The biological activity of hydrogen peroxide VII. l-Histidine increases incorporation of H 2O 2 into cells and enhances formation of 8-oxodeoxyguanosine by UV-C plus H 2O 2 but not by H 2O 2 alone

Abstract

l-Histidine ( l-His) enhances the clastogenic effects of hydrogen peroxide (H 2O 2). We previously suggested the involvement of active transport in the efficient influx of an l-His–H 2O 2 adduct into cells (Oya-Ohta et al. [1]). In this study, we detected intracellular H 2O 2 by monitoring formation of 2′,7′-dichlorofluorescein (DCF) from its precursor. More fluoroproduct accumulated dose-dependently in cells treated with a mixture of l-His and H 2O 2 (mixture) than with H 2O 2 alone. This observation supports our hypothesis that active transport is involved in the enhanced incorporation of H 2O 2 into cells. Moreover, both mixture and the l-His–H 2O 2 adduct were less active in the generation of hydroxyl radicals ( OH) upon addition of FeCl 2 than was H 2O 2 alone in a cell-free system. This result suggests that the Fenton reaction might occur more effectively around the nucleus in cells. An immunohistochemical assay using 8-oxodG-specific monoclonal antibodies did not reveal whether the accumulation of H 2O 2 generates 8-oxodeoxyguanosine (8-oxodG). No 8-oxodG was evident in cells treated with mixture or with H 2O 2 alone, or even in cells treated with H 2O 2 at high doses up to 20 mM and, in some cases, pre-treated with catalase inhibitors. It appears, therefore, that OH and, specifically, OH derived from intracellular Fenton reactions, might not play a role in the formation of 8-oxodG. However, exposure to UV-C of cells treated with H 2O 2 yielded more 8-oxodG in the presence of l-His than in the absence of l-His. Thus, the previously observed enhancing effects of l-His were also noted during the induction of formation of 8-oxodG by UV-C plus H 2O 2. The formation of 8-oxodG in response to UV-C alone was very limited and, hence, H 2O 2 seemed to be an effective source of OH only in the presence of UV-C. It is suggested that the OH that induces formation of 8-oxodG is not OH formed via intracellular Fenton reactions but is OH formed via the dissociation of H 2O 2 under UV-C.