Introduction

The photoreactivity of bilirubin was probably observed for the first time by Saeki in 1932 (referred from Girotti 1975). Saeki exposed red blood cells to bilirubin and light and found that this combination caused photohemolysis. Fischer and Herle in 1938 found that solutions of bilirubin that were left in the light gradually lost the typical yellow colour of the tetrapyrrole pigment.

Later, the finding of Fischer and Herle was used as a model to explain the observation that newborns suffering from neonatal jaundice, recovered from the condition more rapidly if they were exposed to visible light (Cremer et al. 1958). The loss of bilirubin observed, either by optical absorption or by biochemical analysis, was thought to be due to the photooxygenation or dehydrogenation of the pigment to biliverdin or some other pigment. As late as in 1971 when phototherapy of jaundice had become an established and preferred mode of treatment, photooxidation was a leading theory (McDonagh 1971). However, bilirubin can undergo several other photoreactions, notably photoisomerisation (McDonagh et al. 1979) and today it is believed that formation of photoisomers is the main photochemical mechanism behind the effect of phototherapy.

Photochemical reactions in the presence of bilirubin normally proceed via excitation of the molecule to the first excited singlet state. The two ends of the molecule are not exactly similar, and the excitation energy will become distributed to either end somewhat dependent on the wavelength of the light source (Agati and Fusi 1990, Troup et al. 1997). From the excited state a number of reaction pathways are possible (see reaction schemes in Landen et al. 1982). Among these are electron or hydrogen transfer from the first excited singlet state and intersystem crossing to the first excited triplet state. Further reaction of the triplet state with molecular oxygen and subsequent formation of singlet oxygen is possible. "Self-sensitised" photooxygenation of bilirubin has been observed, but it is debated whether this reaction involves singlet molecular oxygen (Meisel et al. 1980, Landen et al. 1982). It is clear that bilirubin will react with singlet oxygen (Matheson 1979) formed by irradiation of common photosensitisers both in vitro and in vivo (Knobloch et al. 1991), and that a large number of reaction products are formed. The third class of bilirubin photosensitised reactions is therefore the possible reaction between biomolecules and products formed by the photodegradation of bilirubin. In fact such reactions have been found, but the detailed reaction mechanisms are for a great part unknown. Our group concluded that human cells could be inactivated and growth inhibited and that their DNA could be damaged after exposure to bilirubin and light in vitro (Christensen 1984). The effects were due to a product with a lifetime of several hours and the level of which could be reduced by addition of catalase. These conclusions are supported by the observations made by Rosenstein et al. (1983) who observed that DNA-strand breaks formed in cells could partly be attributed to hydrogen peroxide formed by irradiated bilirubin and partly to some unknown breakdown product. Later studies have shown that the DNA-damage may lead to mutations (Christensen et al. 1988) and that bleaching of bilirubin bound to cells is accompanied by the induction of membrane damage in nucleate cells (Böhm et al. 1995). The occurrence of damage to the outer cell membrane of red blood cells by photoactivated bilirubin has, as mentioned above, been known for many years. Several authors have covered this subject, but the studies of Girotti’s group are of particular interest (Girotti 1975,1976, 1978, Deziel and Girotti 1980). One of their main conclusions was that peroxidation of unsaturated fatty acids could be attributed to the formation of singlet oxygen, while damage to proteins as the Na⁺ K⁺ -ATPase and the formation of protein crosslinks apparently were induced by a different mechanism.

The objective of this communication is to report our first studies aimed at defining the mechanism of injuries to a mammalian cell line by bilirubin and light. Mouse 308 epidermal cells were used for two reasons: Their reactions to this mode of treatment are partly known (Christensen et al. 1994, 1996) and it is regarded as relevant to study the effect on skin cells when the effects in vitro are going to be compared to possible effects of phototherapy in newborns. Data on the reproductive cell death and DNA-damage in the cells will be presented together with observation of their morphology as a function of time after irradiation. Previous findings of cellular effects of bilirubin and light will be summarised and discussed on the background of our data.

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