We investigated mechanisms of mitochondrial
phototoxicity caused by the cationic cyanine
dye N,N'-bis(2-ethyl-1,3-dioxylene)kryptocyanine (
EDKC), examining the role of the mitochondrial membrane potential on the
dye uptake by
carcinoma cells in vitro, and both the dark and
photosensitizing effects of the
dye on the function of isolated mouse liver mitochondria. When human bladder
carcinoma cells (EJ) were pretreated with
2,4-dinitrophenol or
nigericin, cellular uptake of
EDKC decreased or increased, respectively, consistent with
dye uptake that is dependent on membrane potentials. In isolated liver mitochondria, during
NADH linked substrate oxidation (using
glutamate plus
malate or
beta-hydroxybutyrate as substrates), low concentrations of the
dye (0.25-0.5 microM) sensitized mitochondria to illumination with long wavelength light and inhibited both basal and
ADP-stimulated respiration. Similar effects were observed during
succinate oxidation, but only at higher concentrations of
EDKC (greater than 5 microM) and at 10-fold greater light doses.
NADH coenzyme Q reductase (Complex I) activity was inhibited by
dye with or without light to an extent comparable to the inhibition of
glutamate plus
malate oxidation. Activity of
cytochrome c oxidase, the terminal
enzyme in the electron transport chain, was photosensitized with high
dye doses (greater than 5 microM) and light, but the extent of inhibition was much less than the inhibition of respiration with
succinate as substrate.
ATP synthetase (
F0F1 ATPase) activity was minimally affected by 4.0 microM
EDKC with or without 24 J/cm2 light. We conclude that at low concentrations of
dye,
respiratory Complex I is a primary target for
EDKC dark and light-induced toxicities. If Complex I is bypassed by using
succinate as a respiratory substrate, the mitochondria can tolerate much higher
dye concentrations and light doses.