Synergistic effects of photoactivated tetra(4-sulfonatophenyl)porphine and nocodazole on microtubule assembly, accumulation of cells in mitosis and cell survival.

Human carcinoma cells of the line NHIK 3025 were incubated with meso-tetra(4-sulfonatophenyl)porphine (TPPS4) for 18 h and exposed to light in the absence or presence of nocodazole. Nocodazole (1 microgram ml-1) was applied to the cells 15 min prior to light exposure and washed off the cells immediately afterwards. The presence of nocodazole during photoactivation of TPPS4-loaded cells leads to a significantly reduced ability of tubulin to repolymerize after withdrawal of nocodazole, an increased accumulation of the cells in mitosis with a larger fraction in c-metaphase and a higher yield of photoactivated cells. A higher proportion of the cells accumulating in mitosis 6-12 h after exposure to light is unable to form colonies when exposed to light in the presence of nocodazole than in its absence. The present results are consistent with a specific TPPS4-induced photodamage to the unpolymerized form of the microtubule components.     

Kinetics of Photobleaching of Protoporphyrin IX in the Skin of Nude Mice Exposed to Different Fluence Rates of Red Light

Abstract— The purpose of the present study was to determine the kinetics and the fluence rate dependency of the photo-bleaching of protoporphyrin IX (PpIX) in normal skin of Balb/c nude mice after systemic and topical application of 5-aminolevulinic acid (ALA). ALA was administered systemically (200 mg/kg body weight, i.p.) and topically (20% w/w ALA cream) to the mice. Fluences of up to 40 J/cm² were delivered by a dye laser (636 nm) at fluence rates of 37.5, 75, 150, 300 and 500 mW/cm². The photo-bleaching rate was constant within this range of fluence rates. This result suggests that there is no oxygen effect for PpIX photobleaching in this region for the skin of Balb/c nude mice. During light exposure the fluorescence decay followed neither first- nor second-order kinetics. The decay rate was slightly faster after systemic application than after topical application of ALA, but did not depend on the time (1–8 h) between application and analysis.     

PHOTOSENSITIZING EFFICIENCIES, TUMOR- and CELLULAR UPTAKE OF DIFFERENT PHOTOSENSITIZING DRUGS RELEVANT FOR PHOTODYNAMIC THERAPY OF CANCER

Abstract Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)-di-hexyl-ether, HP-di-ethyl-ether, tetra (3-hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS₄) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e₆) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X-114/H₂0 partition coefficients at different pH-values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption-, fluorescence-excitation- and fluorescence-emission spectra of the cell-bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes. The following main conclusions were drawn: (1) 3THPP was the best and most selective tumor localizer of the dyes tested, followed by AIPCTS, TPPS₄, PII and Chi e.,. (2) The Triton X-114/H₂0 partition coefficient of most of the dyes decreased with increasing pH. (3) The cellular uptake of the dyes (18 h incubation in medium with 3% serum) increased with increasing Triton X-114/H₂0 partition coefficient. (4) HP-di-hexyl-ether had the highest quantum yields both for photoinactivation of cells and degradation of cell-bound DPBF, followed by the other lipophilic porphyrins and Chi e₆. The water-soluble dyes TPPS₄ and AIPCTS had quantum yields of the order of ten times lower than those of the lipophilic porphyrins. (5) There was a clear correlation between the quantum yields for cell-inactivation and those for photodegradation of DPBF, suggesting that the same reactive photoinduced species is involved in both processes. This suggestion was strengthened by the observation that DPBF reduced the quantum yield of cell inactivation. Thus, all the tested dyes seem to act via a type II process. (6) All of the dyes, even the water-soluble TPPS₄ and AIPCTS, are aggregated in aqueous solutions, and the cells bind both monomers and aggregates. (7) A significant fraction of the cell-bound dyes was located close to tryptophan-containing proteins. (8) Cell-bound Chi e,₆ had the highest fluorescence quantum yield of the dyes.     

Study of the conformation of polyelectrolytes in dilute solutions by elastic neutron scattering

Experiments using small angle neutron scattering have been performed on dilute solutions of polyelectrolytes without added salt; they allow to show that this method is very suitable for measuring the radius of gyration and determining the conformation of polyions in solution.     

Induction of multinucleated cells caused by UVA exposure in different stages of the cell cycle

Fibroblasts of the line 3T3 from swiss albino mice were exposed to ultraviolet A (UVA) irradiation. The cells were synchronized by treatment with nocodazole and mitotic shake-off, and then exposed to UVA irradiation in different stages of the cell cycle. Their photosensitivity varied through the cell cycle, being greatest in the G2 phase. UVA irradiation was found to induce the formation of multinucleated cells. Cells in the G1 phase were found to be most prone to multinucleation 15 min after UVA irradiation, while cells exposed to UVA irradiation in S and G2 phases contained the largest fractions of multinucleated cells 24 h after treatment. The present results indicate that multinucleated cells are formed by fusion of two or more cells shortly after UVA irradiation of early G1 cells, while impairment of cytokinesis is a possible explanation for the delayed formation of multinucleated cells after irradiation in S and G2.     

