Hydroxyl radical production via NADPH oxidase in rat striatum due to carbon monoxide poisoning

Severe poisoning induced by carbon monoxide (CO) at 3000 ppm, but not 1000 ppm, enhances hydroxyl radical (OH) production in rat striatum, which is greatly susceptible to inhibitors of NADPH oxidase (NOX), including diphenyleneiodonium (DPI), but not xanthine oxidase. The quantitative real-time PCR confirmed the previous microarray finding that CO at 3000 ppm, but not 1000 ppm, enhanced mRNA expression of dual oxidase 2 (DUOX2), but not DUOX1, in rat striatum, both of which are NOX family members producing reactive oxygen species. However, the protein levels of DUOX2 and DUOX1 were decreased by 3000 ppm CO. The CO-induced OH production was resistant to chelerythrine and SB230580, inhibitors of protein kinase C and p38MAPK, respectively, which are reported to mediate activation of DUOX1 and DUOX2, respectively. Deprivation of Ca²⁺, which is required for activation of both DUOXs, failed to suppress the CO-induced OH production. The CO-induced OH production was strongly suppressed by EHT1864, an inhibitor of Rac (Ras-related C3 botulinum toxin substrate), which is a factor for activation of NOX1, NOX2 and NOX3 (the role of Rac on Nox3 activation is controversial) as much as that was suppressed by DPI. In addition, EHT1864 in combination with DPI further suppressed the CO-induced OH production. There were no significant changes in the protein levels of NOX1 through NOX4 and Rac1. It is likely that the CO-induced OH production is mediated through the activation of Rac-dependent NOX enzymes, such as Nox1, Nox2, and Nox3.