A micro-computed tomography study of the sinus tympani variation in humans.

BACKGROUND: The posterior part of the tympanic cavity comprises a depression called the sinus tympani (ST). The said structure is of outmost importance, e.g. in surgical procedures involving the middle ear, as a pathology (microbial biofilm or cholesteatoma) present in this difficult to access location might hinder its effective treatment. The aim of the study was to evaluate anatomical variants of the ST in human adult petrous bones. For this purpose, three-dimensional (3D) models of the ST were recreated from micro-computed tomography (CT) scans of 44 dry petrous bone samples (19 female, 25 male), applying 3D Slicer, Meshmixer and MeshLab software. MATERIALS AND METHODS: Anatomical variants of the ST were classified in terms of both shape and surface configuration. The internal configuration of the ST was classified as heterogeneous - containing small bony trabeculae and crests up to 1.0 mm in size, contrasting to homogeneous ST that characterizes a relatively smooth interior, or mere presence of minor depressions and mild folds. Female STs were more bowl-shaped (57.9%) than saccular (42.1%), and had heterogeneous surface configuration (52.6%) compared to homogeneous (47.4%). On the contrary, male STs were more saccular (52.0%) rather than bowl-shaped (48.0%), and predominantly had a heterogeneous surface (84.0%) over homogeneous (16.0%). RESULTS AND CONCLUSIONS: A complex combination of ST features comprised of a saccular shape and heterogeneous surface occurred in 52.0% of males and in 15.8% of females (a statistically significant difference; p = 0.0254, Fisher's exact test) seems to be clinically important because of its potential negative implication on health outcomes after surgery in the case of, for example, cholesteatoma, and it may also favour chronic pathological processes.

A new method to estimate 3D cell parameters from 2D microscopy images.

Optical microscopy has been a basic and standard technique in cell biology research for decades. Microscopy techniques function well for thin, optically transparent cultures and allow for the imaging of thicker biological specimens. There is no better method of in vitro cell observation and analysis, hence microscopic techniques are extensively used and constitute an optimal tool for cell culture studies. This paper proposes an original methodology of optical microscopy data processing based on the phase contrast technique during cell culture monitoring. By exploiting images recorded during cell proliferation, a surface reconstruction was performed based on assumption, it can be considered that the local brightness of the image depends on the cells' thickness and thus the obtained results can be interpreted in the form of a surface that represents a three-dimensional structure, which allowed for a quantitative description of the cell evolution. The 3D data obtained enabled the investigation of parameters describing the morphology of the cells and the topology of their proliferation. These parameters included cell sizes in plane but also in the direction perpendicular to it, cell volume changes, their spatial distribution, as well as anisotropy and directivity. The method presented provides data carrying information similar to that obtained using a holographic microscope, e.g. A HoloMonitor (Phase Holographic Imaging PHI Inc.), or from confocal scanning microscopy with the "z-stack" mode. The techniques of bright field or phase contrast cell observation are, however, much cheaper, and widely available when compared to holographic microscopy, for instance. Besides, these also enable monitoring of cell activity over time, i.e. the study and quantitative description of dynamic changes in the cells. The proposed approach uses generally available free tools such as ImageJ software with BoneJ and Particle Analyzer plugins. The methodology is suitable for even a basic microscope, it can be easily implemented as a script, and thus data processing can be significantly shortened, the methodology can be automated, and also applied for data processing in real time.

Micro-imaging of implanted scaffolds using combined MRI and micro-CT.

RATIONALE AND OBJECTIVES: Thanks to the advanced studies in biomaterial engineering a panoply of polymers can be used to manufacture porous scaffolds for bone tissue regeneration. Suitability of the scaffold for its purpose is determined by factors like size of the pores, its orientation and shape, as well as biocompatibility of the material. Even though a variety of analysis methods is available for in vitro studies, investigating the process of bone reconstruction on implanted scaffold meets with difficulties. METHODS AND MATERIALS: Polylactide porous sponges imbued in hydroxyapatite were implanted into long bones of white New Zealand rabbits for 3 months. The bones obtained from the animals were subjected to MRI and µCT imaging. The obtained images were subsequently fused together. RESULTS AND CONCLUSIONS: Combined MRI and µCT resulted in high resolution diagnostic images which allow for: implant positioning, inflammation divulgement, rating degree of implant resorption, observation of newly formed trabeculae, texture analysis and other quantitative measurements.

Genetic algorithms as a useful tool for trabecular and cortical bone segmentation.

