Ascorbic acid and β-carotene as modulators of oxidative damage

Renata Cozzi1,3, Ruggero Ricordy2, Tania Aglitti1, consequently reducing their mutagenic activity in Chinese Valentina Gatta2, Paolo Perticone2 and Rosella De Salvia2 hamster ovary (CHO*) cells in vitro. Among vitamins, whose antioxidant activity is thought to 1Dipartimento di Biologia, Università degli Studi ‘Roma Tre’, and 2Centro offer protection to DNA from oxidative free radical damage, di Genetica Evoluzionistica, c/o Dipartimento di Genetica e Biologia carotenoids and vitamin C have been extensively studied (3,4). Molecolare, Università ‘La Sapienza’, Roma, Italy While much literature is available concerning the reducing 3To whom correspondence should be addressed: Dipartimento di Genetica e activity of ascorbic acid against mutagens such as ethylmethanBiologia Molecolare, Università degli Studi ‘La Sapienza’, P. le A. Moro 5, I-00185 Roma, Italy sulfonate and N-methyl-N-nitrosoguanidine (5,6) and UV and 4-nitroxyquinoline (7), in 1984 Shamberger (8) summarized Naturally occurring antioxidants are extensively studied the genotoxic effects of vitamin C in different test systems, for their capacity to protect organisms and cells from according to its capacity, at higher doses, to function as a prodamage induced by oxygen reactive species. In fact, oxidaoxidant, itself generating oxygen radicals. tive stress is considered a cause of aging, degenerative Experimental and epidemiological studies are also present disease and cancer. We have focused our attention on two in literature about the anticarcinogenic and chemopreventive agents, ascorbic acid and β-carotene, commonly considered activities of ascorbic acid, although the data are often conto be antioxidants, but whose protective activity against flicting and inconclusive. Recently Drake et al. (9) have cancer is insufficiently known. This paper reports on the proposed that high dietary ascorbic acid intake protects against ability of these agents to act against damage induced gastric cancer. by H2O2 and bleomycin, in Chinese hamster ovary cells cultivated in vitro. Cytogenetic and cytofluorimetric β-Carotene, an important pro-vitamin A, is considered an analyses were performed. Both vitamins proved effective efficient antioxidant (3) acting as a quencher of singlet oxygen in reducing H2O2-induced sister chromatid exchanges, but and free radicals. Experiments in animals have also suggested increased H2O2and bleomycin-induced chromosomal that β-carotene is able to reduce DNA and chromosomal aberrations. Cytofluorimetric data, in contrast, showed damage induced by alkylating agents such as ethylnitrosourea that ascorbic acid and β-carotene act as scavengers of (10) and methylmethanesulfonate (11), and also by proendogeneous and H2O2-induced oxygen species. carcinogens as benzo[a]pyrene (12) and cyclophosphamide (13). β-Carotene has been extensively used in cancerchemopreventive studies, in which it displayed suppressing activity against oral and colon tumors (14,15). Numerous exogenous agents and endogenous processes are The aim of this paper is to investigate β-carotene and capable of generating free radicals in vivo. Among these ascorbic acid scavenging activity, and their effectiveness as molecules the reactive oxygen species are produced not only modulators of DNA damage induced by two oxidants (H2O2 as a consequence of normal metabolism, but also during and bleomycin), in order to find a rational basis for a potential irradiation or metabolic activation of specific chemicals (1). use of such vitamins in cancer therapeutic approaches. Many defence mechanisms within the organism have evolved Cytogenetic and cytofluorimetric experiments were perto limit the levels of damage induced by oxidative stress. formed in CHO cells cultivated in vitro. CHO cells are routinely Among these mechanisms are enzymes (superoxide dismutase, cultured in our laboratory (2). For sister chromatid exchanges catalase, glutathione peroxidase) and also dietary components, (SCE) and chromosomal aberration (ChAb) analysis, cultures such as vitamins, which exhibit scavenging or chelating were treated with the appropriate concentration of ascorbic activity against oxidant molecules. As oxidation phenomena acid or β-carotene; after 15 min in combined treatment, H2O2 in biological systems are considered a cause of aging, was added. This treatment, performed in NaCl (0.9%), lasted degenerative diseases and cancer, particular attention has 30 min. The cultures were then incubated in fresh complete been focused on the possibility of modulating the detrimental medium containing ascorbic acid or β-carotene and bromoeffects of oxidative stress through the use of free radical deoxyuridine (BrdUrd) (for sister chromatid differentiation) at scavengers able to minimize cellular injury, particularly DNA damage. a final concentration of 5310–6 M. Bleomycin treatment was Epidemiological studies and experimental analyses have performed in complete medium and lasted 1 h. All agents been addressed to the identification of natural dietary constituwere prepared immediately before each experiment. Ascorbic ents capable of favourably modulating carcinogenic and mutaacid (Sigma) and β-carotene (Roche) were dissolved in genic processes. We have recently postulated (2) that ellagic distilled water. H2O2 was dissolved in NaCl (0.9%) from a acid, a naturally occurring phenolic lactone, is able to counter30% stock solution. Bleomycin (Rhone-Poulenc) was dissolved act active oxygen species produced by known mutagens, in phosphate-buffered saline (PBS) lacking Ca21 and Mg21. 29,7’-Dichlorofluorescein diacetate (DCFH-DA) was dissolved *Abbreviations: BrdUrd, bromodeoxyuridine; DCFH, dichlorofluorescein; in ethanol. (For concentration of reagents see figures and PBS, phosphate-buffered saline; CHO cells; Chinese hamster ovary cells; tables.) The cells were fixed 26 and 30 h after BrdUrd treatment ChAb, chromosomal aberrations; SCE, sister chromatid exchanges; MI, mitotic index; DCFH-DA, 29,7’-dichlorofluorescein diacetate; a.u., arbitrary units. for SCE analysis and 16 h after for ChAb analysis. Colchicine