Photodynamic action of merocyanine 540 on artificial and natural cell membranes: involvement of singlet molecular oxygen.

Abstract

The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H20, and identification of the cholesterol product 5a-cholest-6-ene-3,8,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to-K-562 leukemia cells in 2H20 than in H20. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye. (4)(5)(6)(7)(8). In this way leukemic cells have been selectively purged from bone marrow in vitro, and attempts to exploit this differential photosensitivity in clinical situations are underway (9).Although the effectiveness of MC 540 in killing neoplastic cells has been demonstrated (5-8), little is known about the mechanism of cell killing, and those factors on which the selectivity of MC 540 for cancer cells is based are not understood. However, the fact that uptake and toxicity of MC 540 in leukemic cells are augmented in the presence of visible light and oxygen (2) suggests that photodynamic processes are involved. A preliminary report has indicated that singlet oxygen (102) is one of the reactive intermediates (10). The first detectable event following the production of this species is rapid consumption of molecular oxygen, which can occur as a result of reactions with biological constituents. Thus, we have used the highly sensitive spin label oximetric approach (11, 12) to monitor oxygen consumption during MC 540-sensitive photooxidation. Using this approach, we now report additional evidence for the production of 102 by the photoactivated dye. The involvement of 102 in the killing of leukemic cells and in the peroxidation of cell membrane phospholipids and cholesterol is also reported. MATERIALS AND METHODSOximetry Studies Using Model Systems. Sample preparation. MC 540 obtained from Eastman was dissolved in a 50:50 mixture of ethanol and water and frozen until further use. Arachidonic acid (Sigma) was dissolved in ethanol. All other chemicals were used as received.Sample irradiation. Samples for irradiation typically contained 90 ,uM MC 540 in deionized water, pH 7.0. 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy(CTPO; Aldrich) was added to give a final concentration of 1.2 X 10-4 M. Samples, following saturation with a mixture of 50% nitrogen and 50% air from calibrated flow meters (Matheson) were introduced into a Pasteur pipette, which was sealed with capillary tube sealant (Miniseal, American Scientific Products, Stone Mountain, GA) and subsequently positioned inside the electron spin resonance (ESR) cavity.Samples were irradiated in the ES...

Photodynamic inactivation of infectivity of human immunodeficiency virus and other enveloped viruses using hypericin and rose bengal: inhibition of fusion and syncytia formation.

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Quantitative assessment of antioxidant properties of natural colorants and phytochemicals: carotenoids, flavonoids, phenols and indigoids. The role of β‐carotene in antioxidant functions

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The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria

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