Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 micros in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only approximately 220 nm during three lifetimes (approximately 5% 1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo-oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 'O2 occur in cells.

Aging and photoaging cause distinct changes in skin cells and extracellular matrix. Changes in hairless mouse skin as a function of age and chronic UVB exposure were investigated by fluorescence excitation spectroscopy. Fluorescence excitation spectra were measured in vivo, on heat-separated epidermis and dermis, and on extracts of mouse skin to characterize the absorption spectra of the emitting chromophores. Fluorescence excitation spectra obtained in vivo on 6 wk old mouse skin had maxima at 295, 340, and 360 nm; the 295 nm band was the dominant band. Using heat separated tissue, the 295 nm band predominantly originated in the epidermis and the bands at 340 and 360 nm originated in the dermis. The 295 nm band was assigned to tryptophan fluorescence, the 340 nm band to pepsin digestable collagen cross-links fluorescence and the 360 nm band to collagenase digestable collagen cross-links fluorescence. Fluorescence excitation maxima remained unchanged in chronologically aged mice (34-38 wk old), whereas the 295 nm band decreased in intensity with age and the 340 nm band increased in intensity with age. In contrast, fluorescence excitation spectra of chronically UVB exposed mice showed a large increase in the 295 nm band compared with age-matched controls and the bands at 340 and 350 nm were no longer distinct. Two new bands appeared in the chronically exposed mice at 270 nm and at 305 nm. These reproducible changes in skin autofluorescence suggest that aging causes predictable alterations in both epidermal and dermal fluorescence, whereas chronic UV exposure induces the appearance of new fluorphores.

p38 mitogen-activated protein kinase is activated and involved in cleavage of caspase-3 during apoptosis induced by a number of stimuli. However, the signaling events triggered by p38 that result in caspase-3 activation are still unknown. In human leukemia cells, two reactive oxygen species, singlet oxygen and hydrogen peroxide (H 2 O 2 ), selectively stimulated the phosphorylation of p38. Preincubation of cells with SB203580, a specific inhibitor of p38, dose dependently inhibited DNA fragmentation induced by singlet oxygen but not by H 2 O 2 . Protection from apoptosis by SB203580 correlated with inhibition of caspase-3, and several events that are associated with caspase-3 activation, including Bid cleavage, decrease in mitochondrial transmembrane potential and release of cytochrome c from mitochondria, whereas caspase-8 cleavage was not affected by this inhibitor. In contrast, blockade of caspase-8 with Ile-Glu-Thr-Asp-fluoromethyl ketone is sufficient to prevent formation of DNA fragments and to inhibit all the above signaling events, with exception of p38 phosphorylation, in both singlet oxygen-and H 2 O 2 -treated cells. These data suggest that caspase-3 activation is regulated through redundant signaling pathways that involve p38 and caspase-8 acting upstream of Bid during singlet oxygen-induced apoptosis, whereas the activation of caspase-3 by H 2 O 2 is only governed by a caspase-8-mediated apoptotic pathway.