Doina Gazdaru

Chromatin structure modification in an excimer laser field

Chromatin is the complex of deoxyribonucleic acid (DNA) with proteins that exists in eukaryotic cell nuclei. Chromatin was extracted from livers of Wistar rats and subjected to a 248-nm excimer laser radiation, in doses of 0.5-3 MJ/m 2 . An UV excimer laser Iofan 1701, with 40-mJ dose/pulse and frequency of 30 Hz was used. The radiolysis of chromatin was analyzed by (1) 1 H-NMR spectroscopy, (2) steady-state fluorescence, (3) time-resolved fluorescence, and (4) fluorescence resonance energy transfer (FRET) methods. The laser action on chromatin determines bigger values of the transverse relaxation time (T 2 ), which indicates less bound water in the chromatin structure, therefore a more injured one. The chromatin intrinsic fluorescence decreases on laser action, proving the destruction of the chromatin protein structure. By the time-resolved fluorescence we established that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with the laser dose. This denotes single- and double-strand breaks produced in DNA structure. By the FRET method, the energy transfer efficiency and the distance between dansyl chloride and acridine orange coupled at chromatin were determined. The distance increases with laser action. The determination of the chromatin structure modification in an excimer laser field can be of real interest in medical applications.

Optical properties of cytostatic drugs used in cancer treatment

A spectroscopical characterization of methotrexate, cytostatic drug used frequently in cancer therapy, was performed. The absorption, emission and excitation spectra were measured for methotrexate solutions in natural saline and sodium hydroxide at concentration in the range 10-5 M -10-6 M and pH 8.4. The absorption bands are noticed in the spectral range 250 nm - 450 nm. The fluorescence excitation was made at 340 nm and 370 nm; the fluorescence emission was detected in the spectral range 400 nm - 500 nm with a maximum at 450 nm. The behavior of absorption and fluorescence spectra of methotrexate solution exposed to uv-visible light was investigated. The irradiation was made using an Xe lamp (emission between 325 nm and 420 nm and power density of 11 mW/cm2). The exposure time was between 15 min. and 3 h. Major modifications on absorption bands for irradiation times longer than 1 hour were observed. Furthermore, the methotrexate solutions become strongly fluorescent after irradiation. The observed changes are not linear with the exposure time indicating complex photochemical processes which implies, at least, one intermediate product.

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0–250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (Dc = 80 ± 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O2 removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A0) increases in O2‐free buffer (98 ± 13) and in 10% ethanol‐containing buffer (74 ± 34) compared to control conditions (23 ± 5). Varying the dose rate between 1–10 Gy/min at a constant dose of 50 Gy, an inverse dose‐rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright

The modification of spectral characteristics of cytostatics by optical beams

Besides the biochemical action of methotrexate (MTX) and 5-fluorouracil (FU) their effect in destroying cancer tumours could be enhanced by exposure to light at different doses. Absorption, excitation and emission spectra of 10-4M - 10-5M MTX solutions in natural saline and sodium hydroxide at pH = 8.4 were measured, while their exposure to coherent and uncoherent light in the visible and near ultraviolet (UV) spectral ranges was made (Hg lamps and Nitrogen pulsed laser radiation were used). Absorption spectra exhibit spectral bands in the range 200 nm - 450 nm. The 200 - 450 nm excitation spectra were measured with emission centered on 470 nm; MTX fluorescence excitation was measured at 390 nm and the emission was detected between 400 nm and 600 nm showing a maximum at 470 nm. Spectra modifications, nonlinearly depending on exposure time (varying from 1 min to 20 min), evidenced MTX photo-dissociation to the fluorescent compound 2,4 diamino-formylpteridine. In the 5-FU case the absorption spectra exhibit bands between 200 nm and 450 nm. The emission fluorescence spectra were measured between 400 nm and 600 nm, with λex = 350 nm for UV Hg lamp and with λex = 360 nm for laser irradiated samples; at irradiation with N2 laser emitted radiation the excitation spectra were measured in the range of 200 nm - 400 nm, with λem = 440 nm. New vascularity rapid destruction was observed for conjunctive impregnated with 5-FU solution whilst exposed to incoherent UV and visible light.

