PHOTOPRODUCTION AND DIRECT SPECTRAL DETECTION OF SINGLET MOLECULAR-OXYGEN (O-1(2)) IN KERATINOCYTES STAINED WITH ROSE-BENGAL

In vivo, keratinocyte skin cells are exposed to photooxidative process es, some of which can be mediated by singlet molecular oxygen (O-1(2)) , a species that is very difficult to detect spectrally in cells. We p hotosensitized O-1(2) in cultured HaCaT keratinocytes stained with ros e bengal (RB) that localizes exclusively inside the keratinocyte hydro phobic regions, as evidenced by strongly red-shifted absorbance and in tense fluorescence. We used keratinocytes grown in a monolayer on a pl astic coverslip and in suspension. The phosphorescence spectrum (1200- 1350 nm) from O-1(2) was strongest when the coverslip containing RE-st ained keratinocytes was irradiated in air. The spectral intensity decr eased when the coverslip was immersed in D2O during irradiation and wa s almost completely quenched when it was irradiated while immersed in water. Water not only shortens the O-1(2) lifetime but also reabsorbs part of the O-1(2) phosphorescence, processes that do not occur when O -1(2) is produced in a keratinocyte layer exposed to air. Because the RE was inside keratinocytes, singlet oxygen must also be produced insi de the keratinocytes. However, the sensitivity to the extracellular en vironment suggests that most of the detectable O-1(2) phosphorescence originates from those O-1(2) molecules that escaped from the cell thro ugh its membrane into D2O or into the air, where O-1(2) has longer lif etimes. Our results confirm directly that O-1(2) is indeed photosensit ized in living cells by RE. They also suggest that keratinocyte monola yers may be a good cell model to examine in vitro the production of O- 1(2) by other photosensitizers of environmental and photomedical inter est.