Rosmarinic acid, a natural polyphenol, has a potential pro-oxidant risk via NADH-mediated oxidative DNA damage

Rosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid.

RA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in ³²P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H₂O₂ scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than ^•OH) but not by typical ^•OH scavengers and SOD, indicating the involvement of H₂O₂, Cu(I), and ROS other than ^•OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than ^•OH, because ^•OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H₂O₂ generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA.

In this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH.