Generation of free radicals during photosensitization of hypocrellin A and their effects on cardiac membranes.

Hypocrellin A (HA), a peryloquinone derivative, has recently been isolated from a fungus Hypocrella bambusae. This lipid soluble pigment, in combination with phototherapy, has been used to treat many skin diseases including the keloids caused by scalding and burns. We have studied the effects of photosensitized HA on biomembranes using pig heart microsomes. Photosensitization of HA was found to peroxidize the membrane lipids in the cardiac microsomes. The photodamage imposed by HA depended not only on the concentration of HA but also on the time of irradiation and pH of the system. Superoxide dismutase (SOD), ascorbic acid, beta-carotene and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) inhibited the lipid peroxidation approximately 50, approximately 50, approximately 30 and approximately 97%, respectively. Spin trapping in combination with EPR spectroscopic techniques was used to identify the reactive free radicals during the photoreaction. Formation of superoxide anion radical, (O2-.), was identified by the SOD-inhibitable DMPO-O2- EPR spectrum. Both SOD and ascorbic acid inhibited the EPR signal intensity in a dose-dependent manner with rate constants of 6.78 x 10(8) M-1 s-1 and 1.82 x 10(4) M-1 s-1, respectively. The lifetime of O2-., under these conditions, was found to be 1.1 s. Photoirradiation of HA yielded a HA free radical with a g = 2.002 which was not suppressed by SOD but in the presence of reductants such as ascorbic acid and catechol the septum was completely suppressed. The increase of the EPR signal intensity and malondialdehyde formation with increasing pH may be due, in part, to the production of predominant *HA- species at high pH which would be more reactive with oxygen to yield O2-.. These results indicate that the lipid peroxidation of the cardiac membranes observed during photooxidation of HA may arise, in part, from the interaction of membrane lipids with reactive species of oxygen and HA free radical produced during the photo-irradiation.