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Oxidative DNA Damage by Vitamin A and Its Derivative via Superoxide Generation

Oxidative DNA Damage by Vitamin A and Its Derivative via Superoxide Generation

Recent intervention studies revealed that β-carotene supplement to smokers resulted in a higher incidence of lung cancer. However, the causal mechanisms remain to be clarified. We reported here that vitamin A (retinol) and its derivative (retinal) caused cellular DNA cleavage detected by pulsed field gel electrophoresis. Retinol and retinal significantly induced 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in HL-60 cells but not in H2O2-resistant HP100 cells, suggesting the involvement of H2O2 in cellular DNA damage. Experiments using 32P-labeled isolated DNA demonstrated that retinol and retinal caused Cu(II)-mediated DNA damage, which was inhibited by catalase. UV-visible spectroscopic and electron spin resonance-trapping studies revealed the generation of superoxide and carbon-centered radicals, respectively. The superoxide generation during autoxidation of retinoids was significantly correlated with the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, although the yield of carbon-centered radicals was not necessarily related to the intensity of DNA damage. These findings suggest that superoxide generated by autoxidation of retinoids was dismutated to H2O2, which was responsible for DNA damage in the presence of endogenous metals. Retinol and retinal have prooxidant abilities, which might lead to carcinogenesis of the supplements of β-carotene.

Many studies have addressed the role of antioxidant vitamins A, C, and E in protection against cancers and cardiovascular diseases (1). It has been suggested that the antioxidant potency of vitamin A and β-carotene may scavenge oxygen radicals and protect against cancer occurrence (1-3). The Alpha-tocopherol, Beta-carotene Cancer Prevention (ATBC) study group (4) and the Beta-carotene and Retinol Efficacy Trial (CARET) (5) supplied β-carotene and/or vitamin A to smokers and asbestos-exposed workers, who were high risk groups for lung cancer. After following up for several years, surprisingly, higher incidences of lung cancer were observed in the intervention groups than the placebo groups. The CARET researchers have suggested that β-carotene can function as a prooxidant under the free-radical-rich atmosphere produced by the chemicals in cigarette smoke and the resultant inflammatory response in the lung (6). The ATBC researchers suspected that β-carotene acts as a promoter of pre-existing latent lung cancers in smokers (7). However, attempts to use retinoids and cartenoids for cancer chemoprevention and therapy are ongoing (8-11). Therefore, the causal mechanisms should be elucidated to establish safe approaches in cancer chemoprevention.

Every antioxidant, including vitamin antioxidants, is in fact a redox (reduction-oxidation) agent, protecting against free radicals in some circumstances and promoting free radical generation in others (12). Several studies have revealed prooxidant effects of vitamin E (13, 14) and vitamin C (15-17) under certain circumstances. Other antioxidants such as N-acetylcysteine (18) and quercetin (19, 20) can cause oxidative damage to cellular and isolated DNA. β-Carotene may also act as both an antioxidant and a prooxidant under various oxygen partial pressures (21-23). Although vitamin A is a good acceptor and donor of electrons in chemical reactions, its properties appear to be very carefully protected by retinol-binding proteins and other endogenous antioxidants in vivo (24). However, pharmacological amounts of the supplements above physiological amounts may perturb key physiological processes.

We report here that low concentrations of vitamin A (retinol) and its derivative (retinal; vitamin A aldehyde) caused cellular DNA cleavage. Furthermore, induction of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)1 by retinol and retinal was investigated in HL-60 cells and its H2O2-resistant clone HP100 cells. To clarify the mechanism of DNA damage, we performed experiments using32P-labeled DNA isolated from the human p53tumor suppressor gene and c-Ha-ras-1 protooncogene. In addition, the time course of 8-oxodG formation was measured by HPLC-ECD, and the reactive species was also detected using UV-visible and ESR spectroscopies.
We report here that both retinol and retinal caused oxidative damage to cellular and isolated DNA. Retinoids significantly induced 8-oxodG formation in HL-60 cells but did not significantly increase 8-oxodG in H2O2-resistant HP100 cells. It is suggested that the generation of H2O2 plays a critical role in oxidative DNA damage by retinol and retinal. When the isolated DNA was used, the existence of Cu(II) was required for the DNA damage. Copper exists in the nucleus and is closely associated with chromosomes and DNA bases in vivo (37). In cell-free systems, catalase and bathocuproine inhibited Cu(II)-mediated DNA damage, suggesting that H2O2 reacts with Cu(I) to produce active species causing DNA damage. DNA-bound Cu(II) can undergo Cu(II)/Cu(I) redox cycling; also, O2 is reduced to O⨪2, which is dismutated to H2O2,resulting in the DNA-Cu(I)-H2O2 complex (33). Typical free ·OH scavengers showed little or no inhibitory effect on the DNA damage. Therefore, it is considered that the DNA damage is caused by a reactive oxygen species such as Cu(I)OOH rather than free ·OH. Site-specific DNA cleavage supports this hypothesis, because it is known that free ·OH causes DNA damage without site specificity (38). Thus, it is speculated that H2O2 reacts with endogenous metals to form a metal-oxygen complex causing oxidative DNA damage in vivo.
It is noteworthy to find that the addition of such low doses of retinoids can induce cellular DNA damage. β-Carotene is converted to two molecules of retinal principally by central cleavage. Retinal is further oxidized to retinoic acid or reduced to retinol. We confirmed using isolated DNA that other derivatives of vitamin A such as retinoic acid and β-ionone and β-carotene itself induced slight DNA damage, including 8-oxodG in the presence of Cu(II), but not efficiently in comparison with retinol and retinal (data not shown). If excessive intake or supplements of vitamin A and β-carotene saturate binding protein, free compounds may have cytotoxicity. This has been suggested by several studies (43 44). Excess amounts of vitamin A increased chromosomal aberration in a lymphocyte culture system (43). Rats treated with retinol had increased incidence of pheochromocytomas in a dose-related manner (44). The supplements of β-carotene and retinol resulted in higher incidences of lung cancer in the intervention groups (4, 5).

It is known that reactive oxygen species are related to tumor-promoting potencies (45, 46). Reid and Loeb (30) indicated that oxidative stress caused tandem double CC → TT mutations. It is very interesting that retinoids strongly generated piperidine-labile sites at the CC sequences in the p53 tumor suppressor gene observed in our study with 32P-labeled DNA fragment. On the basis of the finding that excessive retinol and retinal induced oxidative DNA damage via O⨪2 generation, it is suggested that the oxidative DNA damage may be responsible for initiation and/or tumor promotion/progression in multistage carcinogenesis. Retinol and retinal, β-carotene metabolites, have a stronger ability to induce DNA damage and to generate O⨪2 than β-carotene itself. Therefore, it is suggested that both retinol and retinal play important roles in carcinogenesis in the intervention studies using excess amounts of β-carotene. Further studies will be required for evaluation of safety and efficacy before recommending use of retinoid supplements for cancer chemoprevention.

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