REDUCTION OF OXIDATIVE DNA DAMAGE WITH N-ACETYLCYSTEINE Student Researcher: Coralis Rodrı ´guez, Thomas Armstrong Toro High School Mentors: Eugenio Mun ˜oz; Wilfredo Herna ´ndez, Ponce School of Medicine, Ponce, Puerto Rico Cancer is thought to be the result of accumu- lated mutations in the DNA. A major contrib- utor to these mutations is the damage due to oxidative stress caused by free radicals. Strat- egies that reduce oxidative stress on cells should result in less damage to DNA, less mutations, and thus less cancer. Prevention of cancer could include the consumption of antioxidants, both in foods and in supple- ments. 1 Our studies aim to gather quantitative data to support the notion that antioxidant supplementation reduces cancer by protecting the DNA from oxidative damage. Our hypoth- esis was that an antioxidant supplement can reduce the damage to DNA caused by oxidative free radicals. To test our hypothesis, we chose to subject cell cultures to oxidative damage with H 2 O 2 . Both the control and the experimental culture received 1 mm H 2 O 2 for 4 h, but the exper- imental was preincubated for 24 h with 6 mM N-acetylcysteine (NAC), a potent sulphur- containing antioxidant. At the end of the oxidative stress period, DNA was extracted and analyzed for damage. We also ran controls for no-treatment and treatment with NAC alone. DNA damage was assessed to detect abasic (AP) sites in DNA, which, in the extracted DNA, is a direct measurement of damage. Our preliminary data suggest that cultures exposed to NAC show less AP sites and that NAC reduced the number of AP sites resulting from insult with H 2 O 2 . BACKGROUND The current paradigm of carcino- genesis presents it as the result of accumulated mutations in the DNA. These mutations come from damage to the DNA from several sources. Oxida- tive stress caused by free radicals derived mainly from oxygen metabo- lism is considered a major contributor to the DNA damage that eventually gives rise to many cancers. It follows that strategies that reduce oxidative stress on cells should result in less damage to DNA, less mutations, and thus less cancer. Consumption of antioxidants, in foods or in supple- ments should prevent cancer. 1 Sales of antioxidant supplements on the as- sumption that they will protect from cancer form the basis of a multi- million dollar business. The data published is full of controversy but relatively empty of quantitative data. Our studies aim to gather quantitative data to support the notion that anti- oxidant supplementation reduces can- cer by protecting the DNA from oxidative damage. Our hypothesis was that an antioxidant supplement could reduce the damage to DNA caused by oxidative free radicals. N-acetylcysteine (NAC) is a potent and popular sulphur-containing antiox- idant. It is an immediate precursor of glutathione, a very important natural antioxidant. NAC has been shown to scavenge hydroxyl radicals. 2,3 Our pre- liminary data show that NAC reduces the oxidative damage to DNA, as judged by a reduction of abasic (AP) sites, after insult to cell cultures with H 2 O 2 . METHODS Cell cultures Rat cells (Big Blue, Stratagene, California) were cultured in 50 mL flasks according to the instructions of the supplier. The cells were cultured for about 3 days, until confluent, prior to the experimentation. Experimental cul- tures were exposed for 24 hours with 6 mM NAC. All control cultures were mock treated. Preliminary tolerance tests To adjust the doses of H 2 O 2 to a level where most cells would survive, cultures were treated with several con- centrations of the peroxide. After the treatment, remaining live cells were determined using the MTT assay. 4 We demonstrated that 1 mm H 2 O 2 for 4 hours would kill about 30% of the cells. Peroxide treatment To prevent NAC from reacting with the incoming peroxide in the medium, all cultures were washed with phosphate saline and replenished with fresh medi- um just prior to exposure to peroxide. In this way, the relevant NAC is intracellular. The cells were treated with 1 mm H 2 O 2 for 4 hours, at 37uC in the incubator. At the end of the exposure period, the cells were washed and their DNA collected. Extraction of DNA DNA was extracted with the Gen- Elute kit from Sigma (St. Louis, Mo), following the instructions of the kit. The kit is based on silica adsorption in spin columns. DNA concentration was determined spectrophotometrically. S1-30 Ethnicity & Disease, Volume 18, Spring 2008