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Mechanisms of Mitochondrial Photosensitization by the Cationic Dye, N,N'-Bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC): Preferential Inactivation of Complex I in the Electron Transport Chain¹

Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 [G. A., A. R. O.]; Mitochondrial Physiology Unit, Department of Biology, Tufts University, Medford, Massachusetts 02155 [J. R. A.]; Renal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 [C. D. M., J. V. B.]; Rowland Institute of Science, Cambridge, Massachusetts 02142 [L. C., J. F.]; and Department of Determatology, Tufts Medical School, New England Medical Center, Boston, Massachusetts 02111 [S. B. K., A. R. O]

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 ß-hydroxybutyrate as substrates), low concentrations of the dye (0.25–0.5 µM) 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 (>5 µM) and at 10-fold greater light doses. NADH coenzyme Q reductase (Complex 1) 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 (>5 µM) and light, but the extent of inhibition was much less than the inhibition of respiration with succinate as substrate. ATP synthetase (F_eF₁ ATPase) activity was minimally affected by 4.0 µM 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.

¹ This work was supported in part by a grant from the Arthur O. and Gullan M. Wellman Foundation, by NIH R01 CA-44205 (A. R. O.) and by Office of Naval Research Contract N00014-86-K-0017.

² Present address: Department of Dermatology, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111.

³ To whom requests for reprints should be addressed.

Received 3/ 5/87. Revised 8/25/87. Accepted 9/21/87.