Monitoring of DNA structural changes after incorporation of the phenylpyrazole insecticide fipronil.

The use of insecticides presents a risk to the environment because they can accumulate in the water, soil, air, and organisms, endangering human and animal health. It is therefore essential to investigate the effects of different groups of insecticides on individual biomacromolecules such as DNA. We studied fipronil, which belongs to the group of phenylpyrazole insecticides. The interaction of fipronil with calf thymus DNA was investigated using spectroscopic methods (absorption and fluorescence spectroscopy) complemented with infrared spectroscopy and viscosity measurement. Fluorescence emission spectroscopy showed the formation of a fipronil/DNA complex with a combined static and dynamic type of quenching. The binding constant was 4.15 × 103 L/mol. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. A slight change in DNA viscosity in the presence of fipronil was observed. The phenylpyrazole insecticide does not cause significant conformational changes in DNA structure or increase of its chain length. We hypothesize that fipronil is incorporated into the minor groove of the DNA macromolecule via hydrogen interactions as indicated by FT-IR and CD measurements.

Vibrational characterization of the pesticide molecule Tebuconazole.

Pesticide use worldwide exhibits a positive effect on agricultural production while it may negatively affect organisms living in soil, water or the air. Importantly, numerous negative health effects also occur in humans exposed to (accumulated) pesticides or their metabolites over a long period of time. To prevent both environmental catastrophes and adverse human health impacts, initial studies of the selected pesticides need to be performed together with the constant post-approval control; risk assessment analysis and on site monitoring have to be continuously carried out. Given this, Raman spectroscopy, especially surface-enhanced Raman spectroscopy (SERS), during the last decade has become a powerful analytical technique since it can offer quick, selective, and in situ detection of selected pollutants found in analyzed samples at very low concentrations. Moreover, the structural changes caused by the pollutant-biomacromolecule interaction can also be recognized in the molecule-specific Raman spectral signatures of biomolecules. In this study, we report a vibrational characterization of the fungicide molecule Tebuconazole (TB) which is listed to be a possible carcinogen. Even though its international and common use there is no evidence about the use of Raman/SERS spectroscopy to detect it sensitively and selectively as well as to analyse its impacts on biological systems. Therefore, we have recorded and calculated Raman and infrared spectra of TB. Furthermore, SERS spectra of TB were also registered and comprehensively analysed in view of the employed SERS substrates, dependence on the excitation wavelengths and pH of the analysed molecular systems. The molecule of TB interacts preferentially through the triazole moiety with the colloidal metal nanoparticles (NPs) whereas the silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride resulted to be the most effective ones. Consequently, the limit of detection was determined to be 1.4 µM≈430 ppb. The present paper thus could serve significantly for further investigations focused on both conducting vibrational analyses of structurally related molecules as well as providing a more precise explanation of the mechanism of action of TB and its influence on biological macromolecules.

Chromosomal Aberrations in Cattle.

Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

Interaction of Conazole Pesticides Epoxiconazole and Prothioconazole with Human and Bovine Serum Albumin Studied Using Spectroscopic Methods and Molecular Modeling.

The interactions of epoxiconazole and prothioconazole with human serum albumin and bovine serum albumin were investigated using spectroscopic methods complemented with molecular modeling. Spectroscopic techniques showed the formation of pesticide/serum albumin complexes with the static type as the dominant mechanism. The association constants ranged from 3.80 × 104-6.45 × 105 L/mol depending on the pesticide molecule (epoxiconazole, prothioconazole) and albumin type (human or bovine serum albumin). The calculated thermodynamic parameters revealed that the binding of pesticides into serum albumin macromolecules mainly depended on hydrogen bonds and van der Waals interactions. Synchronous fluorescence spectroscopy and the competitive experiments method showed that pesticides bind to subdomain IIA, near tryptophan; in the case of bovine serum albumin also on the macromolecule surface. Concerning prothioconazole, we observed the existence of an additional binding site at the junction of domains I and III of serum albumin macromolecules. These observations were corroborated well by molecular modeling predictions. The conformation changes in secondary structure were characterized by circular dichroism, three-dimensional fluorescence, and UV/VIS absorption methods.

DNA methylation studies in cattle.

