Materials and methods

Cell cultivation

Cells from the mouse epidermal cell line 308 (Yuspa and Morgan 1981.) were cultivated in Minimal Essential Medium (MEM, Eagles) containing 7 % foetal calf serum. The cells were detached by gentle treatment with trypsin/EDTA and inoculated in flasks. For microscopic observation 20000 cells cm^-2 were used, and for survival measurement 300 cells were inoculated in each of five 25 cm² tissue culture flasks.

Experimental solution

Bilirubin (BR, mixed isomers from bovine gall stones, Sigma) was used without further purification. The bilirubin was dissolved in 0.1 N NaOH as a stock solution of 1600 mM and used within four hours. It was filtered through a 0.45 mm sterile filter and adjusted to pH 7.2-7.4 with 0.1 N HCl. A sterile mixture of phosphate buffered saline (PBS) and foetal calf serum (FCS) at a ratio of 2:1 was prepared, and the BR-stock solution was added to a final concentration of approximately 160 mM BR and 50 mM bovine serum albumin (BSA, included in the calf serum).

Irradiation

Irradiation of the cells was performed in flasks on a plexiglass stand placed above two fluorescent tubes (Philips TL20 W/52) that are frequently used in phototherapy. These lamps emit blue light with a spectral distribution peaking at 450 nm and the spectra do not include ultraviolet radiation to any significant extent. The spectrum of the lamps and dose measurements have been published previously (Christensen et al 1990). The irradiance used in the present experiments was 40 Wm^-2.

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In vitro irradiation of bilirubin

100 mM of the experimental bilirubin solution was used. A test tube (glass with plastic lid) containing 1 ml of the solution was placed in a beaker on top of two fluorescent light tubes as described above (Irradiation section). The solution was gassed with helium for three minutes prior to irradiation in order to reduce the oxygen concentration in the solution and the test tube. Samples for HPLC-analysis at a detection wavelength of 450 nm were taken after 2, 2.5, 3 and 4 hours, respectively (Shimadzu LC-10 AT liquid chromatograph, SPD-10 AV spectrophotometric detector, equipped with a Nucleosil 120-5C18 reversed phase column (250x4.6 mm) (Macherey-Nagel AG). Integration was done with Class LC-10 software). The mobile phase consisted of methanol (HPLC-grade, Rathburn), acetic acid (puriss.p.a., Fluka) and destilled water, and pH was adjusted to 7.7 with dioctylamine (for synthesis, Merck). It was continuously gassed with helium gas during analysis, and the flow was set to 1ml/min. The areas under the peaks of the chromatograms were used in calculating the relative concentrations of the bilirubin isomers. The sum of the areas of the 4Z,15Z IX-a and the photoisomers (4Z,15E), (4E,15Z) and Z-lumirubin prior to irradiation was normalised to 1.0. The extinction coefficients for the structural isomer ( Z-lumirubin) and the geometric isomers 4Z,15E and 4E,15Z relative to 4Z,15Z were calculated as 1.7 and 1.25, respectively (McDonagh et al.1989).

Observation of cellular effects

For microscopic observations cells were inoculated in Slide flasks (Nunc) and irradiated as described above. The appearance of the cells treated with either light, bilirubin or a combination of these was observed in a phase contrast microscope (Nikon) at different times after the end of irradiation. The cell cultures were fixed in methanol/acetic acid and stained vith Giemsa 22 h after irradiation. Images were made with a Hamamatsu CCD camera connected to a PC with Image-Pro software.

Clonogenic survival was assayed by incubating 500 cells per flask and treating the flasks with bilirubin and light as described above. At the end of irradiation the medium was changed and the cells were incubated at 37 ^oC in 5 % CO₂. After one week, the cells were fixed with absolute ethanol and stained with 1 % azur-eosine-methylene blue. The number of visible colonies was counted and compared to the number in untreated controls. Thus the surviving fraction was obtained.

Pulse field gel electrophoresis (PFGE) with a CHEF III apparatus (BioRad) was used to detect double strand breaks in DNA. The gel was electrophoresed for 24 hours at 6V/cm, switch time 60-120s and angle 120° C in 0.5x TBE buffer. DNA from Sacccharomyces cerevisiae was used as a standard. The electrophoretic conditions used made it possible to visualise fragments as large as 2Mbp of DNA entering the gel. In a pilot experiment samples of ca. 5x10⁵ cells prepared in one agarose plug from cultures irradiated with or without additional treatment with BR were compared to untreated controls (the detailed methods to be published).

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