Time-Course of Hypericin Phototoxicity and Effect on Mitochondrial Energies in EMT6 Mouse Mammary Carcinoma Cells

Abstract

Photoactivated hypericin produces singlet oxygen and superoxide anion radical; however, the intracellular events contributing to toxicity are unknown. Clonogenic assays of oxygen-dependent hypericin phototoxicity to EMT6 cells have previously shown that 0.5 μM hypericin + 1.5 J cm −2 fluorescent light is non-toxic and that 1.0 μM hypericin + 1.5 J cm −2 fluorescent light produces LD 40 toxicity. Intracellular events leading to toxicity were revealed at these doses. Lactate dehydrogenase leakage was elevated for both 0.5 μM and 1.0 μM hypericin + light immediately following irradiation. While values eventually returned to control levels for 0.5 μM hypericin + light, leakage increased over time for 1.0 μM hypericin indicating reversible and irreversible toxicity, respectively. Increases in lipid and protein oxidation were measured immediately following irradiation; however, these parameters return to control levels within 0.5 h for both doses. Both total cellular ATP levels and cellular respiration were depressed by approximately 50% of control values for 1.0 μM hypericin + light. These values were unchanged for 0.5 μM hypericin + light. Along with previously reported data demonstrating that light-activated hypericin can inhibit mitochondial succinoxidase in beef heart mitochondria in vitro, these data support oxidative stress-initiated mitochondrial damage as a key target in hypericin phototoxicity.