Studies on activated cytostatic fluorouracil as photosensitizer: to use in eye tumor treatment

Hydroxypyrimidine 5-fluorouracil (5-FU) belongs to the cytostatics group known as antimetabolites. The effect of UV irradiation on 5-FU was investigated by absorption and fluorescence spectroscopy. The study of the photosensitizer properties of 5-FU was made since their effects could be enhanced by exposure to UV radiation at different doses. Solutions 2.5x10-4M in natural saline water (0.8% NaCl), irradiated with optical beams emitted by N2 laser and UV Hg classic lamp, were used. The 5-FU was chosen due to its strong absorption along a large spectral range which makes possible the fluorescence excitation in UV. The absorption spectra exhibit bands between 250 - 450 nm. The emission fluorescence was measured in the 400-550 nm spectral range, with λex=320 and 350 nm for samples irradiated with Hg lamp and with λex=360 nm for samples irradiated with N2 laser. The excitation fluorescence was measured in the spectral range 200-400 nm, with λem=440 nm for samples irradiated with N2 laser. The spectra reveal a fluorescence enhancement with the exposure time, with a maximum at 3 min due to the transformation of 5-FU molecule into a fluorescent tautomeric form. The destruction more rapid than usual of the neovascularisation was observed for conjunctive of rabbit eyes, when they are impregnated with 5-FU solution and exposed to incoherent UV and visible light.

CANCERIGENS OR RADIATION ACTION ON CHROMATIN DETERMINED BY SPECTROFLUORIMETRIC METHODS

Steady state and time resolved fluorescence and fluorescence resonance energy transfer (FRET) methods were used in the determination of cancerigens or radiation action on chromatin -- the complex of deoxyribonucleic acid (DNA) with proteins, that exists in eukaryotic cells nuclei. The results indicated modifications in general chromatin structure, DNA strand breaks with the reduction of the double helix DNA proportion (for radiation action) or the growth of the proportion of available DNA for ligand binding (in cancerigens action), proteins structure destruction and an increase of acidic proteins proportion on cancerigens action. The action of other physical or chemical agents may be analyzed by these high performance optical methods.

Studies on cytostatics used as photosensitizing material in photodynamic therapy

Introduction of the photosensitizer properties of cytostatics drus was made, pointing out that the fact that besides the biochemical action of the cytostatics their effects could be enhanced by the exposure to light at different doses. A spectroscopical characterisation of methotrexate and fluorouracil, cytostatic drugs used frequently in cancer therpy was performed. The absorption, emission and excitation spectra were measured for methotrexate solutions in natural saline and sodium hydroxide at concentration in the range 10-5 -10-6M and pH 8.4. The absorption, emission and excitation spectra were measured for fluorouracil solutions in natural saline at concentration in the range 10-4 -10-5M. The absorption spectrum exhibits spectral bands in the range 250nm -450nm for both drugs. The fluorescence excitatioan for methotrexate was made at 340nm and 370nm, the fluorescence emission was detected in the spectral range 400nm - 500nm with a maximum at 470nm. The fluorescence excitation was measured in teh range 200nm-500nm with the emission centred on 530nm, for Xe lamp irradiation, and 300nm for Hg lamp and laser irradiation. The fluorescence emission spectra was monitored in the spectral range 400nm - 600nm. The effects of irradiation on spectroscopic characteristics of methrotrexate and fluorouracil were investigated. The irraditaion was made using a UV classic lamp with Xe, for the first experimental part and for the second one it was used both a class Hg lamp and a nytorgen pulsed laser.

Contribution to the spectroscopic study of cytostatics molecules

The effect of UV irradiation of methotrexate was investigated by steady state absorption and fluorescence spectroscopy. Major modifications on absorption bands were detected upon irradiation fluence greater than 59J/cm2. In addition the irradiated solutions become strongly fluorescent. The detected changes are not linear with the exposure time suggesting that the photo-induced chemical processes are complex.

Quenching of Chlorophyll a Fluorescence by β-Carotene

Carotenoids have multiple functions in photosynthetic systems. A part of them have been well documented /1,2/. More recently, an additional function has been proposed which involves a specific carotenoid-chlorophyll interaction in their singlet excited states, leading to a quenching in chlorophyll (Chl) fluorescence. This means that carotenoids may play a role in regulating the energy flow of Chl excited states in photosynthetic antenna, being implied in an energy dissipation route. The former reports concerning the quenching of Chl fluorescence by β-carotene have been shown to be contradictory /3–5/. Further studies have shown that β-carotene can quench Chi a fluorescence in organic solvents /6,7/.