Pristine (C60) and hydroxylated [C60(OH)24] fullerene phototoxicity towards HaCaT keratinocytes:: Type I vs type II mechanisms

The increasing use of fullerene nanomaterials has prompted widespread concern over their biological effects. Herein, we have studied the phototoxicity of gamma-cyclodextrin bicapped pristine C-60 [(gamma-CyD)(2)/C-60] and its water-soluble derivative C-60(OH)(24) toward human keratinocytes. Our results demonstrated that irradiation of (gamma-CyD)(2)/C-60 or C-60(OH)(24) in D2O generated singlet oxygen with quantum yields of 0.76 and 0.08, respectively. Irradiation (> 400 nm) of C-60(OH)(24) generated superoxide as detected by the EPR spin trapping technique; superoxide generation was enhanced by addition of the electron donor nicotinamide adenine dinucleotide (reduced) (NADH). During the irradiation of (gamma-CyD)(2)/C-60, superoxide was generated only in the presence of NADH. Cell viability measurements demonstrated that (y-CyD)(2)/ C-60 was about 60 times more phototoxic to human keratinocytes than C-60(OH)(24). UVA irradiation of human keratinocytes in the presence of (gamma-CyD)(2)/C-60 resulted in a significant rise in intracellular protein-derived peroxides, suggesting a type II mechanism for phototoxicity. UVA irradiation of human keratinocytes in the presence of C-60(OH)(24) produced diffuse intracellular fluorescence when the hydrogen peroxide probe Peroxyfluor-1 was present, suggesting a type I mechanism. Our results clearly show that the phototoxicity induced by (gamma-CyD)(2)/C-60 is Mainly mediated by singlet oxygen with a minor contribution from superoxide, while C-60(OH)(24) phototoxicity is mainly due to superoxide.