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Photoproduction and Direct Spectral Detection of Singlet Molecular Oxygen ( 10 2 ) in Keratinocytes Stained with Rose Bengal
Author(s) -
Bilski Piotr,
Kukielczak Barbara M.,
Chignell Colin F.
Publication year - 1998
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1998.tb02528.x
Subject(s) - rose bengal , hacat , singlet oxygen , keratinocyte , irradiation , chemistry , monolayer , fluorescence , absorbance , oxygen , biophysics , photochemistry , phosphorescence , analytical chemistry (journal) , in vitro , optics , biochemistry , biology , chromatography , physics , organic chemistry , nuclear physics
In vivo, keratinocyte skin cells are exposed to photoox‐idative processes, some of which can be mediated by singlet molecular oxygen ( 1 O 2 ), a species that is very difficult to detect spectrally in cells. We photosensitized 1 O 2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red‐shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200–1350nm) from 1 O 2 was strongest when the coverslip containing RB‐stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D 2 O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1 O 2 lifetime but also reabsorbs part of the 1 O 2 phosphorescence, processes that do not occur when 1 O 2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1 O 2 phosphorescence originates from those 1 O 2 molecules that escaped from the cell through its membrane into D 2 O or into the air, where 1 O 2 has longer lifetimes. Our results confirm directly that 1 O 2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1 O 2 by other photo‐sensitizers of environmental and photomedical interest.