Discussion

Previous studies and the present results have shown that there are at least two types of toxicity associated with treatment of cells with bilirubin. Bilirubin is a toxic substance in the dark, and a low light dose reduces its toxicity by converting it to more water soluble and less toxic isomers. Simultaneously, light can photooxidise bilirubin, but with a much lower quantum efficiency (Sloper and Truscott 1982, Ennever and Dresing 1991). It has been debated if singlet oxygen is involved in the oxidation of bilirubin, but it has been established that this reactive oxygen species is produced by bilirubin and light. When present in cells singlet oxygen will probably react with a number of different constituents (see the references in "Introduction"). The photooxygenation of bilirubin will lead to the formation of a number of photoproducts, i.e. mono-, di- and tri-pyrroles (McDonagh 1971) and peroxides that are reactive species (i.e. H₂O₂)(Rosenstein et al. 1983, Christensen 1984, 1986). It should also be mentioned that blue light may react with other chromophores than bilirubin in the cells.

It is not clear which photoproduct(s) plays the major role in the induction of cellular damage after bilirubin and light treatment. Some important types of damage to the cells by light doses compatible with cell survival have been demonstrated in this paper and in previous work. The types are traditionally divided into membrane damage and DNA-damage. Membrane damage leading to plasma membrane leakage is probably not induced, at least not for the first hours after treatment. This conclusion can be drawn from the microscopic appearance of the cells, and the observation that no lactate dehydrogenase was released from cultured human cells during the first 48 h after treatment (Christensen 1986). On the other hand, both single- (Rosenstein et al. 1983, Christensen et al. 1988, 1990, 1994) and double strand DNA-breaks have been demonstrated in cultured cells. It was shown that bilirubin and light could induce mutations in V79 Chinese hamster cells (Christensen et al. 1988). After treatment with moderate doses of bilirubin and light the cells’ ability to multiply was affected (Christensen 1986, Christensen et al. 1996) and a slight increase in the fraction of cells in the G₂/M-phases of the cell cycle has been observed.

The question whether the cells become apoptotic after the treatment is of interest since other photosensitisers have been found to induce apoptosis (see Kessel et al. 1997 and references therein). The sensitisers initiate the cellular processes leading to apoptotic cell death differently depending on their subcellular localisation and also probably depending on the photochemical mechanism. Bilirubin does not induce apoptosis after the doses and times studied in this work, but we cannot exclude the possibility that apoptosis may occur in a small number of cells or at different times after irradiation. The observation of double strand breaks may be taken as an indication of early apoptotic DNA-incision, but may also be due to the existence of unrepaired DNA at 1 h after irradiation. Further studies must be done to distinguish between these two possibilities.

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