Melatonin protects neurons from singlet oxygen‐induced apoptosis

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Abstract
Singlet oxygen (O2[1Δg]) is a very reactive molecule that can be produced by living cells and may contribute to cytotoxicity. The pineal hormone melatonin has been reported to possess potent antioxidant activity, and to be capable of scavenging O2(1Δg). We investigated whether melatonin might reduce the neurotoxic action of O2(lΔg). The cytotoxic effect of singlet oxygen was studied in primary cultures of cerebellar granule neurons pretreated with a photosensitive dye, rose bengal, and exposed to light—a procedure that generates O2(1Δg). We found that this procedure triggers neuronal death, which is preceded by mitochondrial impairment (assayed by the rate of the reduction of MTT, 3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, into formazan), and by DNA fragmentation—a marker of apoptosis. DNA fragmentation was determined in situ by terminal deoxynucleotidyl transferase assay; cell death was assayed with 0.4% trypan blue solution—viable cells with an intact membrane are not permeable to trypan blue; dead cells are, and thus, they are stained blue. Neuroprotection was obtained with the pineal hormone melatonin. In a cell-free system, melatonin also protected the enzyme creatine kinase (EC 2.7.3.2) from the rose bengal-induced injury. The results suggest that melatonin might counteract the cytotoxic action of singlet oxygen. Further studies are needed to clarify the exact role singlet oxygen and melatonin might play in neurodegenerative diseases.