To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.

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Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.