Fast and ultrafast spectroscopic investigation of tetracycline derivatives in organic and aqueous media.

The photophysical properties of seven tetracycline derivatives (tetracycline, oxytetracycline, demeclocycline, chlortetracycline, doxycycline, minocycline and meclocycline) in organic solvents and aqueous solution were studied using steady-state absorption and fluorescence techniques and transient absorption spectroscopies with nanosecond and femtosecond time resolution. The molecular structure, solvent and pH effects on the optical properties of this class of pharmaceutically interesting compounds were investigated in detail. The investigation furnished a complete description of the nature, the spectral and kinetic properties of the excited states formed upon irradiation. All the tetracycline derivatives exhibited a similar behaviour, and the photophysics of these molecules is different in organic solvents and in aqueous medium, where they exhibit a significant pH dependence. In water, compared to organic solvents, these compounds showed a blue-shifted bathochromic absorption band, a red-shifted emission spectrum, an increased Stokes shift and a decreased fluorescence quantum yield. These findings, together with the overall investigated solvent effect, suggested that in aqueous solvent additional fast and non-radiative deactivation processes, responsible for the large Stokes Shift and for the reduced fluorescence efficiency, are present. In fact, in organic media just two transients were observed during the ultrafast time-resolved investigation: the vibrationally hot S(1) state which was quickly stabilized by solvent reorganization to the relaxed S(1) state. This state showed lifetimes of tens of picoseconds and relaxed by fluorescence and internal conversion. No longer-lived transients were detected. In aqueous solution the excited-state deactivation of tetracyclines was found to be more complicated. Different protonated and tautomeric forms of the S(1) state were detected: a component which showed decay times of tens of picoseconds and a component which was longer-lived. A significant pH effect on the nature and number of these components was found. In fact, a remarkable change in the Stokes shift and in the fluorescence efficiency was also observed on going from acidic to basic aqueous solutions. The most important variations in the absorption properties were found in the pH range in which the second acid-base equilibrium takes place. The tetracycline lowest excited triplet state was observed as a 'rest absorption' during the femtosecond-resolved measurements in aqueous solution; through the nanosecond-resolved laser flash photolysis study, lower-energy radical species were detected, characterized by lifetimes of tens of microseconds. The formation of these species may be involved in the observed phototoxicity of the tetracycline drugs.

Detecting hydrogen peroxide in leaves in vivo - a comparison of methods.

Four hydrogen peroxide detecting probes, 3,3'-diaminobenzidine (DAB), Amplex Red (AR), Amplex Ultra Red (AUR) and a europium-tetracycline complex (Eu(3)Tc) were infiltrated into tobacco leaves and tested for sensitivity to light, toxicity, subcellular localization and capacity to detect H(2)O(2) in vivo. In the absence of leaves, in water solutions, AUR was very much sensitive to strong light, AR showed slight light sensitivity, while DAB and Eu(3)Tc were insensitive to irradiation. When infiltrated into the leaves, the probes decreased the photochemical yield (Phi(PSII)) in the following order of effect AR > DAB > AUR > Eu(3)Tc. With the exception of Eu(3)Tc, all probes stimulated the build-up of non-photochemical quenching either temporally (DAB, AUR) or permanently (AR), showing that their presence may already limit the photosynthetic capacity of leaves, even in the absence of additional stress. This should be taken into account when using these probes in plant stress experiments. Confocal laser scanning microscopy studies with the three fluorescent H(2)O(2) probes showed that the localizations of Eu(3)Tc and AUR were mainly intercellular. AR partly penetrated into leaf chloroplasts but probably not into the thylakoid membranes. Photosynthesis-related stress applications of AR seem to be limited by the low availability of internal leaf peroxidases. Applications of AR for kinetic H(2)O(2) measurements would require a co-infiltration of external peroxidase, imposing another artificial modifying factor and thus taking experiments further from ideal, in vivo conditions. Our results suggest that the studied H(2)O(2) probes should be used in leaf studies with caution, carefully balancing benefits and artifacts.

Oxidation photosensitized by tetracyclines.

Irradiation with 365-nm UV light of aerated aqueous solutions of tetracycline gives rise to oxygen uptake when the pH of the solution is above 7.5. The kinetics of the reaction were followed using a polarographic oxygen electrode at a range of pH values for seven currently prescribed tetracyclines. Variation of tetracycline concentration, UV light intensity, and temperature showed the characteristics normally associated with a sensitized photo-oxygenation mechanism rather than a free-radical process. Copper(II) ions inhibited the photo-oxidation of tetracycline, apparently by complex formation. The tetracyclines were tested for photosensitizing capability with oxidizable acceptors. In aqueous solution, no photosensitizing effect could be seen, but methanol solutions of 2,5-dimethylfuran and dl-limonene were oxidized at considerably increased rates when small amounts of tetracyclines were present. This observations has implications for the mechanism of in vivo photosensitivity reactions that occur when tetracyclines are taken internally.

Photosensitization by fluorescein dyes and tetracycline drugs at an oil/water interface.

Measurements were made of the changes that occur in the interfacial tension of isopropylbenzene when floating on water containing between 10(-5) and 10(-3) M of various fluorescein dyes and tetracycline drugs and irradiated for up to 240 min with ultraviolet light. Singlet oxygen was found to be an active intermediate in the reactions. The results are discussed in relation to adverse photosensitization in-vivo due to fluorescein dyes and tetracycline drugs.

Phototoxicity of the tetracyclines: photosensitized emission of singlet delta dioxygen.

The spectroscopic observation of 1268-nm emission of singlet oxygen photosensitized by tetracyclines in oxygenated solutions at room temperature is reported. In the series demeclocycline, tetracycline, and minocycline, the efficiency of singlet oxygen generation is found to parallel the clinical observation of relative frequency of phototoxicity of these antibiotics, suggesting singlet oxygen generation as the origin of their phototoxicity.