Analysis of oxidative DNA damage

Work place: Karolinska Institutet, Department of Biosciences and Nutrition at Novum (Huddinge), Center for Nutrition and Toxicology, Unit for Analytical Toxicology.
Supervisors: Prof. Lennart Möller, and Clara Johansson, PhD student.
Contact person: [email protected], 08-6089232.
Time period: 20 weeks. Starting point is flexible (preferably during the summer), according to agreement.

Introduction:
Reactive chemicals including Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) constantly attack the genome (DNA) in our cells. Often, these insults result in oxidative damage to DNA, but defense systems such as antioxidants and DNA repair systems continuously try to neutralize the damage. Oxidative stress has been defined as “a disturbance in the prooxidant-antioxidant balance in favor of the former”, leading to potential damage; i.e. the level of oxidizing agents (primarily ROS) overwhelm the protective antioxidant defense systems and DNA-repair capacity of the cell. A wide range of biomarkers is used to estimate the level of oxidative stress, as well as cancer risk. Oxidation of 2’-deoxyguanosine (dG) by radical attack at the 8-position, gives the oxidation product 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), which is an established biomarker for oxidative stress in DNA. On-line EC (electrochemical) detection after separation with HPLC (high-performance liquid chromatography) is the most common method for analysis of 8-oxodG. Another method for measurement of DNA damage is the Comet assay, which measures DNA damage in intact cells. The Comet assay measures mainly single-strand breaks (SSB) and alkali-labile sites (ALS). To specifically measure oxidative DNA damage with the Comet assay, an additional step can be added where the cells are treated with the DNA repair enzyme Formamidopyrimidine-DNA glycosylase (FPG), which converts modified bases into strand breaks in situ.

A diet rich in fruits and vegetables, which contains a lot of antioxidants, are known to reduce the cancer risk. However, studies using biomarkers are questioning the antioxidant effect from a number of known antioxidants such as vitamin E, ascorbate and beta-carotene. These results may depend on that the protective effect of antioxidants may be a combinatory effect of several antioxidants or that the delicate balance between antioxidants has been disturbed, which possibly have weakened the antioxidant defence. Further, antioxidants can, depending on the chemical surroundings, act as oxidants.

Research project:
To compare the ability of a variety of known antioxidants to protect DNA against induced DNA damage. Different doses of antioxidants will be used since antioxidants in high doses might act as oxidants. Some antioxidants such as ascorbate (vitamin C) has been reported to drastically enhance redox-cycling of transistion metals due to its reducing capacity and might therefore enhance oxidative DNA damage. Pure dG, isolated DNA and intact cells (cultivated human cell lines) will be used.