The aim of this study was to find a semi-automatic method of bone segmentation on the basis of computed tomography (CT) scan series in order to recreate corresponding 3D objects. So, it was crucial for the segmentation to be smooth between adjacent scans. The concept of graphics pipeline computing was used, i.e. simple graphics filters such as threshold or gradient were processed in a manner that the output of one filter became the input of the second one resulting in so called pipeline. The input of the entire stream was the CT scan and the output corresponded to the binary mask showing where a given tissue is located in the input image. In this approach the main task consists in finding the suitable sequence, types and parameters of graphics filters building the pipeline. Because of the high number of desired parameters (in our case 96), it was decided to use a slightly modified genetic algorithm. To determine fitness value, the mask obtained from the parameters found through genetic algorithms (GA) was compared with those manually prepared. The numerical value corresponding to such a comparison has been defined by Dice's coefficient. Preparation of reference masks for a few scans among the several hundreds of them was the only action done manually by a human expert. Using this method, very good results both for trabecular and cortical bones were obtained. It has to be emphasized that as no real border exists between these two bone types, the manually prepared reference masks were quite conventional and therefore charged with errors. As GA is a non-deterministic method, the present work also contains a statistical analysis of the relations existing between various GA parameters and fitness function. Finally the best sets of the GA parameters are proposed.

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines.

Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells.

Anthrapyridones, a novel group of antitumour non-cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells.

1. Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP-binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation. 2. The search of new compounds able to overcome MDR is of prime importance. 3. Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain. 4. The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells. 5. It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P-gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P-gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake. 6. For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P-gp resistant cells in comparison to sensitive cells. 7. We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.

Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells.

Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux. Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells. We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1. We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells. Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2 x 10(-17) mole cell(-1) s(-1), (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 microM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K(m) approximately 2 microM. In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.

The ability of new formamidine sugar-modified derivatives of daunorubicin to stimulate free radical formation in three enzymatic systems: NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase.

Some sterically hindered N-substituted derivatives of daunorubicin are known to be poor substrates for NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase. In consequence, poor oxygen radical generation by these compounds is observed. In this study we examined a new family of sugar-N-substituted derivatives of daunorubicin bearing a bulky substituent introduced on the nitrogen atom through the amidine spacer. These compounds were found to be very active in radical formation catalyzed by all three studied enzymes. Thus, the introduction of a heterocyclic ring, even if it is bulky but flexible, on the nitrogen atom of daunosamine moiety through the one-atom spacer (amidine group), does not induce the steric hindrance effect on the interaction of daunorubicin derivatives with these flavoprotein enzymes.

Kinetic analysis in living cells of the inhibition of the P-glycoprotein-mediated efflux of anthracyclines by vinca alkaloids.

Cells that overexpress the mdr 1 gene have decreased steady-state accumulation and increased efflux of many anticancer drugs including anthracyclines and vinca alkaloids. The mechanism(s) of P-glycoprotein-mediated efflux of drugs is (are) still poorly understood. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, the cellular accumulation has been examined of pirarubicin, doxorubicin and idarubicin alone and in conjunction with four vinca alkaloid derivatives--vinblastine, navelbine, vindesine and vincristine. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. It has been shown that the P-glycoprotein-mediated efflux of these three anthracyclines can be inhibited by vinca alkaloids derivatives. At pH 7.2, 50% of the P-glycoprotein-mediated efflux of daunorubicin and idarubicin was inhibited by about 40 +/- 10 microM vinblastine and that of pirarubicin by 10 +/- 2 microM vinblastine. The vinblastine concentration required to inhibit 50% of the active efflux of these anthracyclines did not depend on the anthracycline concentrations used, indicating that the inhibition was non competitive. The ability of navelbine, vincristine and vindesine to inhibit the active efflux of pirarubicin was also checked; 15 +/- 3 microM navelbine are required to inhibit 50% of the active efflux but at concentrations lower than 100 microM, neither vincristine nor vindesine were able to inhibit this efflux, indicating that the vinca alkaloids compounds which are the most efficient are the most lipophilic. For the four vinca alkaloids, the concentration required to inhibit 50% of the efflux was lower as the pH was higher. A detailed kinetics analysis of the P-glycoprotein-mediated efflux of pirarubicin in the presence of vinblastine indicates a non competitive inhibition with K(I) = 12 +/- 2 microM.

The role of structural factors of anthraquinone compounds and their quinone-modified analogues in NADH dehydrogenase-catalysed oxygen radical formation.

Anthraquinone compounds belong to the most important class of clinical antitumour agents. However, their use is limited by their peroxidating activity, being the consequence of free radical formation initiated by three oxyreductases. This activity is considered to be the main cause of cardiotoxic effects. The affinity of anthraquinone compounds to these enzymes is an essential factor governing the rate of one-electron transfer and the generation of oxygen radicals. A series of novel derivatives and analogues of natural and synthetic anthraquinones has been examined with the aim of identifying the structural factors essential for the ability to stimulate oxygen radical formation catalysed by NADH dehydrogenase. Functional groups and moieties favouring or disfavouring the interaction of the compounds with the enzyme have been determined. The quinonoid moiety as well as at least two phenolic groups in peri positions favoured the affinity of these compounds for NADH dehydrogenase. The modification of the quinonoid structure to iminoquinonoid or carboquinonoid forms dramatically decreased interaction with the enzyme. The O'-substitution by a bulky group in the sugar moiety of daunorubicin decreased the ability of the derivatives to stimulate oxygen radical formation. It has also been shown that the presence of an ionizable amino group on the sugar moiety of daunorubicin favours interaction with the NADH dehydrogenase. However, its location is not essential for this effect.

The direct reduction of cytochrome c by some anthraquinone antitumor compounds.

The ability of various anthraquinone antitumor agents to undergo oxidative metabolism with concomitant cytochrome c reduction has been examined. The reduction of cytochrome c by the compounds had enzymatic character and occurred without the formation of oxygen radicals. We have found that the presence of at least two phenolic groups in ring A of the compounds studied was indispensable for their oxidative metabolism. It is suggested that these groups are essential for the binding to cytochrome c. Furthermore, it has been shown that the existence of hydroxy groups in side chains of these compounds augments their interaction with this hemoprotein. On the basis of the results obtained for a series of analogs of mitoxantrone, we can conclude that the structural factor directly responsible for cytochrome c reduction is the primary or secondary amino group of the side chains.

Molecular modeling of singlet-oxygen binding to anthraquinones in relation to the peroxidating activity of antitumor anthraquinone drugs.

Anthraquinone derivatives are important anti-cancer drugs possessing, however, undesirable peroxidating and, in consequence, cardiotoxic properties. This results from the mediation by these compounds of the one-electron reduction processes of the oxygen molecule, which produces the highly toxic superoxide anion radical and other active oxygen species. This article summarizes the results of our studies on the molecular aspects of the mechanism of anthraquinone-mediated peroxidation which were carried out using enzymatic-assay, electrochemical, and quantum-mechanical methods.

Cell cycle dependent uptake and release of anthracycline by drug-resistant and drug-sensitive human leukaemic K562 cells.

The appearance of cellular resistance to antitumor drugs is a major problem in cancer chemotherapy. This results from the overexpression of the mdr 1 gene which encodes the 170 kDa P-glycoprotein or multidrug transporter. The uptake and release of 4'-O-tetrahydropyranyladriamycin by drug-sensitive and drug-resistant K562 cells in the different phase of the cycle have been determined. Synchronized cells were obtained by centrifugal elutriation. The kinetics, as well as the amount of drug intercalated inside the nucleus and free in the cytoplasm, have been determined using a spectrofluorometric method that we have developed and that does not compromise cell viability. The kinetics of active efflux of the drug under the effect of P-glycoprotein has been determined. We have calculated that the number of 4'-O-tetrahydropyranyladriamycin molecules, which are actively effluxed per cell and per second, is constant whatever the cell cycle phase.

6-[(aminoalkyl)amino]-substituted 7H-benzo[e]perimidin-7-ones as novel antineoplastic agents. Synthesis and biological evaluation.

A class of chromophore-modified anthracenediones with an additional pyrimidine ring incorporated into the chromophore system has been obtained in an attempt to provide compounds with diminished peroxidation activity and thus potentially lowered cardiotoxicity. Their synthesis was carried out by the reaction of 6-amino- or 6-hydroxy-7H-benzo[e]perimidin-7-one with a number of alkylamines. Potent activity was demonstrated in vitro against murine L1210 leukemia cells (equipotent with ametantrone) as well as against P388 leukemia in vivo (% T/C = 130-255). We observed that the benzoperimidines did not stimulate free radical formation, perhaps due to their poor substrate properties for NADH dehydrogenase.

The essential role of anthraquinones as substrates for NADH dehydrogenase in their redox cycling activity.

Redox potential, superoxide production and NADH dehydrogenase substrate properties of daunorubicin, its four sugar-modified derivatives, 4-demethoxydaunorubicin and ametantrone have been examined. A new method for the determination of substrate properties of anthraquinones for NADH dehydrogenase has been developed. This method is based on the ability of anthraquinones to decrease the amount of enzymatic cytochrome c reduction at low concentrations of NADH. The compounds examined stimulated oxygen radical formation in a very varied manner. However, they had very similar redox properties. On the other hand, the extent of the diminution of cytochrome c reduction by anthraquinones depended strongly on the structure of the compounds examined. We postulate that it is not the redox properties but the enzyme substrate properties of anthraquinones which play the most important role in stimulating free radical formation.

Thermodynamics of the anthracycline-nuclei interactions in drug-resistant and drug-sensitive K562 cells.

Fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to measure the uptake of three anthracycline derivatives: Adriamycin (ADR), 4'-o-tetrahydropyranyl-Adriamycin (THP-ADR) and aclacinomycin (ACM) in drug-sensitive and drug-resistant K562 cells. The concentration of drug bound to the nucleus and free in the cytoplasm, at steady state, as well as the concentration of drug bound to the nucleus at equilibrium state have been determined at temperatures ranging from 6 degrees to 40 degrees. The enthalpies for the binding of ADR, THP-ADR and ACM to nuclei equal -35 +/- 3, -35 +/- 3 and -30 +/- 3 kJ/mol, respectively. These values compare with the enthalpies of binding of these drugs to naked DNA.

Kinetic parameters for the uptake of anthracycline by drug-resistant and drug-sensitive K562 cells.

Fluorescence-emission spectra from anthracycline-treated cells suspended in buffer have been used to measure the uptake of three anthracycline derivatives: adriamycin, 4'-O-tetrahydropyranyladriamycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake and the kinetics of active efflux under the effect of an integral membrane glycoprotein, P-glycoprotein, have been measured as a function of temperature. The activation energies for the passage of the drugs through the plasma membrane have been calculated. In the case of 4'-O-tetrahydropyranyladriamycin, the activation energies for the passive diffusion of the drug equal 45 kJ.mol-1 and 37 kJ.mol-1 for sensitive and resistant cells, respectively. The activation energy for the active efflux of 4'-O-tetrahydropyranyladriamycin equal 25 kJ.mol-1.

Molecular determinants of singlet oxygen binding by anthraquinones in relation to their redox cycling activity.

A series of model anthraquinones with varying symmetry of pi-electron density distribution have been examined to verify our previous hypothesis concerning the essential role of quinone-singlet oxygen complex formation by asymmetric anthraquinones in their peroxidating properties. Comparison of the results of enzymatic studies using NADH dehydrogenase with those of cyclovoltammetric measurements fully confirmed the assumption that one-electron transfer mediation is facilitated by the preceding quinone-oxygen complex formation. To extend the scope of the molecular determinants of oxygen binding found in our previous studies, CNDO/2 and molecular electrostatic field (MEF) calculations have been performed. It has been concluded that the analysis of molecular electrostatic field as well as the dipole moment components has to be taken into account to judge whether a mutual orientation of the quinone and oxygen molecule can be reached which enables binding to occur. The second important factor is the appropriate symmetry of the quinone outer filled orbitals which assures that binding is not forbidden by the Woodward-Hoffman rules. These characteristics also explain the lack of oxygen binding by some asymmetric anthraquinones. The efficient electron transfer mediation be anthraquinones requires, beside the formation of the intermediate quinone-oxygen complex, effective catalysis of this process by oxidoreductase enzyme. The results obtained with model anthraquinones indicated that compounds with more than one phenolic group and an unsubstituted quinone carbonyl are good NADH dehydrogenase substrates. Imino derivatives and compounds with a reduced number or without free phenolic groups exhibit low affinity towards the enzyme.

Synthesis, peroxidating ability, and antineoplastic evaluation of 1-[(aminoalkyl)amino]-4-hydroxy-10-imino-9-anthracenones.

A novel group of cytotoxic anthraquinone derivatives, 1-[(aminoalkyl)amino]-4-hydroxy-10-imino-9-anthracenones, has been synthesized. It has been shown that imino analogues of the anthracenediones exhibit diminished ability to generate oxygen radicals. The cytotoxic activity of iminoanthracenones obtained was lower than that of the related quinone carbonyl analogues. One of the obtained imino compounds showed a moderate antileukemic activity in vivo.

Enzymatic studies of the effect of Cu (II) on oxygen radical production stimulated by daunorubicin and ametantrone.

Daunorubicin can bind Cu (II) in Tris-HCl buffer (pH 7.4) and in the presence of NADH (100 microM). The complex is very stable. Cu (II) is not removed from the complex by cytochrome c reductase. The complexation of Cu (II) to daunorubicin gives rise to a modification of its redox properties. The complex, unlike the free drug, does not stimulate oxygen radical production. Ametantrone can also form a complex with Cu (II) in the conditions of enzymatic assays. Nevertheless, this complex is not stable in the presence of cytochrome c reductase. It dissociates immediately after the addition of the enzyme with releasing the metal ion.