Little is directly known about the influence of the local environment experienced by a
photosensitizer in a
biological system on its photophysics and photochemistry. In this paper, we have addressed this issue by correlating mechanistic studies using
laser flash photolysis with cellular
phototoxicity data, obtained under the same experimental conditions. In particular, we have focused on the interaction between local concentrations of
photosensitizer (
deuteroporphyrin) and
oxygen in determining the mechanism of
phototoxicity in L1210 cells. In cells, as well as in models such as
liposomes and red blood cell ghosts, hypochromicity and a reduction in fluorescence and intersystem crossing yields are observed on increasing the
photosensitizer concentration between 0.5 and 20 microM, which illustrates the onset of a self-association. In aerated cellular preparations, the
phototoxicity is predominantly type II (
singlet oxygen) for all concentrations studied but an
oxygen-independent mechanism occurs at the higher concentrations in deaerated samples. These observations are readily explained by consideration of triplet state kinetics as a function of
oxygen and
photosensitizer concentrations in cells. The rate constant for quenching of the
photosensitizer triplet state by
oxygen in cells was measured as 6.6 x 10(8) M-1 s-1 and by
photosensitizer ground state as approximately 10(6) M-1 s-1 (in terms of local concentration). The latter reaction gave rise to a long-lived species that is presumably responsible for the
oxygen-independent
phototoxicity observed at the higher
photosensitizer concentrations used. This self-quenching of the triplet state is postulated to arise from electron transfer resulting in radical ion formation. Under conditions where no self-quenching contributes, the
phototoxicity measured as a function of
oxygen concentration correlates well with a model based on the determined kinetic parameters, thus, unambiguously proving the intermediacy of
singlet oxygen. These effects should be borne in mind when interpreting
phototoxicity mechanisms from in vitro cell studies. The excellent correlation achieved between
laser flash photolysis data and measured
phototoxicity gives credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in
biological media.