Chemical generation of superoxide in the co-presence of riboflavin derivatives and NADH: evidence for a non-enzymatic redox pathway.

We recently reported that flavin-associated compounds such as riboflavin, flavin mononucleotide, and flavin adenine dinucleotide reduce oxidized coenzyme Q and vitamin K homologues to corresponding reduced forms in the presence of reduced β-nicotinamide adenine dinucleotide (NADH). In these processes, coenzyme Q or vitamin K acts as a terminal electron accepter. If oxygen can function as an electron acceptor instead of those quinones, resulting in the formation of superoxide. In this study, superoxide formation from the system of NADH and flavin-associated compounds was examined using a water-soluble tetrazolium salt (WST-1) and 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) assays. Formation of WST-1 formazan and chemiluminescence from MCLA, both of which are specific to superoxide, increased during co-incubation with NADH and a flavin-associated compound, and significantly suppressed with the addition of superoxide dismutase (SOD). Superoxide is an important reactive oxygen species (ROS) that is converted to other ROS, such as hydrogen peroxide or peroxynitrite. HaCaT keratinocytes, immortalized human keratinocytes, were then cultivated with NADH and flavin-associated compounds. Cell viability declined with increasing NADH concentration but was significantly recovered following the addition of SOD and catalase. We propose a chemical pathway for flavin-associated compound and NADH-induced superoxide generation