Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA—alone or in synergism with metals

Therese Bergstrom 0 Clara Ersson 0 Jan Bergman 0 Lennart Moller 0 0 Department of Biosciences and Nutrition, Karolinska Institutet, Novum , SE-141 83 Huddinge , Sweden TThheeAAuutthhoorr22001122..PPuubblilsishheeddbbyyOOxxfofordrdUUninvievresristiytyPrPersesssononbebheahlafloffotfhethUeKUKEnEvnirvoinromnemnetanltaMl uMtaugtaegneSnoScioectiye.ty. AAllll rriigghhttss rreesseerrvveedd.. FFoorr ppeerrmmiissssiioonnss,,pplleeaasseeee--mmaaiill::jjoouurrnnaallss..ppeerrmmiissssiioonnss@@oouupp..ccoomm.. - *To whom correspondence should be addressed. Tel: 46 8 524 810 75; Fax: 46 8 774 55 38; Email: Received on December 15, 2011; revised on February 14, 2012; accepted on February 22, 2012 Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2#-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkalilabile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, b-carotene, folic acid, a-tocopherol, d-tocopherol or g-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak doseresponse increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins. Introduction Antioxidants have the ability to scavenge radicals and protect biomolecules, such as lipids, proteins and DNA against oxidative damage. On the contrary, their ability to accept and donate electrons also enables them to act as pro-oxidants under certain conditionscausing oxidative damage to biomolecules. Oxidative stress is achieved when oxidising agents such as reactive oxygen species (ROS) overwhelm the protective system of the cell. ROS has been suggested to be linked to many different diseases and they seem to be particularly important to the development of cancer and neurodegenerative diseases, such as Parkinsons disease and Alzheimers disease (1). Many dietary antioxidants are also essential vitamins and crucial for a functioning metabolism. Rich sources of antioxidants include fruits and vegetables, which are generally known to protect against oxidative damage and cancer (2). Dietary supplements containing antioxidants have, on the other hand, not been verified to have the same health beneficial effect. On the contrary, intervention and epidemiological studies have shown ambiguous results. Multivitamins administered to a population with a poor nutritional state has been reported to decrease total cancer and mortality risk (3), while a number of studies on healthy individuals have shown different vitamin and antioxidant supplements to have no significant effect on health (4,5). Supplements containing antioxidants or metals (minerals) have also revealed harmful effects, increasing the incidence of prostate cancer (6), lung cancer (79) and mortality (10,11). In the above-mentioned studies, b-carotene, vitamin A, vitamin C and vitamin E as well as iron (Fe) and copper (Cu) have been subject of investigation, along with other antioxidants, vitamins and metals. The reason for the contradicting results is still not fully understood and the factors that might contribute include initial nutritional status, antioxidant dose, the combination of antioxidants and properties of the immediate surrounding such as oxygen pressure. High concentrations of certain antioxidants have been considered to have pro-oxidant properties (12,13). Whether these pro-oxidants properties are physiologically relevant, in vivo is unclear. Vitamin C and b-carotene have been extensively documented to have pro-oxidant properties in different milieus and a high antioxidant dose and an oxidised environment (such as that of a smokers lung) can promote their pro-oxidant behaviour (12,13). In a recent study, we showed that vitamin A and C compounds permitted in supplements can act as pro-oxidants with different potencies (14). A key site of radical and oxidative attack of DNA is at the 8-position of guanine, and the resulting oxidation product 8-oxoguanine (8-oxoGua) is a pro-mutagenic lesion inducing G:C / T:A transversions and the most commonly studied biomarker for oxidative DNA base lesions (15). The aim of the present study was to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Their ability to cause oxidation in a chemical system, by oxidising the nucleoside deoxyguanosine, dG, was evaluated as well as their capacity to cause damage to DNA of cells in culture, where both DNA breaks and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase (FPG)sensitive sites, were measured using the comet assay. Materials and methods FPG was kindly provided by Prof. A.R. Collins (Department of Nutrition, University of Oslo, Norway). The following vitamin and metal compounds were used: L-ascorbic acid, b-carotene, copper (II) sulfate pentahydrate, folic acid, lycopene from tomato, nicotinic acid, pyridoxine hydrochloride, retinol, (-)-riboflavin, seleno-DL-methionine, thiamine hydrochloride, ( )-a-tocopherol, ()-d-tocopherol, ()-c-tocopherol, vitamin B12 and zinc sulfate heptahydrate (SigmaAldrich, St Louis, MO, USA) and iron (II) sulfate heptahydrate (Merck, Darmstadt, Germany). The Cu, Fe and zinc (Zn) compounds were chosen in forms that are listed by the European Parliament and Council (2002) as compounds allowed in commercial supplements. The term metal is in the present study used loosely to include the Cu, Fe and Zn compounds and their related ions (Fe2, Cu2 and Zn2), and Se refers to the selenium compound used. Physiologically relevant concentrations of vit (...truncated)