Toyoko Arimoto (1), Toshikazu Yoshikawa (1,*), Hirohisa Takano (2) and
Masahiro Kohno (3)
(1) First Department Internal Medicine, Kyoto Prefectural University of Medicine, Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
(2) National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan
(3) ESR Application Laboratory, Application and Research Center, Analytical Instruments Division, JEOL Ltd., Musashino, Akishima, Tokyo 196-0021, Japan
(*) To whom correspondence should be addressed.
Abstract: The generation of the reactive oxygen species during the interaction of diesel exhaust particles (DEP) with NADPH-cytochrome P450 reductase (P450 reductase) was investigated by electron spin resonance using the spin-trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of DEP extract to an incubation mixture of mouse lung microsomes in the presence of NADPH resulted in a time-dependent NADPH oxidation and acetylated-cytochrome c reduction. Using purified P450 reductase as the enzyme source, superoxide radicals which were detected as the spin adduct (DMPO-OOH) while metabolized by P450 reductase were dependent upon both DEP and enzyme concentrations. The ELISA method using a specific monoclonal antibody revealed that DEP produced 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is formed from deoxyguanosine in DNA by hydroxyl radicals, in the culture medium of L1210 cells. Active oxygen scavengers such as superoxide dismutase and catalase effectively blocked the formation of 8-OHdG in culture medium, and deferoxamine, which inhibits hydroxyl radicals production by chelating iron, was also effective in inhibiting the DEP-produced 8-OHdG formation. These results indicate that DEP components produce 8-OHdG through the hydroxyl radical formation via superoxide by redox cycling of P450 reductase.
Keywords: Diesel exhaust particle, Reactive oxygen species, NADPH-cytochrome P450 reductase, Electron spin resonance, 8-Hydroxy-2'-deoxyguanosine