Differences in the generation of 8-hydroxyguanine by cigarette smoke in calf-thymus DNA and in A549 cells
In this report we investigated the generation of 8-hydroxyguanine by cigarette smoke using calf-thymus DNA and A549 cells. At first, we exposed the calf-thymus DNA to cigarette smoke bubbled PBS and measured the amount of 8-OhdG, which is the hydrolysate of 8-hydroxyguanine, so as to determine the generation of 8-hydroxyguanine. By the co-incubation of calf-thymus DNA and cigarette whole smoke bubbled PBS (CWSbP), we could observe the generation of 8-hydroxyguanine in a dose dependent manner. But using cigarette gaseous vapour phase smoke bubbled PBS (GVPbP), the generation of 8-hydroxyguanine was not detected in calf-thymus DNA. Next, we investigated the influence of cigarette smoke on the induction of 8-hydroxyguanine in A549 cells. By contrast to the reactivity in calf-thymus DNA, we observed an increased level of 8-hydroxyguanine upon exposure not only to CWSbP, but also to GVPbP. From these results, the generating mechanism of 8-hydroxyguanine seems to be different in calf-thymus DNA, as compared to A549 cells. 8-hydroxyguanine is thought to be generated from the reaction between guanine residue and the hydroxyradical. In the ESR study, the hydroxyradical found in the cigarette particle phase was reported to be generated via Fenton reaction from the hydroperoxide, which was suggested to arise from the coupling reaction with hydroquinone. In order to confirm the involvement of hydroquinone, we applied it to calf-thymus DNA and observed the increase of 8-hydroxyguanine in a dose dependent manner. Further evidence was obtained from the calf-thymus DNA experiment, which indicates the involvement of Fenton reaction. The increase of 8-hydroxyguanine induced by CWSbP was predominantly observed in the acidic pH-range, while it was inhibited by the iron ion chelator defferoxamine. Different mechanisms for the increase in 8-hydroxyguanine by cigarette smoke inside the cells have been proposed, including indirect pathways.