Hypericin-Induced Phototoxicity in Cultured Fibroblasts and Swine Erythrocytes

Hypericin is a naturally occurring photosensitizer, whose presence in plants has been responsible for cutaneous phototoxicity in grazing animals. The photosensitizing properties of this agent have recently been exploited in models for anti-tumor and anti-viral activity. The cytotoxicity of hypericin and light was assessed in 3T3 mouse fibroblasts using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)] assay and the lactate dehydrogenase (LDH) leakage assay. Membrane damage was assessed in swine erythrocytes using hemolysis, potassium (K⁺) leakage and formation of lipid hydroperoxides. Concentration- and light-dependent decreases in fibroblast viability were seen starting at hypericin concentrations of 1.25 μM and light power flux levels of 24 J/cm² using a visible light source and at 0.417 μM hypericin and a similar light dose using a solar simulator, No LDH leakage was observed at hypericin concentrations up to 30 μM and visible light up to 144 J/cm². Light-and/or concentration-dependent increases in hemolysis, K⁺ leakage and formation of lipid hydroperoxides in red blood cell (RBC) membranes were observed, but at concentrations and light doses much greater than those required to induce cytotoxicity in fibroblasts. Lipid peroxidation and hemolysis occurred at 15 μM hypericin and 24 J/cm² (visible light source). Potassium ion leakage occurred at concentrations and light levels as low as 5 μM and 12 J/cm² or 15 μM and 4.8 J/cm² (visible light source) but was still a less sensitive indicator than fibroblast cytotoxicity. Evidence for both type I and type II reactions was shown in RBC membranes by TLC analysis of cholesterol products. In the absence of light, hypericin appears to be relatively nontoxic in the models tested.