Several xenobiotics cause oxidative DNA damage by reactive oxygen species (ROS) induction. The antioxidant defence system, including antioxidant enzymes, radical scavengers and chain breakers, limits cell injury induced by ROS. In particular the antioxidants such as reduced glutathione (GSH) and ascorbic acid (AA) and the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutatione reductase (GR) are implicated in antioxidant cellular response. Oxidative stress occurs when there is an imbalance between production of ROS and cellular antioxidant capacity or when there is a decrease in this capacity. Failure of antioxidant systems may lead to mutagenic oxidative DNA damage as well as deregulation of cell cycle control, resulting in carcinogenesis. In the last years oxidative DNA damage as consequence of anti-oxidant system reduced activity or inhibition has been widely studied by comet test, a rapid and sensitive technique that allows to evaluate DNA damage and its repair on single whole cell. In particular Comet assay modified with Fpg (formamidopyrimidine glycosylase) or endo III enzymes, that recognize and cut oxidized DNA bases, allows to evaluate oxidative DNA damage measuring oxidized purines and pyrimidines respectively. Comet assay also permits to study oxidative DNA repair evaluating the persistence of oxidative DNA damage in relation to repair time, performing the test on cells immediately after induction of DNA damage and after DNA recovery time. Recent studies indicate that oxidant-generating enzymes such as inducible nitric oxide synthase (iNOS) and the inducible cyclooxygenase- 2 (COX-2) are associated with growth and progression of tumour malignancy acting as mediators of inflammation, angiogenesis inductors and cancer proliferation promoters. It allowed to develop new anticancer treatments based on COX-2 inhibitors also in combination with radiation or antineoplastic drugs that may reduce side effects of cancer therapy.