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 chain.
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.
D019297:2,4-Dinitrophenol; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002232:Carbocyanines / Q000493:pharmacokinetics / Q000494:pharmacology*; D004396:Coloring Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D004140:Dinitrophenols / Q000494:pharmacology; D004579:Electron Transport; D006801:Humans; D008027:Light; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000528:radiation effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000528:radiation effects; D016660:NAD(P)H Dehydrogenase (Quinone); D009550:Nigericin / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D011804:Quinolines / Q000494:pharmacology*; D011808:Quinone Reductases / Q000037:antagonists & inhibitors*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000648:ultrastructure
D002232:Carbocyanines; D004396:Coloring Agents; D004140:Dinitrophenols; D011804:Quinolines; D011838:Radiation-Sensitizing Agents; C051149:N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine; D000255:Adenosine Triphosphate; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D019297:2,4-Dinitrophenol; D009550:Nigericin