Acid-base properties of chlorin e6: relation to cellular uptake

Chlorins are attractive compounds for photodynamic therapy because of their high absorption in the red spectral region. In this study, the absorbance, fluorescence excitation and fluorescence emission spectra of chlorin e6 have been recorded as functions of pH in phosphate-buffered saline (PBS) solution with and without fetal calf serum (FCS). For pure PBS solutions, variation of the pH of the solution results in a shift of both the absorption and the fluorescence spectrum as well as in a decrease of the fluorescence intensity. Spectrophotometric and fluorimetric titration curves, based on observed changes, have been plotted. There is an indication of aggregate formation at low pH values (pH < 5). The presence of 5% FCS results in a shift of the titration curve, from an inflection point at about 6.5 to one at about 7.6. Pronounced spectral changes of the fluorescence emission spectra of protein-bound chlorin e6 (change of spectral shape, decrease of peak intensity) are also observed. The partition coefficients in the 1-octanol-water system increase with decreasing pH. Thus, relatively more of the drug is incorporated in the octanol phase at low pH. Cellular uptake of chlorin e6 in the presence of serum is significantly higher at pH 6.7 as compared with that at 7.3 and 7.6. We conclude that a change in the pH value of the surrounding medium leads to a change in the lipophilicity of chlorin e6. Such a change is likely to influence its binding to the serum proteins as well as its interaction with the plasma membrane of cells and may also be related to the selective tumor uptake of the drug.     

MITOTIC INHIBITION BY PHENYLPORPHINES AND TETRASULFONATED ALUMINIUM PHTHALOCYANINE IN COMBINATION WITH LIGHT

This work relates to studies on modes of phototoxicity by tetrasulfonated aluminium phthalocyanine (AlPcS4), tetrahydroxy- and monosulfonated meso-tetraphenylporphines (3-THPP and TPPS1) on culture cells. Toxicity at moderate light exposures appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with the sensitizers and exposed to light inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. For the first hours after treatment, the mitotic cells were always mainly found in metaphase; generally seen as c-metaphases and three-group metaphases. During this time, anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. The accumulation in mitosis was more extensive after treatment with AlPcS4 and light than after treatment with 3-THPP or TPPS1 and light. This may be related to the great difference in the lipophilic properties of these sensitizers; i.e. AlPcS4 being highly water soluble while TPPS1 and 3-THPP are lipophilic sensitizers. The lipophilicity of several sensitizers has been measured by two different methods, the partition between an aqueous and a lipophilic phase (Triton X-114) and the binding strength to a reverse phase column. The results show that the measured relative lipophilicity of the sensitizers may be influenced by the method of analysis.     

DETECTION OF UVR-INDUCED DNA DAMAGE IN MOUSE EPIDERMIS in vivo USING ALKALINE ELUTION

Abstract— Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320–400 nm) or indirectly by UVB (280–320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kj/m² (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1 -β-D-arabinofuranosylcytosine were administered (intraperi-toneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kj/m² (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kj/m² (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.     

Thermal decomposition kinetics of some aromatic azomonoethers

Thermal analysis of three azomonoether dyes, exhibiting liquid-crystalline properties, was performed in dynamic air atmosphere. Thermal stability studies and the evaluation of the kinetic parameters of each physical or chemical transformations are essential for a full characterization, before attempting accurate thin films’ depositions of such materials used in non-linear optical applications. New synthesized dyes with general formula: where R is a nematogenic group: CN, CF₃ or a highly polarizable group: NO₂ were investigated using TG, DTG, DTA and DSC techniques, under non-isothermal regime. The evolved gases were analyzed by FTIR spectroscopy. The activation energies of the first decomposition step were evaluated for each compound, the obtained results revealing complex mechanisms.     

Colon cancer: prognosis for different latitudes, age groups and seasons in Norway

The survival of colon cancer patients in Norway, as determined three years after diagnosis, is dependent on the season of diagnosis. This has been attributed to seasonal variations of the vitamin D status. Since solar radiation and food are the human sources of vitamin D, we divided Norway in three regions: The southeast region with a high annual dose of ultraviolet (UV) to the population, as evidenced by a high incidence rate of squamous cell carcinoma of the skin (SCC), the midwest region and the north region with low annual UV doses. The latter region is characterized by a high consumption of vitamin D, mainly through fish intake. Vacations to southern latitudes were equally frequent for all the three geographical regions. Two age groups were analyzed separately (< or =65 years and >65 years), since the photosynthesis of vitamin D(3) in skin decreases with age. In all three regions, and in both age groups, the survival was highest for summer and autumn diagnosis. The seasonal effect was slightly, but not significantly, better for the younger than for the older age group. The effect was similar for all three geographical regions, irrespective of SCC incidence.