Publications

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine,5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140°C were raised 8-fold compared to derivatization at 23°C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140°C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured.This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23°C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during the derivatization process.

Abstract Anthracyclines such as doxorubicin remain the first line of treatment for haematological malignancies, and breast cancers. However, the potential risk of cardiac injury by anthracyclines, which may lead to severe myopathy or heart failure, severely limits their application, and remains a challenge to ensuring curative chemotherapy. While the complex interplay between pathological pathways of anthracycline cardiotoxicity is yet to be fully understood, oxidative damage, iron overload-mediated formation of reactive oxygen species (ROS), mitochondrial dysfunction, and inflammation are all believed to be involved. The unique dietary thione, ergothioneine, while not produced in animals and humans, can be avidly absorbed and accumulated in tissues including the heart. Amongst other cytoprotective properties ergothioneine has been shown to scavenge various ROS, decrease proinflammatory mediators, chelate metal cations such as Fe 2+ , preventing them from partaking in redox activities, and may protect against mitochondrial damage and dysfunction. Moreover, low plasma ergothioneine levels are also strongly correlated to risk of cardiovascular events in humans, suggestive of a cardioprotective role. Taken together this highlights the potential for ergothioneine to counteract anthracycline cardiotoxicity. Here we investigate the potential of ergothioneine supplementation to protect against cardiac dysfunction in mice models of doxorubicin-induced cardiotoxicity and found that it had significant protective effects. Moreover, ergothioneine administration in a mouse breast cancer model did not exacerbate the growth of the tumour and did not interfere with the chemotherapeutic efficacy of doxorubicin. These results suggest that ergothioneine could be a viable co-therapy to alleviate the cardiotoxic effects of anthracyclines in the treatment of cancers.

Hypochlorous acid (HOCl), generated by myeloperoxidase released from activated macrophages, is thought to contribute to vascular dysfunction and oxidation of low density lipoproteins (LDLs) in atherogenesis. We have previously shown that HOCl exposure can cause chlorination and oxidation of isolated DNA and that vitamin C protects human arterial smooth muscle cells against oxidized LDL-mediated damage. We report in the present study that vitamin C attenuates HOCl-induced DNA base and protein damage and depletion of intracellular glutathione (GSH) and ATP in human arterial smooth muscle cells. Cells were pretreated in the absence or presence of 100 micromol/L vitamin C (24 hours) and then exposed to HOCl (0 to 500 micromol/L, 0 to 60 minutes) in the absence of vitamin C. Intracellular GSH and ATP levels were depleted by HOCl treatment, and gas chromatography-mass spectroscopy revealed a concentration- and time-dependent increase in DNA base oxidation and protein damage (measured as 3-chlorotyrosine). Pretreatment of smooth muscle cells with vitamin C significantly reduced the extent of HOCl-induced DNA and protein damage and attenuated decreases in intracellular ATP and GSH. Our findings suggest that physiological levels of vitamin C provide an important antioxidant defense against HOCl-mediated injury in atherosclerosis.