Investigation of the role of epigenetics in cattle breeding is gaining importance. DNA methylation represents an epigenetic modification which is essential for genomic stability and maintenance of development. Recently, DNA methylation research in cattle has intensified. The studies focus on the definition of methylomes in various organs and tissues in relation to the expression of genes underlying economically important traits, and explore methylome changes under developmental, environmental, disease, and diet influences. The investigations further characterize the methylation patterns of gametes in connection with their quality, and study methylome alterations in the developing naturally or assisted produced zygotes, embryos, and fetuses, considering their viability. A wide array of technologies developed for accurate and precise analysis of DNA methylation patterns is employed for both single-gene and genome-wide studies. Overall, the research is directed towards the identification of single methylation markers or their combinations which may be useful in the selection and breeding of animals to ensure cattle improvement.

Biophysical Characterization and Anticancer Activities of Photosensitive Phytoanthraquinones Represented by Hypericin and Its Model Compounds.

Photosensitive compounds found in herbs have been reported in recent years as having a variety of interesting medicinal and biological activities. In this review, we focus on photosensitizers such as hypericin and its model compounds emodin, quinizarin, and danthron, which have antiviral, antifungal, antineoplastic, and antitumor effects. They can be utilized as potential agents in photodynamic therapy, especially in photodynamic therapy (PDT) for cancer. We aimed to give a comprehensive summary of the physical and chemical properties of these interesting molecules, emphasizing their mechanism of action in relation to their different interactions with biomacromolecules, specifically with DNA.

Fungicide Tebuconazole Influences the Structure of Human Serum Albumin Molecule.

Studies of interactions between pesticides and target mammalian proteins are important steps toward understanding the pesticide's toxicity. Using calorimetric and spectroscopic methods, the interaction between triazole fungicide tebuconazole and human serum albumin has been investigated. The spectroscopic techniques showed that fluorescence quenching of human serum albumin by tebuconazole was the result of the formation of tebuconazole/human serum albumin complex with the static type as the dominant mechanism. The association constant was found to be 8.51 × 103 L/mol. The thermodynamic parameters were obtained as ΔH = -56.964 kJ/mol, ΔS = -115.98 J/mol·K. The main active interactions forming the tebuconazole/human serum albumin complex were identified as the interplay between hydrogen bonds and/or van der Waals forces, based on thermodynamic experiments. These binding modes were corroborated well by the predictions of molecular modeling. Hydrogen bonding of tebuconazole with Arg222, Ala215 and Ala291 of human serum albumin played a relevant role in binding. The conformation changes in secondary structure were characterized by circular dichroism and 3D fluorescence spectra.

Interaction of a Potential Anticancer Agent Hypericin and its Model Compound Emodin with DNA and Bovine Serum Albumin.

BACKGROUND/AIM: We report the incorporation of prospective anticancer agent hypericin into DNA and bovine serum albumin (BSA), respectively, with emphasis on comparison of the differences in interaction mode between hypericin and its model compound emodin. MATERIALS AND METHODS: Spectrophotometric methods were used for determination of the binding constants of the drug complex with biomacromolecules. Differential scanning calorimetry was applied for evaluation of drug-macromolecule complex thermal stability. RESULTS: The strength of interaction expressed by binding constants was found to be 4.0×104 l/mol for hypericin-DNA and 8.1×104 l/mol for emodin-DNA complex. Both molecules stabilize bovine serum albumin macromolecule and bind into the hydrophobic cavity in IIA subunit but their localization within the molecule is different. CONCLUSION: Anticancer agent hypericin and its derivative emodin interact with DNA with medium strength and are probably incorporated into the groove of DNA by hydrogen bonds. Bovine serum albumin can serve as a transport protein for hypericin since the binding force between both molecules is adequate.

Formation of Large Hypericin Aggregates in Giant Unilamellar Vesicles-Experiments and Modeling.

The incorporation of hypericin (Hyp) from aqueous solutions into giant unilamellar vesicle (GUV) membranes has been studied experimentally and by means of kinetic Monte Carlo modeling. The time evolution of Hyp fluorescence originating from Hyp monomers dissolved in the GUV membrane has been recorded by confocal microscopy and while trapping individual GUVs in optical tweezers. It was shown that after reaching a maximum, the fluorescence intensity gradually decreased toward longer times. Formation of oversized Hyp clusters has been observed on the GUV surface at prolonged time. A simplified kinetic Monte Carlo model is presented to follow the aggregation/dissociation processes of Hyp molecules in the membrane. The simulation results reproduced the basic experimental observations: the scaling of the characteristic fluorescence decay time with the vesicle diameter and the buildup of large Hyp clusters in the GUV membrane.

