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