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Norwegian Radiation Protection Authority |
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| Photosensitizing effects
metalloporphyns in connection with hyperbilirubinemia
Materials and methods
Two established cell lines were used in the present study: Cells from the human glioblastoma line TMG-1 (16) cultivated in RPMI medium in the presence of 10 % foetal bovine serum, and epithelial cells from the mouse epidermal line 308 (17) cultivated in MEM medium in the presence of 7% foetal bovine serum. When the effects of metalloporphyrins on the DNA of TMG-1 cells were to be measured, the porphyrins were added to the tissue culture medium 1 h before trypsin detachment. The cells were detached by gentle treatment with trypsin/EDTA, and equal numbers of cells (0.3-1.106) were transferred to a number of Falcon tissue culture tubes. The cells were resuspended in 1 ml phosphate buffered saline (PBS) at pH 7.2-7.4. When the effects of bilirubin were to be studied, the bilirubin was added directly to the cell suspension in PBS before irradiation. The mouse cells 308 were used for cell survival measurements. They were detached as described above and inoculated in 6 cm plastic petri dishes where the bottoms had been divided into five areas marked with straight lines separated by 1.0 cm. 5.104 cells per dish were spread evenly over the surface of each dish with as constant cell density as possible at different positions of the dish. The cells were incubated overnight before irradiation.
Bilirubin (mixed isomers from bovine gall stones) was purchased from Sigma and used without further purification. L-Tryptophan was purchased from Fluka. The fluorochrome Hoechst 33258 (bisbenzamide) from Calbiochem was dissolved in water as a stock solution at 10-3 M. The stock solution could be used for several months if stored in the dark at 4o C. Tin protoporphyrin (SnPP), zinc protoporphyrin (ZnPP), chromium protoporphyrin (CrPP), chromium mesoporphyrin (CrMP), hematoporphyrin (HP) and protoporphyrin (PP) were purchased from Porphyrin Products (Logan, Utah). The purity of the porphyrins was assayed by HPLC. The porphyrins were separated in the HPLC-system LC-10A from Shimadzu. They were eluted by a gradient mixture of methanol/H2O, ranging from 60/40 to 90/10 after 35 minutes run with 1.5 ml/min flow. All porphyrins eluted as single peaks. Bilirubin was dissolved in 0.1 N NaOH as a stock solution. Only fresh bilirubin solutions were used. The stock solution was diluted to the desired concentration in PBS containing bovine foetal serum albumin. Subsequently the pH of the solution was adjusted to 7.2-7.5 by slowly adding HCl if necessary. The porphyrins were dissolved in 0,1 N NaOH as a stock solution when possible, but if this treatment was unsuccessful they were dissolved in 0,01 N NaOH containing 20 % methanol. The porphyrin solutions were stored frozen in the dark and used within two weeks after preparation.
Two Philips TL 20 W/52 blue fluorescent tubes were mounted parallel to each other with a distance between them of 2 cm. This type of tubes is frequently used in phototherapy, and the spectrum has been published previously (6). The solutions to be irradiated were placed on a plexiglass stand absorbing light with wavelengths below 400 nm. The light irradiance at this position was monitored by a photodiode (United Detector Technology). By application of appropriate correction factors for spectral responsivity of the detector, the irradiance was measured and expressed as the number of photons (moles, Einstein) per unit area (Em-2). At the position of the experimental solutions, the irradiance varied between 0.45 and 0.9 E/m2 h (35-64 W/m2) throughout the experimental period.
One ml of TMG-1 cell suspensions in 12 ml tissue culture tubes were placed on ice above the lamps for different times. The tubes were agitated a few times during irradiation to prevent the cells from settling at the bottom of the tubes. The degree of DNA damage was measured by an alkali unwinding method (18) described in detail elsewhere (6). In brief, a volume of 0.1 N NaOH was added to the tubes after irradiation. After an unwinding period of 30 min., the solution was neutralised and supplemented with 10-6 M Hoechst 33258. This fluorochrome binds to both single and double stranded DNA, but fluoresces more strongly when bound to double stranded DNA. During the unwinding process, more single stranded DNA is formed when there are breaks or alkali labile sites present. Therefore, one can measure the degree of DNA damage by assaying fluorescence from Hoechst 33258 after the unwinding. The cell suspensions were sonicated before the assay and the fluorescence was excited at 353 nm and read at 454 nm. An expression for the fraction double stranded DNA after unwinding (D) was derived by measuring the fluorescence from double stranded DNA in non-alkali treated cells (DS) and completely single stranded DNA (SS) obtained by sonicating the cells before the alkali treatment. If the fluorescence from a sample was F, the expression for D was calculated as: D = (F - SS)/(DS - SS) The determination of DS and SS was based on the mean of two cell suspensions, and the resulting fraction double stranded DNA was the mean of two determinations of D.
Cells of the line 308 attached to petri dishes were irradiated through the bottom of the dishes. The light doses were varied by moving the dishes step-wise into the light, starting with the highest dose and keeping the dark areas shielded by aluminium foil throughout the experiment. In this way every dish contained areas irradiated with three or four different light doses and one or two areas serving as dark controls. The metalloporphyrins were added directly into the tissue culture medium to a final concentration of 5 mM. Addition of metallophorphyrin solutions lead to a slight increase in the pH of the medium (from 7.4 to approx. 7.8) and the cells were incubated for approx. 1 h. in the presence of the metalloporphyrins. By that time the medium was removed and PBS with 10 mM BSA or 10 mM BSA plus 10 mM bilirubin was added. The cells were irradiated immediately. After irradiation the irradiated solutions were removed and the dishes were supplemented with fresh tissue culture medium. The cells were incubated for two days, fixed, stained with methylene blue and the number of cells that had undergone one or more divisions were counted under a microscope at a magnification of 100 x. About 20 cell groups per field were found in untreated dishes. Five fields were counted per dose per dish, and three separate dishes were treated with the same solutions in each experiment. Cells that had divided at least once, forming a micro-colony of at least two cells, were scored as surviving.
Measurement of absorbance and fluorescence A Perkin-Elmer LS 5B fluorescence spectrometer was used with 5 nm slits. The absorbance of samples containing bilirubin was measured by a Perkin-Elmer Lambda 2 spectrophotometer connected to a Copam 286 personal computer with PECSS software (Perkin Elmer Computerised Spectroscopy Software, version 3.2). The concentrations of tetrapyrroles was assayed by reading either absorption spectra or fluorescence spectra. The peak absorbance or peak fluorescence in the Soret band was used. When the absorbance of mixtures of bilirubin and one porphyrin was measured, the peaks were partially overlapping. The overlaps were corrected for by determination of the absorbance in solutions of each substance alone. When absolute concentrations of the porphyrins in cell suspensions were to be determined, known concentrations of porphyrins dissolved in cell suspensions were used as standards. Protein contents in cell samples were determined spectrophotometerically after addition of a protein assay (Bio-Rad). Flourescence micrographs were made by aid of a Nikon inverted microscope with a high sensitivity, cooled CCD camera from Hamamatsu attached to a Dell 486 PC with Image-Pro software.
Photooxidation of L-tryptophan Two mM L-tryptophan was dissolved in water as a stock solution and diluted to 10 mM in a NaOH solution at pH 12 containing 2 mM of the porphyrin-derivatives to be tested. One ml aliquots of the solution were irradiated as described above for different periods ranging from 0 to 120 min. After irradiation the fluorescence from L-tryptophan at 355 nm was measured by exitation at 280 nm.
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