin II appears to determine the photosensitizing efficiency of this porphyrin.
