Results

Figure 1 shows the survival of the cells as a function of irradiation time. The shape of the survival curve indicates that bilirubin in the dark may be somewhat toxic, and that a low dose of light irradiation will decrease the toxicity presumably by converting the bilirubin to less toxic geometric and structural isomers. The dark toxicity of bilirubin is strongly dependent on the bilirubin concentration and pH in the medium (unpublished observations). At a higher dose of light the survival of the cells decreases, probably as a result of the formation of toxic bilirubin photoproducts (Christensen 1986).

Figure 2 shows the total concentration of (4Z,15Z IXa), ( 4Z,15E), (4E,15Z) and Z-lumirubin as a function of irradiation time. The reduction of bilirubin isomers is linear within this time span. After two hours, about 40% of the isomers have disappeared. This observation correlates well with previous experiments performed by our group (Amundsen 1991). The light dose is comparable to the cell studies described above, indicating that measurable cell death takes place only after a relatively large light dose that is able to photooxidise a significant fraction of bilirubin, and not after smaller light doses that are expected to cause mainly photoisomerisation of bilirubin.

No gross morphological changes took place during the first four hours after irradiation. Later, the morphology was different in the various groups as can be seen from Figure 3 showing light micrographs of the cells 22 h after irradiation. Neither the cells treated with light in the absence of bilirubin nor the cells treated with bilirubin in the dark showed changes compared to the control cells. In the cells treated with the combination of bilirubin and light, a number of cells were damaged. The cultures had fewer cells, mitotic figures were missing, but most of the cells were still attached to the substratum. In the dead cells the nuclei did not seem to be fragmented and the cytoplasm was less refractive and more weakly stained with Giemsa.

It is of interest to look for signs of apoptosis in the cells. Therefore both the cells floating in the supernatant and the attached cells were carefully observed, and it was found that signs of typical changes associated with cells undergoing apoptosis (apoptotic bodies) were missing.

The analysis of double strand breaks in the DNA indicated that a significant amount of fragments was formed in cells treated with the combination of bilirubin and 1.75 E/m² blue light. This finding strongly indicates the presence of double strand breaks, but the observation must be reproduced to indicate the extent of cell damage.

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