Deeper insights into the drug defense of glioma cells against hydrophobic molecules.

By means of fluorescence microscopy the intracellular distribution of fluorescent drugs with different hydrophobicity (quinizarin, emodin and hypericin) was studied. Selective photoactivation of these drugs in precisely defined position (nuclear envelope) allowed moderately hydrophobic emodin enter the nucleus. Highly hydrophobic hypericin was predominantly kept in the membranes with no fluorescence observed in the nucleus. The redistribution of quinizarin, emodin and hypericin between lipids, proteins and DNA was studied in solutions and cells. Based on these results was proposed theoretical model of hydrophobic drugs' nuclear internalization after photo-activation. Molecular docking models showed that hypericin has the strongest affinity to P-glycoprotein involved in the cell detoxification. Presence of 10 µM quinizarin, emodin or hypericin increased P-glycoprotein function in U87 MG cells. Moreover, emodin pretreatment allowed quinizarin nuclear internalization without photo-activation, which was not the case for hypericin. The synergy of such pretreatment and photo-activation should lessen the drug doses with simultaneous increase of drug efficacy triggering cell apoptosis/necrosis.

How hydrogen peroxide is metabolized by oxidized cytochrome c oxidase.

In the absence of external electron donors, oxidized bovine cytochrome c oxidase (CcO) exhibits the ability to decompose excess H2O2. Depending on the concentration of peroxide, two mechanisms of degradation were identified. At submillimolar peroxide concentrations, decomposition proceeds with virtually no production of superoxide and oxygen. In contrast, in the millimolar H2O2 concentration range, CcO generates superoxide from peroxide. At submillimolar concentrations, the decomposition of H2O2 occurs at least at two sites. One is the catalytic heme a3-CuB center where H2O2 is reduced to water. During the interaction of the enzyme with H2O2, this center cycles back to oxidized CcO via the intermediate presence of two oxoferryl states. We show that at pH 8.0 two molecules of H2O2 react with the catalytic center accomplishing one cycle. In addition, the reactions at the heme a3-CuB center generate the surface-exposed lipid-based radical(s) that participates in the decomposition of peroxide. It is also found that the irreversible decline of the catalytic activity of the enzyme treated with submillimolar H2O2 concentrations results specifically from the decrease in the rate of electron transfer from heme a to the heme a3-CuB center during the reductive phase of the catalytic cycle. The rates of electron transfer from ferrocytochrome c to heme a and the kinetics of the oxidation of the fully reduced CcO with O2 were not affected in the peroxide-modified CcO.

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA.

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes.

Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.

On the diffusion of hypericin in dimethylsulfoxide/water mixtures-the effect of aggregation.

Hypericin (Hyp) is a natural photosensitizing pigment with a possible application in the photodynamic therapy of cancer. Hyp is readily dissolved in dimethylsulfoxide (DMSO) but forms nonsoluble aggregates in an aqueous environment. Fluorescence spectroscopy and diffusion coefficient measurements are used to investigate the self-association of Hyp molecules in DMSO/water mixtures. Fluorescence measurements reveal that Hyp remains in its monomeric form in DMSO/water mixtures containing up to ∼20-30 wt % water. At higher water concentration, Hyp starts to form nonfluorescent aggregates. To determine the size of the aggregates, the diffusion coefficient of Hyp is determined for different DMSO/water mixtures both experimentally and theoretically. Our data indicate that the size of the aggregates increases as more water is added into DMSO. At 50 wt % water content, the effective diffusion coefficient is about 30% smaller than the calculated value for the stacked Hyp tetramer. The results indicate that in an aqueous environment, Hyp presumably produces large molecular weight stacked H-aggregates. We have also confirmed that in an aqueous environment at alkaline pH, molecules of Hyp remain in the monomeric state.

Differential stability of dimeric and monomeric cytochrome c oxidase exposed to elevated hydrostatic pressure.

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2-3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time- and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, high-performance ion-exchange chromatography, and reversed-phase and SDS-PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure.