Photochemistry and Photobiology, 20**, **: *–* Special Issue Research Article 1 Effects of Light Energy and Reducing Agents on C 60 -Mediated Photosensitizing Reactions † Michael Quinones 1 , Yazhou Zhang 2 , Penelope Riascos 1 , Huey-Min Hwang 3 , Winfred G. Aker 3 , Xiaojia He 3 and Ruomei Gao* 1,2 1 Chemistry and Physics Department, SUNY College at Old Westbury, Old Westbury, NY 2 Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 3 Department of Biology, Jackson State University, Jackson, MS Received 10 August 2013, accepted 28 October 2013, DOI: 10.1111/php.12206 ABSTRACT Many biomolecules contain photoactive reducing agents, such as reduced nicotinamide adenine dinucleotide (NADH) and 6- thioguanine (6-TG) incorporated into DNA through drug metabolism. These reducing agents may produce reactive oxygen species under UVA irradiation or act as electron donors in various media. The interactions of C 60 fullerenes with biological reductants and light energy, especially via the Type-I electron-transfer mechanism, are not fully understood although these factors are often involved in toxicity assess- ments. The two reductants employed in this work were NADH for aqueous solutions and 6-TG for organic solvents. Using steady-state photolysis and electrochemical techniques, we showed that under visible light irradiation, the presence of reducing agents enhanced C 60 -mediated Type-I reactions that generate superoxide anion (O 2 .- ) at the expense of singlet oxygen ( 1 O 2 ) production. The quantum yield of O 2 .- produc- tion upon visible light irradiation of C 60 is estimated below 0.2 in dipolar aprotic media, indicating that the majority of triplet C 60 deactivate via Type-II pathway. Upon UVA irradi- ation, however, both C 60 and NADH undergo photochemical reactions to produce O 2 .- , which could lead to a possible syn- ergistic toxicity effects. C 60 photosensitization via Type-I pathway is not observed in the absence of reducing agents. INTRODUCTION C 60 fullerenes are extensively used in various biomedical appli- cations and produced in large quantities worldwide (1–4) These facts indicate an urgent need for adequate determination of their toxicity in the environment and to humans (5,6). The assessment of C 60 toxicity is hampered by two major obstacles: limited solu- bility and an uncertain toxicity mechanism. The large dissimilari- ties are observed due to the numerous issues involved in toxicity assessment, such as functionalization (7), aggregation (8) and the use of various solvents (9). The fact that C 60 may act as both active oxygen producer and radical scavenger under light illumi- nation further confuses the situation. Various reactive oxygen species (ROS) play different roles in oxidative stress (10–12). It is important to know the factors that affect C 60 photosensitizing reactions that generate ROS. The present work examines the mechanism by which the photoactivity of pristine C 60 is altered by light energy and reducing agents. Pristine C 60 has no acute or subacute toxicity in darkness in a variety of living organisms ranging from human leukocytes, bac- teria and fungi (13). C 60 fullerenes, however, are highly toxic under light exposure due to photosensitized production of ROS (Scheme 1) (14,15). The excited singlet state of C 60 ( 1 C 60 *) pro- duced initially upon irradiation is efficiently converted to the excited triplet state ( 3 C 60 *) by intersystem crossing (ISC) (16). 3 C 60 * is known to undergo energy transfer to ground state oxy- gen ( 3 O 2 ) to produce singlet oxygen ( 1 O 2 ) with high quantum yields (Type-II mechanism) (17). 3 C 60 * may also undergo elec- tron transfer from reductants to produce the radical anion (C 60 ˙ À ) that reduces 3 O 2 to superoxide anion (O 2 ˙ À ) or produces the hydroxyl radical (ÁOH) under Fenton-like conditions (Type-I mechanism) (18,19). The Type-I pathway is favored by the pres- ence of electron donors, such as reduced nicotinamide adenine dinucleotide (NADH). 1 O 2 and O 2 ˙ À are the two primary ROS and play different roles in oxidative damage (10–12). For instance, 1 O 2 oxidizes guanine bases (20,21), whereas the rela- tively unreactive O 2 ˙ À undergoes Fenton reactions to form highly reactive ÁOH, leading to DNA cleavage (22–24). Therefore, it is critical to know the relative amounts of the two primary ROS in assessing C 60 toxicity. Yamakosi et al. reported the first mecha- nistic study of the formation of ROS by underivatized photoex- cited fullerenes in aqueous media in the presence of NADH and Fe 2+ , suggesting that O 2 ˙ À and ÁOH, rather than 1 O 2 , are the active species for DNA cleavage activity (23). DNA cleavage by O 2 ˙ À formed upon visible light irradiation of c-cyclodextrin- bicapped C 60 in the presence of NADH was also demonstrated (24). Unlike 1 O 2 that can be directly observed at its emission wavelength of 1270 nm, the detection of O 2 ˙ À by DNA cleavage tests, spin traps or spectroscopy is indirect and more qualitative. The relative amounts of ROS from C 60 -mediated Type-I and Type-II pathways are usually uncertain. Moreover, biomolecules, such as NADH and 6-thioguanine (6-TG), contain photoactive reducing agents. These reducing agents may facilitate the genera- tion of ROS under UVA irradiation or donate electrons to C 60 fullerenes to alter the photosensitization pathways (19,25–27). *Corresponding author email: gaor@oldwestbury.edu (Ruomei Gao) †This paper is part of the Special Issue honoring the memory of Nicholas J. Turro. © 2013 The American Society of Photobiology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 1 PHP 12206 Dispatch: 16.11.13 CE: Shanoshini Journal Code Manuscript No. No. of pages: 6 PE: Annie -