PhorochemisrT and Photobiology, zyxwvutsrqpon Vol. 62, No. 6, pp. zyxwvutsrq 98CL983, 1995 Printed in the United States. All rights reserved zyxwvutsrq 0031-8655/95 zyx $05.00+0.00 0 1995 American Society for Photobiology BILIRUBIN PHOTOTOXICITY TO HUMAN CELLS BY GREEN LIGHT PHOTOTHERAPY zyx in vitro F. BOHM', F. DRYGALLA', P. CHARLESWORTH,, K. BOHMI, T. G. TRUSCOTT*~ and K. JOKIEL~ 'Humboldt University, Department of Dermatology (Charitk), Schumannstrasse 20/2 1, Berlin 10117, Germany and 2Department of Chemistry, University of Keele, Keele, Staffs. ST5 5BG, UK (Received 27 January 1995; accepted 16 August 1995) Abstract-Phototherapy of newborn infants with blue or green light is the most common treatment of neonatal hyperbilirubinemia. Using bilirubin bound to human lymphoid and basal skin cells we obtained the green light dose dependency of the bilirubin phototoxicity to these cell types. Cells (3-5 zyx X 106/mL) were incubated with bilirubin complexed to human serum albumin (final concentrations 340 FM bili- rubin, 150 albumin). Under these conditions all cells showed maximum binding of bilirubin. Irra- diation with broadband green light (kma = 512 nm) over 24 h led to a light dose-dependent population of cells, which contained no bilirubin on the cell membrane as determined by Nomarski interference microscopy. The light-induced mechanism of the disappearance of bilirubin caused lethal membrane damage to the cells (trypan blue exclusion test). The cell kill rate increased with the irradiation dose and with the fraction of cells with no bilirubin. When 90% of lymphoid cells were bilirubin free, 46% of them were dead (using 480 J cm-I green light). Similar results were obtained with basal skin cells. In addition, bilirubin-induced damage of cell membrane and nuclear membrane was also shown by transmission electron microscopy. Bilirubin (340 CLM) in the dark led to 5% of the cells being killed. Basal skin cells bind 2.5 times more bilirubin molecules than lymphoid cells and showed a different bilirubin disappearance. Irradiation of bilirubin in carbon tetrachloride with 5 14.5 nm laser light showed generation of singlet oxygen via its luminescence at 1270 nm. These results demonstrate that green light phototherapy of hyperbilirubinemia may cause both skin and immune system damage. INTRODUCTION Phototherapy of newborn infants with blue, white or recently blue-green light is no doubt the most common therapy of neonatal hyperbilirubinemia. Introduced into general use in the late 1960s' visible light phototherapy has been used on millions of infants worldwide. Many clinical trials2 have shown the effectiveness of phototherapy. No significant toxicity has been identified, but long-term observations with regard to tumor promotion due to the in- fluence of DNA damage are not available. However, damage of bilirubin (BR)t-containing cells due to blue light irradi- ation in vitro has been rep~rted.~ In the early 1980s the mechanisms of the photoisomerism of ZZ-BR zyxwvutsrq (i.e. cis, cis-BR) to its more excretable forms ap- peared to be well under~tood.~ However, recent work has shown there is a strong wavelength dependence to these mechanisms and to the ZZ-BR H isomer eq~ilibrium.~~~ In clinical phototherapy, the use of blue light is predominant, with these wavelengths leading to the production of config- urational photo isomers such as ZE-BR (cis, trans-BR). This has been shown to be less efficiently excreted from the body than the structural isomer, lumirubin (LR), produced by green light irradiati~n.~.~ Hence, it has been suggested in *To whom correspondence should be addressed ?Abbreviations: BR, bilirubin; DABCO, diazabicyclooctane; HSA, human serum albumin; LR, lumirubin; '0,. singlet oxygen. several publications that green light should replace blue light in clinical phot~therapy.~.~ We suggest the structure of this isomer (LR) is such that it is capable of photosensitizing the production of singlet molecular oxygen (lo2). The genera- tion of singlet oxygen in the vicinity of cells may lead to damage of the cell membrane and the DNA. The long-term effects of such damage is unknown and may have important consequences for the future life of the individual. To investigate any cellular damage due to the bleaching of BR bound to cell membranes by green light irradiation we have studied human lymphoid cells and human skin cells that were coated with BR in vitro. Using a Nomarski inter- ference microscopy technique and UVhisible spectroscopy we measured the disappearance of cellular bound BR from the cell surface. Parallel to this cell staining with trypan blue electron microscopy was performed to show membrane le- sions obtained as a result of the BR loss. The production of '0, during green light irradiation of BR in a cell-free envi- ronment was demonstrated via its 1270 nm luminescence. EXPERIMENTAL Lymphoid cells were taken from healthy human blood and pre- pared as previously described.'O Briefly, we diluted the anti-coagu- lated blood using heparin 1: 1 with Dulbecco buffer (phosphate-buf- fered saline buffer), passed it over a Ficoll' density gradient of 1.076 g/mL (Ficoll-PaqueR, Pharmacia, Uppsala, Sweden) and collected the buffy coat after centrifugation (150 zyx g, 30 min, 22°C) and divided in two. One was treated with BR and the other used as a control. For the cells with BR we incubated for 60 min in the dark at 4°C 980