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BILIRUBIN PHOTOTOXICITY TO HUMAN CELLS BY GREEN LIGHT PHOTOTHERAPY in vitro

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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× 106/mL) were incubated with bilirubin complexed to human serum albumin (final concentrations 340 μM bilirubin, 150 μM albumin). Under these conditions all cells showed maximum binding of bilirubin. Irradiation with broadband green light (Λmax= 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−1 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 μM) 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 514.5 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.

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