The effects of adjuvant arthritis on the myometrial adrenergic functions in the nonpregnant and the late-pregnant rat.

The beneficial effects of pregnancy on the symptoms of inflammatory diseases are well documented. The modulation in the uterine functions in the presence of generalized inflammation, however, is much less characterized. The aim of the present study was to explore the modulatory action of adjuvant arthritis on the adrenergic functions of the uterus in nonpregnant and late pregnant rats. Adjuvant arthritis was induced by the subplantar injection of M. butyricum. Presynaptic functions were characterized by a superfusion technique and by registration of the contractions of isolated uterine rings elicited by electric field stimulation. The functions of the adrenoceptors were characterized by constructing concentration-response curves with agonists for both α- and ß-receptors. Where these curves differed significantly from the control, the expressions of these receptors at the mRNA level were additionally determined. Adjuvant arthritis substantially decreased the uptake and release of [(3)H]noradrenaline in myometrial samples from nonpregnant rats, but caused no change at term. The electrically induced contractions were decreased by inflammation in both gestational states. Arthritis resulted in decreased ß-adrenoceptor-mediated relaxation (in both the nonpregnant and the late-pregnant animals) and an increase in α-mediated contraction at term. It can be concluded that adjuvant arthritis deteriorates the adrenergic innervation of the uterus. The effects of exogenous sympathomimetics are shifted, favoring a state of higher contractility. If similar mechanisms are operative in humans, the present results could imply that ß-adrenoceptor agonists are not ideal tocolytics when pregnancy is complicated by generalized inflammation.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.

Photobiological activity of exogenous and endogenous porphyrin derivatives in Escherichia coli and Enterococcus hirae cells.

Photodynamic treatment, the combined application of a photosensitiser and visible light, represents a new and promising approach for the inactivation of microorganisms. The photosensitising potentials of exogenous zinc-phthalocyanine-tetrasulphonate (ZnPsTS), tetraphenylporphyrins (TPPs) and endogenous porphyrin derivatives were tested and compared on Gram-negative and Gram-positive bacteria, Escherichia coli B. and Enterococcus hirae, respectively. The synthesis of endogenous porphyrins was induced by 5-aminolevulinic acid (delta-ALA). The porphyrin- or delta-ALA-treated cells were irradiated with white light. The photosensitising efficiency of endogenous derivatives on both types of bacteria is ZnPcTS < TP(4-OGluOH)3P < TP(4-OGluOH)4P. However, neither exogenous derivatives exhibit appreciable photosensitising activity for disinfection application. ALA-induced photodynamic treatment showed good potential for the inactivation of Escherichia coli cells, but not towards Enterococcus hirae cells. The failure of photosensitisation of the Enterococcus hirae strain selected indicates that apart from the Gram-positive character, other structural elements of the membrane can influence the result of photodynamic treatments.

Photoinduced inactivation of T7 phage sensitized by symmetrically and asymmetrically substituted tetraphenyl porphyrin: comparison of efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.

In situ detection of ALA-stimulated porphyrin metabolic products in Escherichia coli B by fluorescence line narrowing spectroscopy.

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on delta-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population.

delta-Aminolaevulinic acid-induced porphyrin synthesis and photodynamic inactivation of Escherichia coli B.

The possibility and conditions for the induction of porphyrin synthesis by exogenous delta-aminolaevulinic acid (ALA) and its applicability for the inactivation of Gram-negative bacteria Escherichia coli B. by photodynamic therapy (PDT) have been studied. The bacteria are supplemented with ALA in the log phase of growth, and are grown in a synthetic medium at 37 degrees C in the dark. The efficiency of porphyrin synthesis is detected by fluorescence spectroscopy performed on the isolated bacterial cells and the medium, respectively, and compared with results of high-performance liquid chromatography (HPLC) analysis. ALA stimulates the synthesis of protoporphyrin in the bacteria by a factor of five to six, and an increased amount of the more hydrophilic derivatives with a significant contribution of mesoporphyrin by a factor of two to three is observed in the culturing medium. The optimal conditions of ALA treatment with respect to PDT are 10-15 min of incubation of a bacterial culture of 2 x 10(7) cells ml-1 with (5-9) x 10(-3) mol l-1 ALA. The ALA-treated cells are irradiated by white light of 80 mW cm-2 under growth conditions and a decrease to 0.6% of the number of colony-forming units (CFUs ml-1) is observed after 90 min of irradiation.

Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives. II. Dark and photoinduced effects on T7 phage, yeast and HeLa cells.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives. Part I: Dark effects on T7 phage and HeLa cells.

Several dioxinocoumarin derivatives have been synthesized for photochemotherapeutical purposes. The physicochemical properties of 3,4-benzo-6,7-dioxinocoumarin and its biological activity in the dark were studied with regard to future photobiological applications. It was found that molecular aggregates are formed in aqueous solution at a concentration higher than 10(-5) mol l-1. In the dark, 3,4-benzo-6,7-dioxinocoumarin inactivates T7 phage and inhibits the growth of HeLa cells in a concentration-dependent manner. The dark inactivation of T7 phage was quantitatively characterized. It was found to be higher than that of 8-methoxypsoralen (8-MOP) and approximately equal to 4,6,4'-trimethylangelicin (TMA). From the inactivation kinetics and the lack of a quenching effect of polynucleotides on the fluorescence emission of the drug, it appears that, apart from the induction of DNA damage, other events are implicated in T7 phage dark inactivation. These results are important for the interpretation of the photobiological effects of this type of compound.

Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions.

The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation. These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products. In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation. Under appropriate experimental conditions these reactions can be studied and characterized separately. The quantitative characteristics (e.g. inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens. Some theoretical and practical consequences of the results obtained are discussed.

Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Characterization of new furocoumarin derivatives by their dark and light-mediated action on RNA bacteriophage MS2.

The monofunctional and bifunctional furocoumarin derivatives 8-methyl-3-carbethoxypsoralen (8Me3CPs) and 8-methoxypsoralen (8MOP) as well as their thiosubstituted derivatives (2-thio-8Me3CPs and 2-thio-8MOP) were compared in terms of their reactivities towards a ribonucleoprotein, the bacteriophage MS2. The order of their photoreactivities differed from that measured with nuclear DNA and mitochondrial DNA. Besides their widely investigated photoreactivity, their biological activity in the dark and after pre-irradiation was quantified. A parameter was defined which compares the number of molecules acting in the dark for 1 h with the number of absorbed photons which lead to the same degree of inactivation. The parameter for the furocoumarin derivatives examined, including 3-carbethoxypsoralen (3CPs) and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), was in the following order: AMT greater than 3CPs greater than 8MOP greater than 8Me3CPs greater than 8Me3CPsS approximately 8MOPS. A similar parameter was also determined for the dark effect of pre-irradiated compounds.

Loosening of the phage structure in a low ionic strength environment.

Structural parameters of phage T7 were compared in two frequently used Tris buffers of high and low ionic strength, in order to explain the different biological activity and drug-binding characteristics. Characteristics of the whole phage geometry were obtained by viscosimetry, static and quasi-elastic light-scattering and small-angle X-ray scattering. The latter method revealed dissimilarities in the intraphage DNA compactness, consistent with the findings of the optical absorption melting studies. Alterations in the particle dimensions determined in the same sample by different methods are discussed, and a model is constructed to explain the structural modifications that occur on lowering the ionic strength.

Salt effects on the bacteriophage T7-II structure and activity changes.

Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers. The ionic strength range was studied between 20-140 mmol/1. In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength. Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage. Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.