Mice were randomized to receive either metformin, mgkgd intraperitoneally, for days or PBS of equal volume.On day, mice were injected with mgkg paraquat dichloride. To obtain serum, a subset of animals was sacriced hours after paraquat injection.The cells were sonicated and debris pelleted by centrifugation at C.We observed a modest increase in intracellular ROS levels following exposure to metformin in both cell lines. As expected, minute exposure to HO increased ROS levels, as detected by DCFDA ow cytometry, and this was not altered when cells were pretreated with metformin for hours, regardless of AMPK expression. Exposure to paraquat for minutes increased ROS levels in both cell lines, in keeping with increased endogenous ROS production. Antimycin binds to cytochromecreductase and leads to the formation of large quantities of ROS. The observation that both paraquat and antimycininduced pERK was attenuated by metformin suggests that the drug decreases ROS levels when they are produced by either complex I, the site of action of paraquat. Mechanistically, this is consistent with induction of ROS defenses by metformin, inhibition of ROS production by the mitochondrial electron transport chain, or a direct scavenging action.The fact that metformin itself induces a modest increase in ROS and fails to reduce ROS related to HO exposure argues against a scavenger or increased ROS detoxication mechanisms.In aqueous solutions, metformin is a poor scavenger of hydroxyl radicals and does not react with either HO or superoxide. Hence, the inhibition of ROS production by complex I remains the most plausible mechanism to explain the observed metformin actions.Blocking NADH oxidation by complex I leads to accumulation of NADH. Inhibition of complex I reduces entry of electrons to the electron transport chain, and therefore, would reduce ROS production by both complex I and III, consistent with our observations.These ndings provide evidence that metformin acts not as a classic antioxidant but rather as a mitochondrial regulator that decreases ROS production associated with oxidative phosphorylation but not ROS produced by nonmitochondrial sources that are required for normal cell signaling and cellular defenses. While mitochondrial ROS production would be expected to preferentially damage mitochondrial as compared with nuclear DNA, nuclear DNA mutations are related to ROS levels. This does not necessarily require direct action of ROS on nuclear DNA, as it has been shown that nuclear mutation may arise due to direct oxidation of the nucleotide pool by ROS. C and D, quantication of the change in ROS levels seen under the indicated conditions.Error bars represent SEM; the differences between ROS levels under the paraquat alone condition and the paraquat plus metformin conditions were signicant. To conrm the generality of our ndings obtained in mouse broblasts, we repeated this experiment using primary HMEC.The DNA damage response constitutes a barrier for tumor progression, and tumors arise when these barriers are inactivated by genetic or epigenetic mechanisms.We considered the possibility that cancer risk reduction by metformin could be attributed at least in part to inhibition of mutagenesis.C, DNA damage foci were estimated by quantifying immunouorescence forgHAX.Representative images are shown, and mean number of cells with foci is shown at the bottom right of each panel.